RecFOR Function Is Required for DNA Repair and Recombination in a RecA Loading-Deficient recB Mutant of Escherichia coli
a Department of Molecular Genetics, Ruder Bos kovic Institute, HR-10002 Zagreb, Croatiai^uk, 百拇医药
b Department of Molecular Biology, Faculty of Science, University of Zagreb, HR-10000 Zagreb, Croatiai^uk, 百拇医药
ABSTRACTi^uk, 百拇医药
The RecA loading activity of the RecBCD enzyme, together with its helicase and 5' "->"i^uk, 百拇医药
3' exonuclease activities, is essential for recombination in Escherichia coli. One particular mutant in the nuclease catalytic center of RecB, i.e., recB1080, produces an enzyme that does not have nuclease activity and is unable to load RecA protein onto single-stranded DNA. There are, however, previously published contradictory data on the recombination proficiency of this mutant. In a recF- background the recB1080 mutant is recombination deficient, whereas in a recF+ genetic background it is recombination proficient. A possible explanation for these contrasting phenotypes may be that the RecFOR system promotes RecA-single-strand DNA filament formation and replaces the RecA loading defect of the RecB1080CD enzyme. We tested this hypothesis by using three in vivo assays. We compared the recombination proficiencies of recB1080, recO, recR, and recF single mutants and recB1080 recO, recB1080 recR, and recB1080 recF double mutants. We show that RecFOR functions rescue the repair and recombination deficiency of the recB1080 mutant and that RecA loading is independent of RecFOR in the recB1080 recD double mutant where this activity is provided by the RecB1080C(D-) enzyme. According to our results as well as previous data, three essential activities for the initiation of recombination in the recB1080 mutant are provided by different proteins, i.e., helicase activity by RecB1080CD, 5' "->"
3' exonuclease by RecJ- and RecA-single-stranded DNA filament formation by RecFOR.rd{s, 百拇医药
HOMOLOGOUS recombination is an essential process required for the repair of DNA damage, restoration of disintegrated replication forks, and the production of genetic variability within a population. In wild-type Escherichia coli most recombinational events are dependent on the RecBCD enzyme. RecBCD is a complex and multifunctional enzyme composed of three subunits, each encoded by a separate gene: recB, recC, and recD. Null mutants in recB and recC genes are deficient in recombination, DNA repair, cell viability, and DNA degradation. On the other hand, recD null mutants are deficient in DNA degradation while recombination, DNA repair, and cell viability are similar to wild-type bacteria (CHAUDHURY and SMITH 1984 ; AMUNDSEN et al. 1986 ). The RecBCD enzyme has several biochemical activities: DNA helicase, double-strand (ds) DNA exonuclease, single-strand (ss) DNA exonuclease, ssDNA endonuclease, {chi} -cutting, and ATPase (KUZMINOV 1999 ; KOWALCZYKOWSKI 2000 ; SMITH 2001 ).
The RecBCD substrate is linear dsDNA with blunt or nearly blunt ends. RecBCD binds to a dsDNA end, and its behavior in vitro depends upon the reaction conditions. When ATP is in excess over Mg2+, RecBCD unwinds the DNA up to the -site, an octamer sequence that stimulates recombination and regulates the activity of the RecBCD enzyme (TAYLOR and SMITH 1992 ). Then RecBCD nicks the strand containing the -sequence (5'-GCTGGTGG-3'), continues unwinding DNA to the end of the DNA molecule, and disassembles into its three subunits (TAYLOR and SMITH 1999 ). However, when there is an excess of Mg2+ over ATP, RecBCD simultaneously unwinds and cuts both DNA strands. It behaves as a destructive nuclease, which degrades both strands but preferentially the 3'-end strand. This mode of action lasts until RecBCD encounters a properly oriented {chi} -site. RecBCD then introduces a nick on the 3'-side of the -site and after that its activity becomes modified. It retains its helicase activity and enhances its 5' "->"
3' ss exonuclease activity. On the other hand, its 3' "->"qj$!, http://www.100md.com
5' ss exonuclease activity is attenuated, and consequently a 3'-ssDNA tail is produced (DIXON and KOWALCZYKOWSKI 1993 ; ANDERSON and KOWALCZYKOWSKI 1997A ). This 3'-ssDNA tail is bound by the RecA protein for the initiation of homologous pairing and strand exchange. An additional property of the modified RecBCD enzyme is that it loads RecA protein onto the ssDNA that it produces (ANDERSON and KOWALCZYKOWSKI 1997B ).qj$!, http://www.100md.com
Modification of the RecBCD enzyme by a -site may be connected with inactivation of the RecD subunit (AMUNDSEN et al. 2000 ). Consequently, RecBC, an enzyme reconstituted in vitro from just RecB and RecC subunits (BOEHMER and EMMERSON 1991 ), and RecBC(D-), an enzyme from recD mutants (AMUNDSEN et al. 2000 ), do not have nuclease activities, do not interact with -sites, do have helicase activity (although weaker than the wild-type enzyme), and are able to constitutively load RecA protein onto ssDNA (CHURCHILL et al. 1999 ). Consistent with this, the RecBC(D-) enzyme promotes {chi} -independent recombination in vivo (CHAUDHURY and SMITH 1984 ).
If the RecBCD enzyme is nonfunctional, as in the recBC sbcBC(D) mutant, the RecF pathway of recombination is activated (HORII and CLARK 1973 ; SMITH 1989 ). In this case, the 3'-ssDNA end is prepared by recQ (helicase) and recJ (5' "->"2[cgw}[, http://www.100md.com
3' ss exonuclease) gene products (LOVETT and KOLODNER 1989 ; CLARK and SANDLER 1994 ; HARMON and KOWALCZYKOWSKI 1998 ; KOWALCZYKOWSKI 2000 ), and RecA loading activity is provided by the products of the recF, recO, and recR (recFOR) genes (UMEZU et al. 1993 ; UMEZU and KOLODNER 1994 ). However, RecFOR does not load RecA protein directly onto ssDNA as does the RecBCD enzyme. It instead replaces single-strand binding (SSB) protein bound on ssDNA with the RecA protein. The current view is that the RecOR complex stimulates RecA-ssDNA filament formation while the RecFR complex prevents its extension when it reaches dsDNA (WEBB et al. 1997 ; BORK et al. 2001 ).2[cgw}[, http://www.100md.com
The biochemical activities of the RecBCD enzyme are essential for its biological functions: recombination, DNA repair, cell viability, and DNA degradation. For example, the nuclease and helicase activities are involved in DNA degradation (ROSAMOND et al. 1979 ; TELANDER-MUSKAVITCH and LINN 1982 ). On the other hand, the helicase and RecA loading activities are essential for recombination (AMUNDSEN et al. 1990 ; ANDERSON and KOWALCZYKOWSKI 1997B ; AMUNDSEN et al. 2000 ; ARNOLD and KOWALCZYKOWSKI 2000 ). However, according to in vitro data, with Mg2+ in excess to ATP, -modified RecBCD enzyme retains its 5' "->"
3' exonuclease activity, which implies a possible role for this activity in recombination (ANDERSON and KOWALCZYKOWSKI 1997A ). Genetic evidence also supports the requirement of the 5' "->"/7, 百拇医药
3' exonuclease activity in RecBCD-mediated recombination. When the nuclease activities of the RecBCD enzyme are inhibited, e.g., in recD mutants (LLOYD et al. 1988 ; LOVETT et al. 1988 ) or in the presence of Gam protein (MURPHY 1991 ; PASKVAN et al. 2001 ), conjugational recombination and DNA repair are dependent on the recJ gene product, which is a 5' "->"/7, 百拇医药
3' ss exonuclease. Also, -recombination and DNA repair after UV irradiation in a recB nuclease-deficient mutant are recJ dependent (JOCKOVICH and MYERS 2001 )./7, 百拇医药
One particular nuclease-deficient mutant is recBD1080A (recB1080). This mutant contains a single amino acid substitution in the nuclease catalytic center of RecB. In addition, the RecB1080CD enzyme is unable to promote RecA loading onto ssDNA, but it retains its helicase activity in vitro (YU et al. 1998 ; ANDERSON et al. 1999 ; WANG et al. 2000 ). However, contrasting conclusions have been reported on its function in vivo. Smith and co-workers proposed that deficiency in RecA loading is responsible for the recombination-deficient phenotype of the recB1080 mutant (AMUNDSEN et al. 2000 ). In their study conjugational recombination and DNA repair after UV irradiation were measured, and the recB1080 mutation was in a recF- genetic background. In contrast, a recent study presented evidence that the recB1080 mutant is recombination proficient for {lambda} -recombination and UV recovery in a recF+ background (JOCKOVICH and MYERS 2001 ). A possible explanation for the high recombination proficiency of the recB1080 mutant in a recF+ background could be that an alternative mechanism of RecA-ssDNA filament formation mediated by RecFOR replaces the RecA loading defect of the RecB1080CD enzyme. In this study we tested this hypothesis and investigated whether conjugational recombination and DNA repair after -irradiation in a recB1080 mutant are dependent on RecFOR and on the RecJ-mediated 5' "->"
3' exonuclease.7', 百拇医药
Using three different assays in vivo, we show that the repair and recombination deficiency of the RecB1080CD enzyme is rescued by the recFOR gene products. We also show that the repair and recombination are independent of RecFOR in the recB1080 recD double mutant. In addition, we found that conjugational recombination and DNA repair after {gamma} -irradiation in the recB1080 mutant require RecJ-dependent 5' "->"7', 百拇医药
3' exonuclease.7', 百拇医药
MATERIALS AND METHODS7', 百拇医药
Bacterial strains and bacteriophages:7', 百拇医药
The list of bacterial strains used in this study is presented in 1. P1 vir phage used for the construction of some bacterial strains was kindly provided by R. G. Lloyd from University of Nottingham, England. Transductions were carried out according to MILLER 1992 .7', 百拇医药
fig.ommitted7', 百拇医药
Table 1. Bacterial strains used in this study
Media and growth conditions:^+umu4, http://www.100md.com
Bacteria were grown in a high salt Luria broth (LB) medium composed of 10 g bacto-tryptone, 5 g yeast extract, and 10 g NaCl, and water was added to 1000 ml. Solid media for plates were supplemented with 16 g/liter of agar. M9 medium contained 0.5 g of NaCl, 1 g of NH4Cl, 3 g of KH2PO4, 7.5 g of Na2HPO4 x 2H2O, 4 g of glucose, 120 mg of MgSO4, and 10 mg of CaCl2, and water was added to 1000 ml. For minimal selective plates, M9 medium was supplemented with appropriate amino acids, 1 mg of thiamine, and 16 g of agar (MARSIC et al. 1993 ). All experiments were done with exponentially growing cells at 37°.^+umu4, http://www.100md.com
Cell survival after - and UV irradiation:^+umu4, http://www.100md.com
For cell survival after -irradiation, 0.1-ml aliquots of the appropriate dilutions of the bacterial culture were plated on LB plates. Surviving cells formed visible colonies during overnight incubation at 37°, and colonies were counted the next day. Cell survival is the ratio of the number of viable cells in a culture after appropriate -dose and the number of viable cells in the control culture. For -irradiation a 60Co source with a dose rate of 11.4 Gy/sec as measured by ferrous sulfate dosimetry was used. Bacteria were irradiated at 0°. For UV irradiation, a 30-W Philips low-pressure Hg germicidal lamp at a distance of 1 m was used. The incident dose was ~ 0.25 mW/cm2, as determined by a UV dosimeter VLX-3W (Bioblock, Illkrich, France). Cell survival after UV irradiation was measured according to AL-DEIB et al. 1996 . Bacteria were irradiated at room temperature.
Conjugational crosses:&)z4m, http://www.100md.com
The procedures for conjugational crosses were those described previously (MILLER 1992 ). Hfr strain KL96 was used as a donor, and the selected marker was His+. Matings were performed in LB medium for 30 min and mixed in a 1:10 donor-to-recipient ratio using recipient and donor cells grown to an OD650 of 0.4. The exconjugant mixture was interrupted by vigorous agitation, serially diluted, and plated on appropriate minimal agar containing 100 µg/ml of streptomycin to counterselect donor cells. Measurements of cell viability relate to the number of colony-forming units in the recipient cultures at an OD650 of 0.4, as determined with nonselective LB agar (RYDER et al. 1994 ). The frequency of conjugational recombination for each experiment was corrected for the recipient's viability relative to wild type.&)z4m, http://www.100md.com
RESULTS AND DISCUSSION&)z4m, http://www.100md.com
Evidence that alternative RecFOR-mediated RecA-ssDNA filament formation rescues the RecA loading deficiency of the RecB1080CD enzyme:
To test whether alternative RecA-ssDNA filament formation by RecFOR replaces the RecA loading defect of the RecB1080CD enzyme, we compared repair and recombination proficiencies of recB1080, recO, recR, and recF single mutants and recB1080 recO, recB1080 recR, and recB1080 recF double mutants. We supposed that the high recombination proficiency of a recB1080 mutant (in a recF+ background) might be caused by RecA-ssDNA filament formation via the RecFOR system if double mutants were strongly recombination deficient. It is known that -recombination in a recB1067 mutant, which shows the same phenotypes as recB1080, is independent of the recR gene product (JOCKOVICH and MYERS 2001 ). This led the authors to propose that both 5' "->"@0x$, 百拇医药
3' exonuclease and RecA loading activities are dependent on the RecJ protein (CORRETTE-BENNETT and LOVETT 1995 ). However, this was not tested for chromosomal recombination. Since - and chromosomal recombination are not identical processes, they may not have the same genetic requirements. Thus, we studied three types of chromosomal recombination: DNA repair after -irradiation, DNA repair after UV irradiation, and conjugational recombination.
In 1A the cell survival curves after {gamma} -irradiation of wild type, recB1080, recBCD, and recB270 null mutants are shown. The recB1080 cells were resistant to {gamma} -rays being just slightly more sensitive than wild-type cells. This result is similar to that obtained for DNA repair after UV irradiation (JOCKOVICH and MYERS 2001 ). In addition, we looked for an effect of the recB1080 mutation in recO, recR, and recF genetic backgrounds, as shown in 1B. Interestingly, the double mutants recB1080 recO, recB1080 recR, and recB1080 recF were extremely sensitive to {gamma} -irradiation, although slightly less sensitive than the recBCD and recB270 null mutants and in contrast to recO, recR, and recF single mutants, which were more resistant. This implies that deficiency in RecA loading of the RecB1080CD enzyme can be observed only when the alternative "RecA loading" is abolished by a recO, recR, or recF mutation. The data from 1B are in accordance with results obtained by Smith and co-workers (AMUNDSEN et al. 2000 ), who concluded that the DNA repair defect after UV irradiation is a consequence of RecA loading deficiency in the recB1080 mutant. According to the data in 1, we concluded that RecFOR-mediated RecA-ssDNA filament formation rescues RecA loading deficiency in the RecB1080CD enzyme (also, compare the cell survival of recB1080 with that of recB1080 recO, recB1080 recR, and recB1080 recF double mutants).
fig.ommittedn*rv1v, 百拇医药
Figure 1. Effect of the recB1080 mutation on DNA repair after -irradiation in a wild-type background (A) and in recO, recR, and recF genetic backgrounds (B). The measure for efficiency of DNA repair was cell survival after different doses of -rays. The experiments were performed with exponentially growing cells at 37°. The values are the means of at least three independent experiments. (A) , wild type (wt; strain AB1157); , recB1080 (RIK174); , recB270 (N4634), recBCD (V2570). (B) •, recR (AM208); , recO (IRB103); , recF (WA576); , recB1080 recR (IIB283); , recB1080 recO (IIB282); , recB1080 recF (IIB289);recBCD (V2570). For more details see the text and 1.n*rv1v, 百拇医药
An additional approach was to study DNA repair proficiency after UV irradiation. Again, double mutants were extremely sensitive, even more sensitive than recBCD and recB270 null mutants (2). The single mutants recB1080, recO, recR, and recF were much more resistant than double mutants. These results are in agreement with the data of DNA repair after -irradiation. The only difference between - and UV repair was observed when the cell survival of double mutants was compared with that of recB null mutants.
fig.ommitted'l-f51f, 百拇医药
Figure 2. Effect of the recB1080 mutation on DNA repair after UV irradiation in a wild-type (wt; A) and in recO, recR, and recF genetic backgrounds (B). The measure for efficiency of DNA repair was cell survival after different UV doses. The experiments were performed with exponentially growing cells at 37°. The values are the means of at least three independent experiments. (A) wt (strain AB1157); recB1080 (RIK174); recB270 (N4634);recBCD (V2570). (B) •, recR (AM208); , recO (IRB103); , recF (WA576); recB1080 recR (IIB283); recB1080 recO (IIB282); recB1080 recF (IIB289); recBCD (V2570). For more details see the text and 1.'l-f51f, 百拇医药
In the case of DNA repair after {gamma} -irradiation, the cell survival of double mutants was similar to the survival of recB null mutants, whereas after UV irradiation the double mutants were more sensitive than recB null mutants. This was perhaps caused by different requirements for the RecFOR function in each particular type of repair. DNA repair after -irradiation is actually double-strand end repair because the most abundant lesions, i.e., double-strand breaks and disintegrated replication forks, have double-strand ends. This repair is almost completely dependent on RecBCD function (KUZMINOV 1999 ; KOWALCZYKOWSKI 2000 ). If the RecA loading deficiency of RecB1080CD is not rescued by the function of RecFOR, as was the case in double mutants, then the consequence is that their maximal recombination deficiency is as in recB null mutants.
DNA repair after UV irradiation is more complex. Two types of recombinational repair operate in UV-treated cells. One type is RecBCD dependent and is thought to be responsible for the repair of disintegrated replication forks, whereas the second is RecFOR mediated and is thought to lead to the repair of single-strand gaps (SSG), which occur in daughter strands after reinitiation of DNA synthesis downstream from a noncoding lesion (GANESAN and SEAWELL 1975; TSENG et al. 1994 ; KUZMINOV 1999 ). The recB null mutant has a deficiency only in the repair of disintegrated replication forks, but the repair of SSGs is normal (KUZMINOV 1999 ). In double mutants, both types of repair are abolished, and consequently these cells are more sensitive than recB null mutants.1d, 百拇医药
In the third approach, we studied the proficiency of conjugational recombination (2). The recB1080 mutant had a moderate recombination defect (relative recombination proficiency was 0.17), while recB270 and {Delta} recBCD were much more recombination deficient (0.028 and 0.005, respectively). The recO and recF single mutants had no effect on conjugational recombination, while recR had a small effect (0.47). This is in agreement with the fact that recombination in these cells is predominantly dependent on RecBCD function (KOWALCZYKOWSKI et al. 1994 ; CROMIE et al. 2001 ). In this respect, conjugational recombination is similar to double-strand end repair. However, the double mutants recB1080 recO, recB1080 recR, and recB1080 recF were strongly recombination deficient with the values of relative recombination proficiencies between those of recBCD and recB270 null mutants. These data support the view that the moderate recombination proficiency of the recB1080 single mutant is due to RecA-ssDNA filament formation by the RecFOR system. Together with the data concerning DNA repair, these results show that chromosomal recombination events have different genetic requirements from {lambda} -recombination since {lambda} -recombination is not dependent on RecFOR function (JOCKOVICH and MYERS 2001 ).
fig.ommitteda, 百拇医药
Table 2. Recombination frequencies in Hfr-mediated conjugational crossesa, 百拇医药
Recombination pathway operating in recB1080 mutants is not the RecF pathway:a, 百拇医药
Our data presented above show that the recombination pathway in recB1080 cells requires the recFOR gene products. Also, data from Myers' group provided evidence that DNA repair after UV irradiation and -recombination in the similar recB1067 strain is dependent on the recJ gene product (JOCKOVICH and MYERS 2001 ). We wanted to test whether DNA repair after -irradiation and conjugational recombination in the recB1080 mutant is also dependent on the recJ gene product. The recJ mutation in a recB1080 background had a strong effect on cell survival (3A) and recombination proficiency (2), similar to the effect of recFOR mutations. This implies that RecJ-mediated 5' "->"a, 百拇医药
3' ss exonuclease activity is indeed essential for double-strand end repair and conjugational recombination in a recB1080 mutant. This is in agreement with the data for UV repair in a recB1080 recJ double mutant presented in 3B and with the study of JOCKOVICH and MYERS 2001 .
fig.ommittedrf:, http://www.100md.com
Figure 3. Effect of the recJ and recQ mutations on DNA repair after - (A) and UV irradiation (B) in wild-type (wt) and recB1080 genetic backgrounds. The measure for efficiency of DNA repair was cell survival after different doses of - and UV irradiation. The experiments were performed with exponentially growing cells at 37°. The values are the means of at least three independent experiments. , wt (strain AB1157); , recJ (JC12123); •, recQ (IRB101); , recB1080 (RIK174); recB1080 recJ (IIB278); , recB1080 recQ (IIB279);recBCD (V2570). For more details see the text and 1.rf:, http://www.100md.com
According to these data, the recombination pathway operating in a recB1080 mutant requires recF, recO, recR, and recJ gene products. Since all of these genes are specific for the RecF pathway, this could suggest that the RecB1080CD pathway of recombination and double-strand end repair is equivalent to the RecF pathway. However, this is not the case, as supported by several arguments. First, by formal definition, the RecF pathway operates in recBC sbcBC(D) mutants (HORII and CLARK 1973 ). This means that it is independent of RecBCD function and that it does not tolerate the presence of ExoI (LEHMAN and NUSSBAUM 1964 ; KUSHNER et al. 1971 ) and SbcCD nucleases (LLOYD and BUCKMAN 1985 ; GIBSON et al. 1992 ; CONNELLY et al. 1997 ). However, by comparing the recombination proficiencies of recB1080 and recB null mutants (3 and 2), it follows that the RecB1080CD pathway is dependent on some RecBCD function (presumably the helicase) and that it tolerates the presence of ExoI and SbcCD nucleases. The recB null mutants have a recombination-deficient phenotype because the missing activities of the RecBCD enzyme (helicase, nuclease, and RecA loading) cannot be replaced by the equivalent functions of the RecF pathway. Interestingly, our results with the recB1080 mutant demonstrate that RecF pathway specific gene products participate in double-strand end repair and conjugational recombination even in the presence of SbcB and SbcCD nucleases. Obviously, this occurs only if the helicase activity of the RecBCD enzyme is preserved, i.e., in the recB1080 mutant. We presume that this is why in recB null mutants, where the helicase activity of the RecBCD enzyme is abolished, restoration of recombination proficiency requires additional suppressor mutations, i.e., sbcBC(D), and the activity of another helicase, i.e., RecQ. This stresses the critical role of the DNA unwinding activity for the specificity of RecB1080CD and RecF pathways. Thus, the RecB1080CD pathway of recombination is not identical to the RecF pathway although both pathways share recJ, recF, recO, and recR gene products.
To further test whether the RecB1080CD pathway of recombination is different from the RecF pathway, we studied the effect of recQ, another RecF pathway specific gene, on conjugational recombination and DNA repair after {gamma} - and UV irradiation. The recB1080 recQ double mutant had recombination proficiency and cell survival after {gamma} -irradiation similar to that of the recB1080 single mutant (2 and 3A). In the case of UV repair, the recQ mutation had a moderate effect, although small in comparison with the effect of recJ mutation (3B). This indicates that the recQ gene product is not needed for RecB1080CD-mediated recombination, although it is required for the RecF pathway (NAKAYAMA et al. 1984 ; MENDONCA et al. 1995 ). Since RecQ is a helicase (UMEZU et al. 1990 ; HARMON and KOWALCZYKOWSKI 1998 ), this is in agreement with the fact that the RecB1080CD enzyme has helicase activity (YU et al. 1998 ).5, 百拇医药
Recombination in a recB1080 recD double mutant is independent of RecFOR:
To further test that the low recombination proficiencies of recB1080 recO, recB1080 recR, and recB1080 recF cells were due to a deficiency in RecA-ssDNA filament formation by both Rec-B1080CD and RecFOR, we also studied recombination proficiency in the recB1080 recD double mutant and recB1080 recD recO triple mutant. It is known that the RecB1080C(D-) form of the enzyme is able to load RecA protein, since the RecD subunit (an inhibitor of RecA loading) is missing (AMUNDSEN et al. 2000 ). Consequently, we expected that recombination proficiency of a recB1080 recD recO triple mutant would be high and similar to that of a recB1080 recD double mutant. We again used the three approaches described before. As expected, the recB1080 recD recO triple mutant was resistant to {gamma} -irradiation (4). It was slightly more sensitive than wild-type and recB1080 recD cells, but almost as resistant as the recO single mutant. This result shows that in the situation in which RecA loading activity is provided by the RecB1080C(D-) enzyme, the recO mutation does not make a cell recombination deficient. The recB1080 recD recO triple mutant and recO single mutant were more sensitive to -irradiation than were wild-type cells and the recB1080 recD double mutant, because recO mutation has a slight effect on the repair of double-strand breaks mediated by RecBCD (CROMIE and LEACH 2001 ) and RecB1080C(D-) enzymes.
fig.ommittedwe?'k, 百拇医药
Figure 4. Effect of the recO mutation on DNA repair after -irradiation in wild-type (wt), recB1080, and recB1080 recD genetic backgrounds. The values are the means of at least three independent experiments. , wt (AB1157); , recB1080 recD (IIB290); •, recO (IRB103); , recB1080 recD recO (IIB291); , recB1080 recO (IIB282). For more details see legend of 1, 1, and the text.we?'k, 百拇医药
DNA repair proficiencies after UV irradiation are presented in 5. Wild-type cells and the recB1080 recD double mutant were UV resistant, while the recO single mutant and recB1080 recD recO triple mutant were more sensitive, but still significantly more resistant, than the recB1080 recO double mutant. The effect of recO mutation in wild-type and recB1080 recD backgrounds is higher after UV irradiation than after {gamma} -irradiation, due to a deficiency in SSG repair that requires RecFOR function (TSENG et al. 1994 ). The important point is that cell survival after UV irradiation of recO and recB1080 recD recO mutants is similar and that the recO mutation does not cause as strong a deficiency in recombination as it does in the recB1080 background. Similar results were obtained with recR, recB1080 recD recR, and recB1080 recR as well as with recF, recB1080 recD recF, and recB1080 recF mutants (data not shown). It follows that in the recB1080 recD genetic background there is no need for RecFOR function since RecA loading activity is provided by the RecB1080C(D-) enzyme (AMUNDSEN et al. 2000 ).
fig.ommitted9/, http://www.100md.com
Figure 5. Effect of the recO mutation on DNA repair after UV irradiation in wild-type (wt), recB1080, and recB1080 recD genetic backgrounds. The values are the means of at least three independent experiments. , wt (AB1157); , recB1080 recD (IIB290); , recO (IRB103); , recB1080 recD recO (IIB291); , recB1080 recO (IIB282). For more details see legend of Fig 2, Table 1, and the text.9/, http://www.100md.com
Data on the recombination proficiencies after conjugational recombination are presented in 2. The triple mutants recB1080 recD recO, recB1080 recD recR, and recB1080 recD recF had a slight effect on recombination proficiency with values of 0.30, 0.62, and 0.48, respectively. In these strains the RecFOR function was not essential since recombinational processing of free ends is predominantly RecBCD dependent (KOWALCZYKOWSKI et al. 1994 ; CROMIE et al. 2001 ). These and the above data concerning DNA repair strongly suggest that recombination deficiency in recB1080 recO, recB1080 recR, and recB1080 recF double mutants is due to the lack of essential RecA-ssDNA filament formation.
Mechanism for the initiation of recombination in a recB1080 mutant:6gv2q, http://www.100md.com
As was mentioned earlier, the biochemical activities of the RecBCD enzyme essential for recombination are its helicase, 5' "->"6gv2q, http://www.100md.com
3' ss exonuclease and RecA loading activities. According to our results and previous data (YU et al. 1998 ; AMUNDSEN et al. 2000 ; JOCKOVICH and MYERS 2001 ), we propose a model for the initiation of recombination mediated by the RecB1080CD enzyme, as shown in 6. In wild-type cells all of these activities are provided by the RecBCD enzyme itself. The situation is different in the recB1080 recD double mutant, where these activities are provided by two different enzymes. RecB1080C(D-) is responsible for the helicase and RecA loading activities, while RecJ provides a 5' "->"6gv2q, http://www.100md.com
3' ss exonuclease. The most complex situation is in the recB1080 single mutant where these activities are provided by RecB1080CD (helicase), RecJ (5' "->"
3' ss exonuclease), and RecFOR (RecA-ssDNA filament formation). Recombination in the recB1080 mutant is a nice example of sophisticated functional redundancy between recombinational genes in presynapsis. The mechanism of RecA filament formation in the recB1080 mutant is probably different from that in wild-type cells and the recB1080 recD double mutant. The -activated RecBCD enzyme and RecB1080C(D-) enzyme coordinately load RecA protein with DNA unwinding. This enables the RecA protein to bind ssDNA before SSB, which otherwise binds faster than RecA protein (KOWALCZYKOWSKI 2000 ). Since RecB1080CD enzyme does not load RecA protein, SSB preferentially binds to ssDNA. The role of the RecFOR system is to replace SSB with the RecA protein (UMEZU et al. 1993 ; KOWALCZYKOWSKI 2000 ). This feature is also incorporated in the model (6). The outcome of the initiation of recombination (presynapsis) is a recombinogenic (RecA-ssDNA) filament that initiates the search for homology and strand exchange with a homologous DNA molecule (synapsis).
fig.ommittedpky, 百拇医药
Figure 6. Model for the initiation of recombination in a recB1080 mutant compared with the same process in wild-type (wt) cells and a recB1080 recD double mutant. For simplicity RecFOR is represented by a single symbol, although the details of RecF, RecO, and RecR action are not completely clear. The arrowheads represent 3' ends of DNA strands. For a full description see the text.pky, 百拇医药
In conclusion, we have shown that alternative RecA-ssDNA filament formation by RecFOR is essential for recombination in a recB1080 mutant and this explains its high recombination proficiency. Furthermore, we have demonstrated that RecJ-mediated 5' "->"pky, 百拇医药
3' ss exonuclease activity is also essential for conjugational recombination and DNA repair after -irradiation in the recB1080 mutant.pky, 百拇医药
ACKNOWLEDGMENTSpky, 百拇医药
We are grateful to R. S. Myers (University of Miami School of Medicine), to G. R. Smith (Fred Hutchinson Cancer Research Center, Seattle), to W. Wackernagel (University of Oldenburg, Germany), and to R. G. Lloyd (University of Nottingham, UK) for sending us bacterial strains. We are also grateful to Milan Blaz evic (Ru&d\mbox|<|--|>|;er Bos kovic Institute) for technical assistance on -irradiation, to Zoran Tadi (University of Zagreb) for help in drawing figures, and to Mary Sopta (Ru&d\mbox|<|--|>|;er Bos kovi Institute) for correcting the English. This work was supported by the Croatian Ministry of Science (grant 0098 1001).
Manuscript received July 28, 2002; Accepted for publication November 14, 2002.lw], 百拇医药
LITERATURE CITEDlw], 百拇医药
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AMUNDSEN, S. K., A. F. TAYLOR, A. M. CHAUDHURY, and G. R. SMITH, 1986 recD: the gene for an essential third subunit of exonuclease V. Proc. Natl. Acad. Sci. USA 83:5558-5562.lw], 百拇医药
AMUNDSEN, S. K., A. M. NEIMAN, S. M. THIBODEAUX, and G. R. SMITH, 1990 Genetic dissection of the biochemical activities of RecBCD enzyme. Genetics 126:25-40.lw], 百拇医药
AMUNDSEN, S. K., A. F. TAYLOR, and G. R. SMITH, 2000 The RecD subunit of the Escherichia coli RecBCD enzyme inhibits RecA loading, homologous recombination, and DNA repair. Proc. Natl. Acad. Sci. USA 97:7399-7404.lw], 百拇医药
ANDERSON, D. G. and S. C. KOWALCZYKOWSKI, 1997a The recombination hotspot is a regulatory element that switches the polarity of DNA degradation by the RecBCD enzyme. Genes Dev. 11:571-581.
ANDERSON, D. G. and S. C. KOWALCZYKOWSKI, 1997b The translocation RecBCD enzyme stimulates recombination by directing RecA protein onto ssDNA in a {chi} -regulated manner. Cell 90:77-86.6e!), 百拇医药
ANDERSON, D. G., J. J. CHURCHILL, and S. C. KOWALCZYKOWSKI, 1999 A single mutation recBD1080A eliminates RecA protein loading but not chi recognition by RecBCD enzyme. J. Biol. Chem. 274:27139-27144.6e!), 百拇医药
ARNOLD, D. A. and S. C. KOWALCZYKOWSKI, 2000 Facilitated loading of RecA protein is essential to recombination by RecBCD enzyme. J. Biol. Chem. 275:12261-12265.6e!), 百拇医药
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MENDONCA, V. M., H. D. KLEPIN, and S. W. MATSON, 1995 DNA helicases in recombination and repair: construction of a uvrD helD recQ mutant deficient in recombination and repair. J. Bacteriol. 177:1326-1335.?, 百拇医药
MILLER, J. H., 1992 Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.?, 百拇医药
MURPHY, K. C., 1991 {lambda} Gam protein inhibits the helicase and {chi} -stimulated recombination activities of Escherichia coli RecBCD enzyme. J. Bacteriol. 173:5808-5821.?, 百拇医药
NAKAYAMA, H., K. NAKAYAMA, R. NAKAYAMA, N. IRINO, and Y. NAKAYAMA et al., 1984 Isolation and genetic characterization of a thymineless death-resistant mutant of Escherichia coli K 12: identification of a new mutation (recQ1) that blocks the RecF recombination pathway. Mol. Gen. Genet. 195:474-480.?, 百拇医药
PAs KVAN, I., E. SALAJ-S MIC, I. IVANc Ic -BAc E, K. ZAHRADKA, and . TRGOVc EVIc et al., 2001 The genetic dependence of RecBCD-Gam mediated double strand end repair in Escherichia coli.. FEMS Microbiol. Lett. 205:299-303.
ROSAMOND, J., K. M. TELANDER, and S. LINN, 1979 Modulation of the action of the RecBC enzyme of Escherichia coli K-12 by Ca2+. J. Biol. Chem. 254:8646-8652./m(l|, http://www.100md.com
RYDER, L., M. C. WHITBY, and R. G. LLOYD, 1994 Mutation of recF, recJ, recO, recQ, or recR improves Hfr recombination in resolvase-deficient ruv recG strains of Escherichia coli.. J. Bacteriol. 176:1570-1577./m(l|, http://www.100md.com
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TAYLOR, A. F. and G. R. SMITH, 1992 RecBCD enzyme is altered upon cutting DNA at a chi recombinational hotspot. Proc. Natl. Acad. Sci. USA 89:5226-5230./m(l|, http://www.100md.com
TAYLOR, A. F. and G. R. SMITH, 1999 Regulation of homologous recombination: chi inactivates RecBCD enzyme by disassembly of the three subunits. Genes Dev. 13:890-900.
TELANDER-MUSKAVITCH, K. M. and S. LINN, 1982 A unified mechanism for the nuclease and unwinding activities of the RecBC enzyme of Escherichia coli.. J. Biol. Chem. 257:2641-2648.6], http://www.100md.com
TSENG, Y. C., J. L. HUNG, and T. C. V. WANG, 1994 Involvement of the RecF pathway recombination genes in postreplication repair in UV-irradiated Escherichia coli cells. Mutat. Res. 315:1-9.6], http://www.100md.com
UMEZU, K. and R. D. KOLODNER, 1994 Protein interactions in genetic recombination in Escherichia coli. Interactions involving RecO and recR overcome the inhibition of RecA by single-stranded DNA binding protein. J. Biol. Chem. 269:30005-30013.6], http://www.100md.com
UMEZU, K., K. NAKAYAMA, and H. NAKAYAMA, 1990 Escherichia coli RecQ protein is a DNA helicase. Proc. Natl. Acad. Sci. USA 87:5363-5367.6], http://www.100md.com
UMEZU, K., N. W. CHI, and R. D. KOLODNER, 1993 Biochemical interaction of the Escherichia coli RecF, RecO and RecR proteins with RecA protein and single-stranded DNA binding protein. Proc. Natl. Acad. Sci. USA 90:3875-3879.6], http://www.100md.com
WANG, J., R. CHEN, and D. A. JULIN, 2000 A single nuclease active site of the Escherichia coli RecBCD enzyme catalyzes single-stranded DNA degradation in both directions. J. Biol. Chem. 275:507-513.6], http://www.100md.com
WEBB, B. L., M. M. COX, and R. B. INMAN, 1997 Recombinational DNA repair: the RecF and RecR proteins limit the extension of RecA filaments beyond single-strand DNA gaps. Cell 91:347-356.6], http://www.100md.com
YU, M., J. SOUAYA, and D. A. JULIN, 1998 Identification of the nuclease active site in the multifunctional RecBCD enzyme by creation of a chimeric enzyme. J. Mol. Biol. 283:797-808.(Ivana Ivani-Bae Petra Peharec Sunana Moslavac Nikolina krobota, Erika Salaj-mic and Krunoslav Bri-Ko)
b Department of Molecular Biology, Faculty of Science, University of Zagreb, HR-10000 Zagreb, Croatiai^uk, 百拇医药
ABSTRACTi^uk, 百拇医药
The RecA loading activity of the RecBCD enzyme, together with its helicase and 5' "->"i^uk, 百拇医药
3' exonuclease activities, is essential for recombination in Escherichia coli. One particular mutant in the nuclease catalytic center of RecB, i.e., recB1080, produces an enzyme that does not have nuclease activity and is unable to load RecA protein onto single-stranded DNA. There are, however, previously published contradictory data on the recombination proficiency of this mutant. In a recF- background the recB1080 mutant is recombination deficient, whereas in a recF+ genetic background it is recombination proficient. A possible explanation for these contrasting phenotypes may be that the RecFOR system promotes RecA-single-strand DNA filament formation and replaces the RecA loading defect of the RecB1080CD enzyme. We tested this hypothesis by using three in vivo assays. We compared the recombination proficiencies of recB1080, recO, recR, and recF single mutants and recB1080 recO, recB1080 recR, and recB1080 recF double mutants. We show that RecFOR functions rescue the repair and recombination deficiency of the recB1080 mutant and that RecA loading is independent of RecFOR in the recB1080 recD double mutant where this activity is provided by the RecB1080C(D-) enzyme. According to our results as well as previous data, three essential activities for the initiation of recombination in the recB1080 mutant are provided by different proteins, i.e., helicase activity by RecB1080CD, 5' "->"
3' exonuclease by RecJ- and RecA-single-stranded DNA filament formation by RecFOR.rd{s, 百拇医药
HOMOLOGOUS recombination is an essential process required for the repair of DNA damage, restoration of disintegrated replication forks, and the production of genetic variability within a population. In wild-type Escherichia coli most recombinational events are dependent on the RecBCD enzyme. RecBCD is a complex and multifunctional enzyme composed of three subunits, each encoded by a separate gene: recB, recC, and recD. Null mutants in recB and recC genes are deficient in recombination, DNA repair, cell viability, and DNA degradation. On the other hand, recD null mutants are deficient in DNA degradation while recombination, DNA repair, and cell viability are similar to wild-type bacteria (CHAUDHURY and SMITH 1984 ; AMUNDSEN et al. 1986 ). The RecBCD enzyme has several biochemical activities: DNA helicase, double-strand (ds) DNA exonuclease, single-strand (ss) DNA exonuclease, ssDNA endonuclease, {chi} -cutting, and ATPase (KUZMINOV 1999 ; KOWALCZYKOWSKI 2000 ; SMITH 2001 ).
The RecBCD substrate is linear dsDNA with blunt or nearly blunt ends. RecBCD binds to a dsDNA end, and its behavior in vitro depends upon the reaction conditions. When ATP is in excess over Mg2+, RecBCD unwinds the DNA up to the -site, an octamer sequence that stimulates recombination and regulates the activity of the RecBCD enzyme (TAYLOR and SMITH 1992 ). Then RecBCD nicks the strand containing the -sequence (5'-GCTGGTGG-3'), continues unwinding DNA to the end of the DNA molecule, and disassembles into its three subunits (TAYLOR and SMITH 1999 ). However, when there is an excess of Mg2+ over ATP, RecBCD simultaneously unwinds and cuts both DNA strands. It behaves as a destructive nuclease, which degrades both strands but preferentially the 3'-end strand. This mode of action lasts until RecBCD encounters a properly oriented {chi} -site. RecBCD then introduces a nick on the 3'-side of the -site and after that its activity becomes modified. It retains its helicase activity and enhances its 5' "->"
3' ss exonuclease activity. On the other hand, its 3' "->"qj$!, http://www.100md.com
5' ss exonuclease activity is attenuated, and consequently a 3'-ssDNA tail is produced (DIXON and KOWALCZYKOWSKI 1993 ; ANDERSON and KOWALCZYKOWSKI 1997A ). This 3'-ssDNA tail is bound by the RecA protein for the initiation of homologous pairing and strand exchange. An additional property of the modified RecBCD enzyme is that it loads RecA protein onto the ssDNA that it produces (ANDERSON and KOWALCZYKOWSKI 1997B ).qj$!, http://www.100md.com
Modification of the RecBCD enzyme by a -site may be connected with inactivation of the RecD subunit (AMUNDSEN et al. 2000 ). Consequently, RecBC, an enzyme reconstituted in vitro from just RecB and RecC subunits (BOEHMER and EMMERSON 1991 ), and RecBC(D-), an enzyme from recD mutants (AMUNDSEN et al. 2000 ), do not have nuclease activities, do not interact with -sites, do have helicase activity (although weaker than the wild-type enzyme), and are able to constitutively load RecA protein onto ssDNA (CHURCHILL et al. 1999 ). Consistent with this, the RecBC(D-) enzyme promotes {chi} -independent recombination in vivo (CHAUDHURY and SMITH 1984 ).
If the RecBCD enzyme is nonfunctional, as in the recBC sbcBC(D) mutant, the RecF pathway of recombination is activated (HORII and CLARK 1973 ; SMITH 1989 ). In this case, the 3'-ssDNA end is prepared by recQ (helicase) and recJ (5' "->"2[cgw}[, http://www.100md.com
3' ss exonuclease) gene products (LOVETT and KOLODNER 1989 ; CLARK and SANDLER 1994 ; HARMON and KOWALCZYKOWSKI 1998 ; KOWALCZYKOWSKI 2000 ), and RecA loading activity is provided by the products of the recF, recO, and recR (recFOR) genes (UMEZU et al. 1993 ; UMEZU and KOLODNER 1994 ). However, RecFOR does not load RecA protein directly onto ssDNA as does the RecBCD enzyme. It instead replaces single-strand binding (SSB) protein bound on ssDNA with the RecA protein. The current view is that the RecOR complex stimulates RecA-ssDNA filament formation while the RecFR complex prevents its extension when it reaches dsDNA (WEBB et al. 1997 ; BORK et al. 2001 ).2[cgw}[, http://www.100md.com
The biochemical activities of the RecBCD enzyme are essential for its biological functions: recombination, DNA repair, cell viability, and DNA degradation. For example, the nuclease and helicase activities are involved in DNA degradation (ROSAMOND et al. 1979 ; TELANDER-MUSKAVITCH and LINN 1982 ). On the other hand, the helicase and RecA loading activities are essential for recombination (AMUNDSEN et al. 1990 ; ANDERSON and KOWALCZYKOWSKI 1997B ; AMUNDSEN et al. 2000 ; ARNOLD and KOWALCZYKOWSKI 2000 ). However, according to in vitro data, with Mg2+ in excess to ATP, -modified RecBCD enzyme retains its 5' "->"
3' exonuclease activity, which implies a possible role for this activity in recombination (ANDERSON and KOWALCZYKOWSKI 1997A ). Genetic evidence also supports the requirement of the 5' "->"/7, 百拇医药
3' exonuclease activity in RecBCD-mediated recombination. When the nuclease activities of the RecBCD enzyme are inhibited, e.g., in recD mutants (LLOYD et al. 1988 ; LOVETT et al. 1988 ) or in the presence of Gam protein (MURPHY 1991 ; PASKVAN et al. 2001 ), conjugational recombination and DNA repair are dependent on the recJ gene product, which is a 5' "->"/7, 百拇医药
3' ss exonuclease. Also, -recombination and DNA repair after UV irradiation in a recB nuclease-deficient mutant are recJ dependent (JOCKOVICH and MYERS 2001 )./7, 百拇医药
One particular nuclease-deficient mutant is recBD1080A (recB1080). This mutant contains a single amino acid substitution in the nuclease catalytic center of RecB. In addition, the RecB1080CD enzyme is unable to promote RecA loading onto ssDNA, but it retains its helicase activity in vitro (YU et al. 1998 ; ANDERSON et al. 1999 ; WANG et al. 2000 ). However, contrasting conclusions have been reported on its function in vivo. Smith and co-workers proposed that deficiency in RecA loading is responsible for the recombination-deficient phenotype of the recB1080 mutant (AMUNDSEN et al. 2000 ). In their study conjugational recombination and DNA repair after UV irradiation were measured, and the recB1080 mutation was in a recF- genetic background. In contrast, a recent study presented evidence that the recB1080 mutant is recombination proficient for {lambda} -recombination and UV recovery in a recF+ background (JOCKOVICH and MYERS 2001 ). A possible explanation for the high recombination proficiency of the recB1080 mutant in a recF+ background could be that an alternative mechanism of RecA-ssDNA filament formation mediated by RecFOR replaces the RecA loading defect of the RecB1080CD enzyme. In this study we tested this hypothesis and investigated whether conjugational recombination and DNA repair after -irradiation in a recB1080 mutant are dependent on RecFOR and on the RecJ-mediated 5' "->"
3' exonuclease.7', 百拇医药
Using three different assays in vivo, we show that the repair and recombination deficiency of the RecB1080CD enzyme is rescued by the recFOR gene products. We also show that the repair and recombination are independent of RecFOR in the recB1080 recD double mutant. In addition, we found that conjugational recombination and DNA repair after {gamma} -irradiation in the recB1080 mutant require RecJ-dependent 5' "->"7', 百拇医药
3' exonuclease.7', 百拇医药
MATERIALS AND METHODS7', 百拇医药
Bacterial strains and bacteriophages:7', 百拇医药
The list of bacterial strains used in this study is presented in 1. P1 vir phage used for the construction of some bacterial strains was kindly provided by R. G. Lloyd from University of Nottingham, England. Transductions were carried out according to MILLER 1992 .7', 百拇医药
fig.ommitted7', 百拇医药
Table 1. Bacterial strains used in this study
Media and growth conditions:^+umu4, http://www.100md.com
Bacteria were grown in a high salt Luria broth (LB) medium composed of 10 g bacto-tryptone, 5 g yeast extract, and 10 g NaCl, and water was added to 1000 ml. Solid media for plates were supplemented with 16 g/liter of agar. M9 medium contained 0.5 g of NaCl, 1 g of NH4Cl, 3 g of KH2PO4, 7.5 g of Na2HPO4 x 2H2O, 4 g of glucose, 120 mg of MgSO4, and 10 mg of CaCl2, and water was added to 1000 ml. For minimal selective plates, M9 medium was supplemented with appropriate amino acids, 1 mg of thiamine, and 16 g of agar (MARSIC et al. 1993 ). All experiments were done with exponentially growing cells at 37°.^+umu4, http://www.100md.com
Cell survival after - and UV irradiation:^+umu4, http://www.100md.com
For cell survival after -irradiation, 0.1-ml aliquots of the appropriate dilutions of the bacterial culture were plated on LB plates. Surviving cells formed visible colonies during overnight incubation at 37°, and colonies were counted the next day. Cell survival is the ratio of the number of viable cells in a culture after appropriate -dose and the number of viable cells in the control culture. For -irradiation a 60Co source with a dose rate of 11.4 Gy/sec as measured by ferrous sulfate dosimetry was used. Bacteria were irradiated at 0°. For UV irradiation, a 30-W Philips low-pressure Hg germicidal lamp at a distance of 1 m was used. The incident dose was ~ 0.25 mW/cm2, as determined by a UV dosimeter VLX-3W (Bioblock, Illkrich, France). Cell survival after UV irradiation was measured according to AL-DEIB et al. 1996 . Bacteria were irradiated at room temperature.
Conjugational crosses:&)z4m, http://www.100md.com
The procedures for conjugational crosses were those described previously (MILLER 1992 ). Hfr strain KL96 was used as a donor, and the selected marker was His+. Matings were performed in LB medium for 30 min and mixed in a 1:10 donor-to-recipient ratio using recipient and donor cells grown to an OD650 of 0.4. The exconjugant mixture was interrupted by vigorous agitation, serially diluted, and plated on appropriate minimal agar containing 100 µg/ml of streptomycin to counterselect donor cells. Measurements of cell viability relate to the number of colony-forming units in the recipient cultures at an OD650 of 0.4, as determined with nonselective LB agar (RYDER et al. 1994 ). The frequency of conjugational recombination for each experiment was corrected for the recipient's viability relative to wild type.&)z4m, http://www.100md.com
RESULTS AND DISCUSSION&)z4m, http://www.100md.com
Evidence that alternative RecFOR-mediated RecA-ssDNA filament formation rescues the RecA loading deficiency of the RecB1080CD enzyme:
To test whether alternative RecA-ssDNA filament formation by RecFOR replaces the RecA loading defect of the RecB1080CD enzyme, we compared repair and recombination proficiencies of recB1080, recO, recR, and recF single mutants and recB1080 recO, recB1080 recR, and recB1080 recF double mutants. We supposed that the high recombination proficiency of a recB1080 mutant (in a recF+ background) might be caused by RecA-ssDNA filament formation via the RecFOR system if double mutants were strongly recombination deficient. It is known that -recombination in a recB1067 mutant, which shows the same phenotypes as recB1080, is independent of the recR gene product (JOCKOVICH and MYERS 2001 ). This led the authors to propose that both 5' "->"@0x$, 百拇医药
3' exonuclease and RecA loading activities are dependent on the RecJ protein (CORRETTE-BENNETT and LOVETT 1995 ). However, this was not tested for chromosomal recombination. Since - and chromosomal recombination are not identical processes, they may not have the same genetic requirements. Thus, we studied three types of chromosomal recombination: DNA repair after -irradiation, DNA repair after UV irradiation, and conjugational recombination.
In 1A the cell survival curves after {gamma} -irradiation of wild type, recB1080, recBCD, and recB270 null mutants are shown. The recB1080 cells were resistant to {gamma} -rays being just slightly more sensitive than wild-type cells. This result is similar to that obtained for DNA repair after UV irradiation (JOCKOVICH and MYERS 2001 ). In addition, we looked for an effect of the recB1080 mutation in recO, recR, and recF genetic backgrounds, as shown in 1B. Interestingly, the double mutants recB1080 recO, recB1080 recR, and recB1080 recF were extremely sensitive to {gamma} -irradiation, although slightly less sensitive than the recBCD and recB270 null mutants and in contrast to recO, recR, and recF single mutants, which were more resistant. This implies that deficiency in RecA loading of the RecB1080CD enzyme can be observed only when the alternative "RecA loading" is abolished by a recO, recR, or recF mutation. The data from 1B are in accordance with results obtained by Smith and co-workers (AMUNDSEN et al. 2000 ), who concluded that the DNA repair defect after UV irradiation is a consequence of RecA loading deficiency in the recB1080 mutant. According to the data in 1, we concluded that RecFOR-mediated RecA-ssDNA filament formation rescues RecA loading deficiency in the RecB1080CD enzyme (also, compare the cell survival of recB1080 with that of recB1080 recO, recB1080 recR, and recB1080 recF double mutants).
fig.ommittedn*rv1v, 百拇医药
Figure 1. Effect of the recB1080 mutation on DNA repair after -irradiation in a wild-type background (A) and in recO, recR, and recF genetic backgrounds (B). The measure for efficiency of DNA repair was cell survival after different doses of -rays. The experiments were performed with exponentially growing cells at 37°. The values are the means of at least three independent experiments. (A) , wild type (wt; strain AB1157); , recB1080 (RIK174); , recB270 (N4634), recBCD (V2570). (B) •, recR (AM208); , recO (IRB103); , recF (WA576); , recB1080 recR (IIB283); , recB1080 recO (IIB282); , recB1080 recF (IIB289);recBCD (V2570). For more details see the text and 1.n*rv1v, 百拇医药
An additional approach was to study DNA repair proficiency after UV irradiation. Again, double mutants were extremely sensitive, even more sensitive than recBCD and recB270 null mutants (2). The single mutants recB1080, recO, recR, and recF were much more resistant than double mutants. These results are in agreement with the data of DNA repair after -irradiation. The only difference between - and UV repair was observed when the cell survival of double mutants was compared with that of recB null mutants.
fig.ommitted'l-f51f, 百拇医药
Figure 2. Effect of the recB1080 mutation on DNA repair after UV irradiation in a wild-type (wt; A) and in recO, recR, and recF genetic backgrounds (B). The measure for efficiency of DNA repair was cell survival after different UV doses. The experiments were performed with exponentially growing cells at 37°. The values are the means of at least three independent experiments. (A) wt (strain AB1157); recB1080 (RIK174); recB270 (N4634);recBCD (V2570). (B) •, recR (AM208); , recO (IRB103); , recF (WA576); recB1080 recR (IIB283); recB1080 recO (IIB282); recB1080 recF (IIB289); recBCD (V2570). For more details see the text and 1.'l-f51f, 百拇医药
In the case of DNA repair after {gamma} -irradiation, the cell survival of double mutants was similar to the survival of recB null mutants, whereas after UV irradiation the double mutants were more sensitive than recB null mutants. This was perhaps caused by different requirements for the RecFOR function in each particular type of repair. DNA repair after -irradiation is actually double-strand end repair because the most abundant lesions, i.e., double-strand breaks and disintegrated replication forks, have double-strand ends. This repair is almost completely dependent on RecBCD function (KUZMINOV 1999 ; KOWALCZYKOWSKI 2000 ). If the RecA loading deficiency of RecB1080CD is not rescued by the function of RecFOR, as was the case in double mutants, then the consequence is that their maximal recombination deficiency is as in recB null mutants.
DNA repair after UV irradiation is more complex. Two types of recombinational repair operate in UV-treated cells. One type is RecBCD dependent and is thought to be responsible for the repair of disintegrated replication forks, whereas the second is RecFOR mediated and is thought to lead to the repair of single-strand gaps (SSG), which occur in daughter strands after reinitiation of DNA synthesis downstream from a noncoding lesion (GANESAN and SEAWELL 1975; TSENG et al. 1994 ; KUZMINOV 1999 ). The recB null mutant has a deficiency only in the repair of disintegrated replication forks, but the repair of SSGs is normal (KUZMINOV 1999 ). In double mutants, both types of repair are abolished, and consequently these cells are more sensitive than recB null mutants.1d, 百拇医药
In the third approach, we studied the proficiency of conjugational recombination (2). The recB1080 mutant had a moderate recombination defect (relative recombination proficiency was 0.17), while recB270 and {Delta} recBCD were much more recombination deficient (0.028 and 0.005, respectively). The recO and recF single mutants had no effect on conjugational recombination, while recR had a small effect (0.47). This is in agreement with the fact that recombination in these cells is predominantly dependent on RecBCD function (KOWALCZYKOWSKI et al. 1994 ; CROMIE et al. 2001 ). In this respect, conjugational recombination is similar to double-strand end repair. However, the double mutants recB1080 recO, recB1080 recR, and recB1080 recF were strongly recombination deficient with the values of relative recombination proficiencies between those of recBCD and recB270 null mutants. These data support the view that the moderate recombination proficiency of the recB1080 single mutant is due to RecA-ssDNA filament formation by the RecFOR system. Together with the data concerning DNA repair, these results show that chromosomal recombination events have different genetic requirements from {lambda} -recombination since {lambda} -recombination is not dependent on RecFOR function (JOCKOVICH and MYERS 2001 ).
fig.ommitteda, 百拇医药
Table 2. Recombination frequencies in Hfr-mediated conjugational crossesa, 百拇医药
Recombination pathway operating in recB1080 mutants is not the RecF pathway:a, 百拇医药
Our data presented above show that the recombination pathway in recB1080 cells requires the recFOR gene products. Also, data from Myers' group provided evidence that DNA repair after UV irradiation and -recombination in the similar recB1067 strain is dependent on the recJ gene product (JOCKOVICH and MYERS 2001 ). We wanted to test whether DNA repair after -irradiation and conjugational recombination in the recB1080 mutant is also dependent on the recJ gene product. The recJ mutation in a recB1080 background had a strong effect on cell survival (3A) and recombination proficiency (2), similar to the effect of recFOR mutations. This implies that RecJ-mediated 5' "->"a, 百拇医药
3' ss exonuclease activity is indeed essential for double-strand end repair and conjugational recombination in a recB1080 mutant. This is in agreement with the data for UV repair in a recB1080 recJ double mutant presented in 3B and with the study of JOCKOVICH and MYERS 2001 .
fig.ommittedrf:, http://www.100md.com
Figure 3. Effect of the recJ and recQ mutations on DNA repair after - (A) and UV irradiation (B) in wild-type (wt) and recB1080 genetic backgrounds. The measure for efficiency of DNA repair was cell survival after different doses of - and UV irradiation. The experiments were performed with exponentially growing cells at 37°. The values are the means of at least three independent experiments. , wt (strain AB1157); , recJ (JC12123); •, recQ (IRB101); , recB1080 (RIK174); recB1080 recJ (IIB278); , recB1080 recQ (IIB279);recBCD (V2570). For more details see the text and 1.rf:, http://www.100md.com
According to these data, the recombination pathway operating in a recB1080 mutant requires recF, recO, recR, and recJ gene products. Since all of these genes are specific for the RecF pathway, this could suggest that the RecB1080CD pathway of recombination and double-strand end repair is equivalent to the RecF pathway. However, this is not the case, as supported by several arguments. First, by formal definition, the RecF pathway operates in recBC sbcBC(D) mutants (HORII and CLARK 1973 ). This means that it is independent of RecBCD function and that it does not tolerate the presence of ExoI (LEHMAN and NUSSBAUM 1964 ; KUSHNER et al. 1971 ) and SbcCD nucleases (LLOYD and BUCKMAN 1985 ; GIBSON et al. 1992 ; CONNELLY et al. 1997 ). However, by comparing the recombination proficiencies of recB1080 and recB null mutants (3 and 2), it follows that the RecB1080CD pathway is dependent on some RecBCD function (presumably the helicase) and that it tolerates the presence of ExoI and SbcCD nucleases. The recB null mutants have a recombination-deficient phenotype because the missing activities of the RecBCD enzyme (helicase, nuclease, and RecA loading) cannot be replaced by the equivalent functions of the RecF pathway. Interestingly, our results with the recB1080 mutant demonstrate that RecF pathway specific gene products participate in double-strand end repair and conjugational recombination even in the presence of SbcB and SbcCD nucleases. Obviously, this occurs only if the helicase activity of the RecBCD enzyme is preserved, i.e., in the recB1080 mutant. We presume that this is why in recB null mutants, where the helicase activity of the RecBCD enzyme is abolished, restoration of recombination proficiency requires additional suppressor mutations, i.e., sbcBC(D), and the activity of another helicase, i.e., RecQ. This stresses the critical role of the DNA unwinding activity for the specificity of RecB1080CD and RecF pathways. Thus, the RecB1080CD pathway of recombination is not identical to the RecF pathway although both pathways share recJ, recF, recO, and recR gene products.
To further test whether the RecB1080CD pathway of recombination is different from the RecF pathway, we studied the effect of recQ, another RecF pathway specific gene, on conjugational recombination and DNA repair after {gamma} - and UV irradiation. The recB1080 recQ double mutant had recombination proficiency and cell survival after {gamma} -irradiation similar to that of the recB1080 single mutant (2 and 3A). In the case of UV repair, the recQ mutation had a moderate effect, although small in comparison with the effect of recJ mutation (3B). This indicates that the recQ gene product is not needed for RecB1080CD-mediated recombination, although it is required for the RecF pathway (NAKAYAMA et al. 1984 ; MENDONCA et al. 1995 ). Since RecQ is a helicase (UMEZU et al. 1990 ; HARMON and KOWALCZYKOWSKI 1998 ), this is in agreement with the fact that the RecB1080CD enzyme has helicase activity (YU et al. 1998 ).5, 百拇医药
Recombination in a recB1080 recD double mutant is independent of RecFOR:
To further test that the low recombination proficiencies of recB1080 recO, recB1080 recR, and recB1080 recF cells were due to a deficiency in RecA-ssDNA filament formation by both Rec-B1080CD and RecFOR, we also studied recombination proficiency in the recB1080 recD double mutant and recB1080 recD recO triple mutant. It is known that the RecB1080C(D-) form of the enzyme is able to load RecA protein, since the RecD subunit (an inhibitor of RecA loading) is missing (AMUNDSEN et al. 2000 ). Consequently, we expected that recombination proficiency of a recB1080 recD recO triple mutant would be high and similar to that of a recB1080 recD double mutant. We again used the three approaches described before. As expected, the recB1080 recD recO triple mutant was resistant to {gamma} -irradiation (4). It was slightly more sensitive than wild-type and recB1080 recD cells, but almost as resistant as the recO single mutant. This result shows that in the situation in which RecA loading activity is provided by the RecB1080C(D-) enzyme, the recO mutation does not make a cell recombination deficient. The recB1080 recD recO triple mutant and recO single mutant were more sensitive to -irradiation than were wild-type cells and the recB1080 recD double mutant, because recO mutation has a slight effect on the repair of double-strand breaks mediated by RecBCD (CROMIE and LEACH 2001 ) and RecB1080C(D-) enzymes.
fig.ommittedwe?'k, 百拇医药
Figure 4. Effect of the recO mutation on DNA repair after -irradiation in wild-type (wt), recB1080, and recB1080 recD genetic backgrounds. The values are the means of at least three independent experiments. , wt (AB1157); , recB1080 recD (IIB290); •, recO (IRB103); , recB1080 recD recO (IIB291); , recB1080 recO (IIB282). For more details see legend of 1, 1, and the text.we?'k, 百拇医药
DNA repair proficiencies after UV irradiation are presented in 5. Wild-type cells and the recB1080 recD double mutant were UV resistant, while the recO single mutant and recB1080 recD recO triple mutant were more sensitive, but still significantly more resistant, than the recB1080 recO double mutant. The effect of recO mutation in wild-type and recB1080 recD backgrounds is higher after UV irradiation than after {gamma} -irradiation, due to a deficiency in SSG repair that requires RecFOR function (TSENG et al. 1994 ). The important point is that cell survival after UV irradiation of recO and recB1080 recD recO mutants is similar and that the recO mutation does not cause as strong a deficiency in recombination as it does in the recB1080 background. Similar results were obtained with recR, recB1080 recD recR, and recB1080 recR as well as with recF, recB1080 recD recF, and recB1080 recF mutants (data not shown). It follows that in the recB1080 recD genetic background there is no need for RecFOR function since RecA loading activity is provided by the RecB1080C(D-) enzyme (AMUNDSEN et al. 2000 ).
fig.ommitted9/, http://www.100md.com
Figure 5. Effect of the recO mutation on DNA repair after UV irradiation in wild-type (wt), recB1080, and recB1080 recD genetic backgrounds. The values are the means of at least three independent experiments. , wt (AB1157); , recB1080 recD (IIB290); , recO (IRB103); , recB1080 recD recO (IIB291); , recB1080 recO (IIB282). For more details see legend of Fig 2, Table 1, and the text.9/, http://www.100md.com
Data on the recombination proficiencies after conjugational recombination are presented in 2. The triple mutants recB1080 recD recO, recB1080 recD recR, and recB1080 recD recF had a slight effect on recombination proficiency with values of 0.30, 0.62, and 0.48, respectively. In these strains the RecFOR function was not essential since recombinational processing of free ends is predominantly RecBCD dependent (KOWALCZYKOWSKI et al. 1994 ; CROMIE et al. 2001 ). These and the above data concerning DNA repair strongly suggest that recombination deficiency in recB1080 recO, recB1080 recR, and recB1080 recF double mutants is due to the lack of essential RecA-ssDNA filament formation.
Mechanism for the initiation of recombination in a recB1080 mutant:6gv2q, http://www.100md.com
As was mentioned earlier, the biochemical activities of the RecBCD enzyme essential for recombination are its helicase, 5' "->"6gv2q, http://www.100md.com
3' ss exonuclease and RecA loading activities. According to our results and previous data (YU et al. 1998 ; AMUNDSEN et al. 2000 ; JOCKOVICH and MYERS 2001 ), we propose a model for the initiation of recombination mediated by the RecB1080CD enzyme, as shown in 6. In wild-type cells all of these activities are provided by the RecBCD enzyme itself. The situation is different in the recB1080 recD double mutant, where these activities are provided by two different enzymes. RecB1080C(D-) is responsible for the helicase and RecA loading activities, while RecJ provides a 5' "->"6gv2q, http://www.100md.com
3' ss exonuclease. The most complex situation is in the recB1080 single mutant where these activities are provided by RecB1080CD (helicase), RecJ (5' "->"
3' ss exonuclease), and RecFOR (RecA-ssDNA filament formation). Recombination in the recB1080 mutant is a nice example of sophisticated functional redundancy between recombinational genes in presynapsis. The mechanism of RecA filament formation in the recB1080 mutant is probably different from that in wild-type cells and the recB1080 recD double mutant. The -activated RecBCD enzyme and RecB1080C(D-) enzyme coordinately load RecA protein with DNA unwinding. This enables the RecA protein to bind ssDNA before SSB, which otherwise binds faster than RecA protein (KOWALCZYKOWSKI 2000 ). Since RecB1080CD enzyme does not load RecA protein, SSB preferentially binds to ssDNA. The role of the RecFOR system is to replace SSB with the RecA protein (UMEZU et al. 1993 ; KOWALCZYKOWSKI 2000 ). This feature is also incorporated in the model (6). The outcome of the initiation of recombination (presynapsis) is a recombinogenic (RecA-ssDNA) filament that initiates the search for homology and strand exchange with a homologous DNA molecule (synapsis).
fig.ommittedpky, 百拇医药
Figure 6. Model for the initiation of recombination in a recB1080 mutant compared with the same process in wild-type (wt) cells and a recB1080 recD double mutant. For simplicity RecFOR is represented by a single symbol, although the details of RecF, RecO, and RecR action are not completely clear. The arrowheads represent 3' ends of DNA strands. For a full description see the text.pky, 百拇医药
In conclusion, we have shown that alternative RecA-ssDNA filament formation by RecFOR is essential for recombination in a recB1080 mutant and this explains its high recombination proficiency. Furthermore, we have demonstrated that RecJ-mediated 5' "->"pky, 百拇医药
3' ss exonuclease activity is also essential for conjugational recombination and DNA repair after -irradiation in the recB1080 mutant.pky, 百拇医药
ACKNOWLEDGMENTSpky, 百拇医药
We are grateful to R. S. Myers (University of Miami School of Medicine), to G. R. Smith (Fred Hutchinson Cancer Research Center, Seattle), to W. Wackernagel (University of Oldenburg, Germany), and to R. G. Lloyd (University of Nottingham, UK) for sending us bacterial strains. We are also grateful to Milan Blaz evic (Ru&d\mbox|<|--|>|;er Bos kovic Institute) for technical assistance on -irradiation, to Zoran Tadi (University of Zagreb) for help in drawing figures, and to Mary Sopta (Ru&d\mbox|<|--|>|;er Bos kovi Institute) for correcting the English. This work was supported by the Croatian Ministry of Science (grant 0098 1001).
Manuscript received July 28, 2002; Accepted for publication November 14, 2002.lw], 百拇医药
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