Kin-Hoe Chow

DNA lesions that block replication are a primary cause of rearrangements, mutations, and lethality in all cells. After ultraviolet (UV)-induced DNA damage in Escherichia coli, replication recovery requires RecA and several other recF pathway proteins. To characterize the mechanism by which lesion-blocked replication forks recover, we used two-dimensional agarose gel electrophoresis to show that replication-blocking DNA lesions induce a transient reversal of the replication fork in vivo. The reversed replication fork intermediate is stabilized by RecA and RecF and is degraded by the RecQ-RecJ helicase-nuclease when these proteins are absent. We propose that fork regression allows repair enzymes to gain access to the replication-blocking lesion, allowing processive replication to resume once the blocking lesion is removed.

Abstract Glioma stem cells (GSC) and epithelial–mesenchymal transition (EMT) are strongly associated with therapy resistance and tumor recurrence, but the underlying mechanisms are incompletely understood. Here, we show that S100A4 is a novel biomarker of GSCs. S100A4+ cells in gliomas are enriched with cancer cells that have tumor-initiating and sphere-forming abilities, with the majority located in perivascular niches where GSCs are found. Selective ablation of S100A4-expressing cells was sufficient to block tumor growth in vitro and in vivo. We also identified S100A4 as a critical regulator of GSC self-renewal in mouse and patient-derived glioma tumorspheres. In contrast with previous reports of S100A4 as a reporter of EMT, we discovered that S100A4 is an upstream regulator of the master EMT regulators SNAIL2 and ZEB along with other mesenchymal transition regulators in glioblastoma. Overall, our results establish S100A4 as a central node in a molecular network that controls stemness and EMT in glioblastoma, suggesting S100A4 as a candidate therapeutic target. Cancer Res; 77(19); 5360–73. ©2017 AACR.

In Escherichia coli, recF and recR are required to stabilize and maintain replication forks arrested by UV-induced DNA damage. In the absence of RecF, replication fails to recover, and the nascent lagging strand of the arrested replication fork is extensively degraded by the RecQ helicase and RecJ nuclease. recO mutants are epistatic with recF and recR with respect to recombination and survival assays after DNA damage. In this study, we show that RecO functions with RecF and RecR to protect the nascent lagging strand of arrested replication forks after UV-irradiation. In the absence of RecO, the nascent DNA at arrested replication forks is extensively degraded and replication fails to recover. The extent of nascent DNA degradation is equivalent in single, double, or triple mutants of recF, recO, or recR, and the degradation is dependent upon RecJ and RecQ functions. Because RecF has been shown to protect the nascent lagging strand from degradation, these observations indicate that RecR and RecO function with RecF to protect the same nascent strand of the arrested replication fork and are likely to act at a common point during the recovery process. We discuss these results in relation to the biochemical and cellular properties of RecF, RecO, and RecR and their potential role in loading RecA filaments to maintain the replication fork structure after the arrest of replication by UV-induced DNA damage. In Escherichia coli, recF and recR are required to stabilize and maintain replication forks arrested by UV-induced DNA damage. In the absence of RecF, replication fails to recover, and the nascent lagging strand of the arrested replication fork is extensively degraded by the RecQ helicase and RecJ nuclease. recO mutants are epistatic with recF and recR with respect to recombination and survival assays after DNA damage. In this study, we show that RecO functions with RecF and RecR to protect the nascent lagging strand of arrested replication forks after UV-irradiation. In the absence of RecO, the nascent DNA at arrested replication forks is extensively degraded and replication fails to recover. The extent of nascent DNA degradation is equivalent in single, double, or triple mutants of recF, recO, or recR, and the degradation is dependent upon RecJ and RecQ functions. Because RecF has been shown to protect the nascent lagging strand from degradation, these observations indicate that RecR and RecO function with RecF to protect the same nascent strand of the arrested replication fork and are likely to act at a common point during the recovery process. We discuss these results in relation to the biochemical and cellular properties of RecF, RecO, and RecR and their potential role in loading RecA filaments to maintain the replication fork structure after the arrest of replication by UV-induced DNA damage. The failure to accurately replicate the genomic template in the presence of DNA damage is believed to be a primary cause of mutagenesis, genomic rearrangements, and lethality in all cells. Irradiation with near-UV (254 nm) light induces DNA lesions that block replication (1Smith K.C. Mutat. Res. 1969; 8: 481-495Google Scholar, 2Setlow R.B. Swenson P.A. Carrier W.L. Science. 1963; 142: 1464-1466Google Scholar). In wild-type Escherichia coli, replication is inhibited after a moderate dose of UV irradiation, but it efficiently recovers at a time correlating with the removal of the lesions by the nucleotide excision repair proteins (2Setlow R.B. Swenson P.A. Carrier W.L. Science. 1963; 142: 1464-1466Google Scholar, 3Setlow R.B. Carrier W.L. Proc. Natl. Acad. Sci. U. S. A. 1963; 51: 226Google Scholar, 4Courcelle J. Crowley D.J. Hanawalt P.C. J. Bacteriol. 1999; 181: 916-922Google Scholar). The recovery of replication in E. coli requires RecA to stabilize and maintain the integrity of replication forks after arrest by DNA lesions. Mutants lacking RecA fail to recover replication after UV-irradiation and exhibit a rapid and eventually complete degradation of the chromosome (5Courcelle J. Carswell-Crumpton C. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3714-3719Google Scholar, 6Horii Z.I. Suzuki K. Photochem. Photobiol. 1968; 8: 93-105Google Scholar). The degradation initiates at the blocked replication forks and regresses back from these points (6Horii Z.I. Suzuki K. Photochem. Photobiol. 1968; 8: 93-105Google Scholar). In vitro, RecA proteins will bind to form a filament on single-strand DNA, and they pair the singlestrand region with homologous duplex DNA (7Shan Q. Bork J.M. Webb B.L. Inman R.B. Cox M.M. J. Mol. Biol. 1997; 265: 519-540Google Scholar, 8Konforti B.B. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 690-694Google Scholar, 9Cox M.M. Lehman I.R. J. Biol. Chem. 1982; 257: 8523-8532Google Scholar), an activity which would be consistent with maintaining the DNA at blocked replication forks (10Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Google Scholar, 11Roca A.I. Cox M.M. Prog. Nucleic Acid Res. Mol. Biol. 1997; 56: 129-223Google Scholar). Similar to recA, recF, and recR mutants also fail to maintain replication forks that are blocked by DNA damage and do not recover replication (4Courcelle J. Crowley D.J. Hanawalt P.C. J. Bacteriol. 1999; 181: 916-922Google Scholar, 5Courcelle J. Carswell-Crumpton C. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3714-3719Google Scholar). In contrast to recA mutants, however, the DNA degradation is less extensive and is limited to ∼50% of the nascent DNA localized at the blocked replication fork (4Courcelle J. Crowley D.J. Hanawalt P.C. J. Bacteriol. 1999; 181: 916-922Google Scholar, 5Courcelle J. Carswell-Crumpton C. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3714-3719Google Scholar). In vitro, RecF, RecO, and RecR interact with and stabilize RecA filaments bound to DNA (12Umezu K. Chi N.W. Kolodner R.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3875-3879Google Scholar), a role that would be consistent with the in vivo observation of limiting DNA degradation at the replication fork. Mutants lacking RecF and RecR also exhibit a delayed induction of the SOS response, consistent with the idea that these genes may help stabilize the RecA filaments which are required for SOS induction (7Shan Q. Bork J.M. Webb B.L. Inman R.B. Cox M.M. J. Mol. Biol. 1997; 265: 519-540Google Scholar, 13Thoms B. Wackernagel W. J. Bacteriol. 1987; 169: 1731-1736Google Scholar, 14Hegde S. Sandler S.J. Clark A.J. Madiraju M.V. Mol. Gen. Genet. 1995; 246: 254-258Google Scholar, 15Whitby M.C. Lloyd R.G. Mol. Gen. Gent. 1995; 246: 174-179Google Scholar). The nascent DNA degradation that occurs in recF mutants has been shown to result from the combined action of RecQ, a 3′-5′ helicase, and RecJ, a 5′-3′ exonuclease (16Courcelle J. Hanawalt P.C. Mol. Gen. Gent. 1999; 262: 543-551Google Scholar, 17Courcelle J. Donaldson J.R. Chow K.H. Courcelle C.T. Science. 2003; 299: 1064-1067Google Scholar). Based upon the extent of nascent DNA degradation in recF mutants (5Courcelle J. Carswell-Crumpton C. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3714-3719Google Scholar), the polarity of the helicase and nuclease in vitro (18Lovett S.T. Kolodner R.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2627-2631Google Scholar, 19Umezu K. Nakayama K. Nakayama H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5363-5367Google Scholar), and the preferential loss of the nascent lagging strand DNA at the fork (16Courcelle J. Hanawalt P.C. Mol. Gen. Gent. 1999; 262: 543-551Google Scholar), RecJ and RecQ are thought to degrade the nascent lagging strand of blocked replication forks prior to the recovery of replication, as depicted in the model shown in Fig. 6. In the absence of either gene product, the nascent DNA degradation does not occur irrespective of whether RecF is present to protect the lagging strand DNA (16Courcelle J. Hanawalt P.C. Mol. Gen. Gent. 1999; 262: 543-551Google Scholar), and the frequency of illegitimate recombination is altered (20Hanada K. Ukita T. Kohno Y. Saito K. Kato J. Ikeda H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3860-3865Google Scholar, 21Ukita T. Ikeda H. J. Bacteriol. 1996; 178: 2362-2367Google Scholar), suggesting that these enzymes may affect the frequency with which replication resumes accurately when it is disrupted. recO is classified with recF, recR, recJ, and recQ as genes belonging to the recF pathway. Like recF and recR, recO mutants exhibit a similar delay in SOS induction, hypersensitivity to UV irradiation, reduced plasmid recombination, reduced conjugational recombination in a recBCsbcBC background, and the persistence of daughter-strand gaps in the nascent DNA of UV-irradiated uvrA mutants (22Horii Z. Clark A.J. J. Mol. Biol. 1973; 80: 327-344Google Scholar, 23Kolodner R. Fishel R.A. Howard M. J. Bacteriol. 1985; 163: 1060-1066Google Scholar, 24Mahdi A.A. Lloyd R.G. Mol. Gen. Genet. 1989; 216: 503-510Google Scholar). Based upon these pheno-types, recF, recR, and recO have been suggested to form an epistatic group, RecFOR. In vitro, RecO physically interacts with both RecF and RecR to form either RecFO, RecRO, or RecFRO complexes, a role which is believed to be important for RecA stabilization (7Shan Q. Bork J.M. Webb B.L. Inman R.B. Cox M.M. J. Mol. Biol. 1997; 265: 519-540Google Scholar, 12Umezu K. Chi N.W. Kolodner R.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3875-3879Google Scholar, 25Umezu K. Kolodner R.D. J. Biol. Chem. 1994; 269: 30005-30013Google Scholar, 26Hedge S.P. Qin M.H. Li X.H. Atkinson M.A.L. Clark A.J. Rajagopalan M. Madiraju M.V.V.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14468-14473Google Scholar). These observations, taken together, suggest that RecO may be required with RecF and RecR to maintain arrested replication forks and promote the resumption of DNA synthesis after arrest. However, to date, the structures that RecO and RecR act upon at DNA damage-blocked replication forks in vivo have not been examined. To identify the relationship between RecF, RecO, and RecR at arrested replication fork structures in vivo, we have characterized the role that RecO plays during the recovery of replication after UV-induced DNA damage to determine when and where this protein functions during the recovery process. Bacterial Strains—SR108 is a thyA36 deoC2 derivative of W3110 (27De-Lucia P. Cairns J. Nature. 1969; 224: 1164-1166Google Scholar). HL946 (SR108 recF332::Tn3), CL544 (SR108 recR6212::cat883), and HL973 (SR108 recF332::Tn3; recJ284::Tn10) have been reported previously (4Courcelle J. Crowley D.J. Hanawalt P.C. J. Bacteriol. 1999; 181: 916-922Google Scholar, 16Courcelle J. Hanawalt P.C. Mol. Gen. Gent. 1999; 262: 543-551Google Scholar, 17Courcelle J. Donaldson J.R. Chow K.H. Courcelle C.T. Science. 2003; 299: 1064-1067Google Scholar). CL584 (SR108 recO1504::Tn5) was made by P1 transduction of the recO1504::Tn5 alleles from RDK1541 (23Kolodner R. Fishel R.A. Howard M. J. Bacteriol. 1985; 163: 1060-1066Google Scholar) into SR108. CL546 (SR108 recF332::Tn3; recR6212::cat883) and CL588 (SR108 recF332::Tn3; recO1504::Tn5) were made by P1 transduction of the recR6212::cat883 and recO1504::Tn5 alleles from strains TP647 (28Murphy K.C. Campellone K.G. Poteete A.R. Gene. 2000; 246: 321-330Google Scholar) and RDK1541 (23Kolodner R. Fishel R.A. Howard M. J. Bacteriol. 1985; 163: 1060-1066Google Scholar), into HL946. CL592 (SR108 recR6212::cat883; recO1504::Tn5) was made by P1 transduction of the recO1504::Tn5 allele from RDK1541 (23Kolodner R. Fishel R.A. Howard M. J. Bacteriol. 1985; 163: 1060-1066Google Scholar) into CL544. CL628 (SR108 recF332::Tn3; recQ6215::cat883) was made by P1 transduction of the recF332::Tn3 allele from HL946 (4Courcelle J. Crowley D.J. Hanawalt P.C. J. Bacteriol. 1999; 181: 916-922Google Scholar) into CL581 (17Courcelle J. Donaldson J.R. Chow K.H. Courcelle C.T. Science. 2003; 299: 1064-1067Google Scholar). CL691 (SR108 recR6212::cat883; recQ1803::Tn3) was made by P1 transduction of the recR6212::cat883 allele from strain TP647 (28Murphy K.C. Campellone K.G. Poteete A.R. Gene. 2000; 246: 321-330Google Scholar) into HL944 (16Courcelle J. Hanawalt P.C. Mol. Gen. Gent. 1999; 262: 543-551Google Scholar). CL684 (SR108 recR6212::cat883; recJ284::Tn10) was made by P1 transduction of the recR6212::cat883 allele from strain TP647 (28Murphy K.C. Campellone K.G. Poteete A.R. Gene. 2000; 246: 321-330Google Scholar) into HL942 (16Courcelle J. Hanawalt P.C. Mol. Gen. Gent. 1999; 262: 543-551Google Scholar). CL666 (SR108 recO1504::Tn5; recJ284::Tn10) and CL668 (SR108 recO1504::Tn5; recQ1803::Tn3) were made by P1 transduction of the recJ284::Tn10 and recQ1803::Tn3 alleles from HL924 (16Courcelle J. Hanawalt P.C. Mol. Gen. Gent. 1999; 262: 543-551Google Scholar) and HL944 (16Courcelle J. Hanawalt P.C. Mol. Gen. Gent. 1999; 262: 543-551Google Scholar), respectively, into CL584. CL590 (SR108 recF332::Tn3; recO1504::Tn5; recR6212::cat883) was made by P1 transduction of the recO1504::Tn5 allele from CL544 into CL546. UV Irradiation—Cultures were UV-irradiated in DGCthy media in Petri dishes on a rotating orbital shaker using a Sylvania 15-W germicidal light bulb (254 nm; 0.9 J/m2/s). Density Labeling of Replicated DNA—A fresh overnight culture was diluted 1:100 and grown in Davis minimal media with 0.4% glucose, 0.2% casamino acids, and 10 μg/ml thymine (DGCthy media) supplemented with [14C]thymine (0.1 μCi/ml) to an A600 of 0.45 in a 37 °C shaking water bath. The culture was then split into two halves and either mock UV-irradiated or UV-irradiated with 27 J/m2, before the cells were filtered on general filtration 0.45-μm membranes (Fisher-brand) and resuspended in DGC media containing 20 μg/ml 5-bromouracil in place of thymine and 0.5 μCi/ml of [3H]thymine (60.5 Ci/mmol). Cultures were allowed to recover for 1 h at 37 °C in a shaking water bath. Then, two volumes of ice-cold NET buffer (100 mm NaCl, 10 mm EDTA, 10 mm Tris, pH 8.0) were added. Cells were pelleted, resuspended in 150 μl of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and lysed by the addition of 150 μlK2HPO4/KOH (pH 12.5) and 20 μlof20% sarcosyl. The lysate was then subjected to isopycnic alkaline CsCl gradient centrifugation by combining 0.3 g lysate, 3.31 g 0.1 m K2HPO4/KOH (pH 12.5), and 2.23 g CsCl (refractive index = 1.4055) in a and to were on in in and the of and in was in a fresh overnight culture was diluted 1:100 and grown in DGCthy media supplemented with [14C]thymine (0.1 μCi/ml) to an A600 of in a 37 °C shaking Cultures were then with 1 μCi/ml for 10 the nascent DNA at the replication before filtered on 0.45-μm membranes with of NET resuspended in DGCthy UV-irradiated with 27 J/m2, and then in a 37 °C shaking of the culture at time were at and in before filtered on The of in was in a and overnight of cells that the plasmid were grown in the presence of μg/ml The overnight were diluted 1:100 and grown in a shaking at 37 °C to an A600 of 0.5 and UV-irradiated with the time were into of NET (100 mm NaCl, 10 mm Tris, pH 10 mm was pelleted, resuspended in 150 μl of 1 and in TE and lysed at 37 °C for 20 this (10 10 and (10 were and at °C for 1 were then with volumes of with volumes of and for h on on a of were then with with and volumes were the genomic DNA were in the in 0.4% at 1 were and in the in at were to membranes and with that been with by to the by using was and with a and RecO to UV-induced DNA recF and recR are required for the recovery of replication after by UV-induced DNA damage (5Courcelle J. Carswell-Crumpton C. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3714-3719Google Scholar). The of recO to recover replication after UV was with that of recF and recR and by the DNA synthesis with Cultures that were either UV-irradiated with 27 or were allowed to recover for a of in media containing 5-bromouracil in place of that DNA during this would be of a the DNA before The DNA in culture was then from the of the DNA by centrifugation in isopycnic alkaline CsCl and shown in Fig. after irradiation, UV-irradiated wild-type cells as DNA as their that replication this time However, the of DNA in either recF, recR, or recO mutants was inhibited to a similar extent after this dose of UV both and recQ the nascent DNA after UV (16Courcelle J. Hanawalt P.C. Mol. Gen. Gent. 1999; 262: 543-551Google Scholar) and affect the time that replication Chow and J. these genes are not for replication to and of DNA synthesis are in the absence of these gene is of to in an wild-type background, in or recQ do not cells to UV S.T. Clark A.J. J. Bacteriol. Scholar, H. Nakayama K. Nakayama R. Nakayama Y. Hanawalt P.C. Mol. Gen. Genet. Scholar). The of recovery in recF and recR mutants is with a failure to maintain replication forks blocked by DNA damage. be by the that are with arrested replication forks on as using (17Courcelle J. Donaldson J.R. Chow K.H. Courcelle C.T. Science. 2003; 299: 1064-1067Google Scholar, 1995; 262: Scholar). To determine whether recO mutants also fail to maintain the replication we characterized the that on plasmid of after UV with in E. coli by (17Courcelle J. Donaldson J.R. Chow K.H. Courcelle C.T. Science. 2003; 299: 1064-1067Google Scholar) have shown that this dose 0.5 lesions plasmid strand and that of the cells the to form Cells containing the plasmid were and the genomic DNA was with the plasmid of the of and by at the In this as form structures and of their and These form an that from the In wild-type a of the replication fork has been shown to occur on after UV (17Courcelle J. Donaldson J.R. Chow K.H. Courcelle C.T. Science. 2003; 299: 1064-1067Google Scholar). The fork is by the RecF, and RecA proteins a time that with the removal of the lesions by nucleotide excision repair and the recovery of The of the nascent DNA the replication fork structure into a replication that in the These structures in a region the replication with UV-irradiated recF and recR mutants not the region to extent (17Courcelle J. Donaldson J.R. Chow K.H. Courcelle C.T. Science. 2003; 299: 1064-1067Google Scholar). we the in UV-irradiated recO mutants, the replication fork also to that RecO, RecF and is required to maintain the replication fork after arrest by UV-induced DNA damage. RecO with RecF and RecR to the DNA at from by RecQ and failure to recover replication in UV-irradiated recF or recR mutants is with the extensive loss of nascent DNA made prior to (5Courcelle J. Carswell-Crumpton C. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3714-3719Google Scholar). The failure of recO mutants to recover replication indicate that RecO is required at a similar to RecF and RecR and is to the nascent DNA RecO be required at a in the recovery the nascent DNA have been or To between these we the nascent degradation in recO To this were with for 10 to the DNA at replication Then, the culture was to and UV-irradiated with 27 The allowed to the degradation in the to that in the DNA made at replication forks prior to UV shown UV-irradiated wild-type cells degrade of their genomic DNA after However, limited degradation of the nascent DNA was at prior to the recovery of replication 4Courcelle J. Crowley D.J. Hanawalt P.C. J. Bacteriol. 1999; 181: 916-922Google Scholar, 5Courcelle J. Carswell-Crumpton C. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3714-3719Google Scholar, P. of the of Scholar). In contrast to the limited degradation in wild-type recF or recR mutants degraded of the nascent DNA made prior to UV the degradation was in recO mutants, we that the nascent DNA was extensively and that the extent of degradation was similar to that in recF and recR mutants The result RecF and RecO to the DNA at blocked replication forks in UV-irradiated cells. extensive degradation occurs in all recF, recR, and recO mutants, it is that these gene protect of the blocked replication fork in this is the then we would that the nascent DNA degradation would when of these gene is To this we the nascent DNA degradation in mutants of recF recR, recF recO, and recR recO, as as the recF recO recR triple mutants nascent degradation that were similar in extent to the recF or recR mutants the nascent DNA degradation in the triple was also limited to of the nascent DNA suggesting that RecO functions with RecF and RecR to protect the same strand of the blocked replication fork. RecO with RecF and RecR to protect the nascent lagging strand of the arrested replication then the nascent DNA degradation be dependent upon RecJ and RecQ (16Courcelle J. Hanawalt P.C. Mol. Gen. Gent. 1999; 262: 543-551Google Scholar). To this we the nascent DNA degradation in recO mutants that also either the RecQ helicase or RecJ nuclease with this the nascent DNA degradation was reduced to a similar extent in recF, recO mutants when either RecJ or RecQ was These observations indicate that the nascent DNA degradation in recO, recF, and recR mutants results from the same degradation of the lagging strand by RecJ and RecF, RecO, and RecR are to form an epistatic upon biochemical and cells to UV irradiation, the frequency of in or transduction assays in or and delay the induction of the SOS after DNA damage S. Sandler S.J. Clark A.J. Madiraju M.V. Mol. Gen. Genet. 1995; 246: 254-258Google Scholar, 15Whitby M.C. Lloyd R.G. Mol. Gen. Gent. 1995; 246: 174-179Google Scholar, 23Kolodner R. Fishel R.A. Howard M. J. Bacteriol. 1985; 163: 1060-1066Google Scholar, 24Mahdi A.A. Lloyd R.G. Mol. Gen. Genet. 1989; 216: 503-510Google Scholar). suggested that the UV hypersensitivity of recO is with a delayed recovery of replication similar to recF and recR S. R. Mol. Microbiol. Scholar). The results indicate that RecF, and RecO function to maintain replication forks arrested by UV-induced DNA damage. In all proteins are required to the degradation of the nascent lagging strand by RecJ and RecQ after UV In vitro, all proteins bind DNA, and RecO has been shown to promote between homologous DNA C. J. Mol. Biol. 1994; Scholar). RecO and RecR promote RecA at the of and stabilize RecA filaments to their (7Shan Q. Bork J.M. Webb B.L. Inman R.B. Cox M.M. J. Mol. Biol. 1997; 265: 519-540Google Scholar, 25Umezu K. Kolodner R.D. J. Biol. Chem. 1994; 269: 30005-30013Google Scholar). of RecF and RecR bind DNA and DNA and RecA filaments are to into B.L. Cox M.M. Inman R.B. 1997; Scholar). the proteins in have been shown to RecA loading DNA (12Umezu K. Chi N.W. Kolodner R.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3875-3879Google Scholar, K. S.C. Mol. 2003; Scholar). These in vitro are consistent with the in vivo observations that RecF, RecO, and RecR may and bind to nascent DNA at blocked replication forks and to stabilize the RecA filaments at these as previously (5Courcelle J. Carswell-Crumpton C. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3714-3719Google Scholar, J. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. Scholar). The and in indicate that by these proteins the nascent degradation on the lagging strand by the in vivo However, to date, the and activity of these gene has not been on replication structures in role for RecF, RecO, and RecR in RecA filaments at blocked replication forks is also consistent with recF, recO, and recR mutants exhibit a delayed SOS induction S. Sandler S.J. Clark A.J. Madiraju M.V. Mol. Gen. Genet. 1995; 246: 254-258Google Scholar, 15Whitby M.C. Lloyd R.G. Mol. Gen. Gent. 1995; 246: 174-179Google Scholar). Because RecA filaments bound to single-strand DNA function as the for of the SOS the delay in of SOS genes may the reduced of RecA to bind to the replication fork in the absence of RecFOR. alleles of RecA that to bind DNA are to the for and the UV of recF, recR, and recO mutants M.V. S.C. Clark A.J. Scholar, M.V. A. Clark A.J. Proc. Natl. Acad. Sci. U. S. A. Scholar, Mutat. Res. 1993; Scholar). To these observations, we have at the nascent lagging strand of the block replication fork However, the and of this will and it that RecF, RecO, and RecR bind and of the replication fork structure to of limiting the nascent lagging strand degradation and of RecA filaments at arrested replication fork is by that show of RecR or RecO the UV of recF mutants S.J. Mol. Gen. Genet. 1994; Scholar, S.J. Clark A.J. J. Bacteriol. 1994; Scholar). However, the and of the on the nascent lagging strand an important that has not been is from this and that these genes are required for of replication forks blocked at DNA lesions a time to when the lesions are and replication In the absence of DNA damage when replication is not of RecF, RecO, or RecR does not to affect the or of E. coli However, in the that replication before the of the has been RecF, RecO, and RecR a role in arrested fork structures as a and in the stabilization of the RecA filament to protect and promote the resumption of replication, the of the chromosome to be We Poteete and for strains in this