Genetics (2004)166:1631-40
- Abstract: Ultraviolet light induces DNA lesions that block the progression of the replication machinery. Several models speculate that the resumption of replication following disruption by UV-induced DNA damage requires regression of the nascent DNA or migration of the replication machinery away from the blocking lesion to allow repair or bypass of the lesion to occur. Both RuvAB and RecG catalyze branch migration of three- and four-stranded DNA junctions in vitro and are proposed to catalyze fork regression in vivo. To examine this possibility, we characterized the recovery of DNA synthesis in ruvAB and recG mutants. We find that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis resumes with kinetics that are similar to that in wild-type cells. The data presented here indicate that RecG- or RuvAB-catalyzed fork regression is not essential for DNA synthesis to resume following arrest by UV-induced DNA damage in vivo.
RecO acts with RecF and RecR to protect and maintain replication forks blocked by UV-induced DNA damage in escherichia coli
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Chow KC; Courcelle J
J Biol Chem. (2004) 279:3492-6
- Abstract: 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 following DNA damage. In this study, we show that RecO functions with RecF and RecR to protect the nascent lagging strand of arrested replication forks following 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 on RecJ and RecQ functions. Since 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 following the arrest of replication by UV-induced DNA damage.
RecA-dependent recovery of arrested DNA replication forks
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Courcelle J; Hanawalt PC
Annu Rev Genet. (2003) 37:611-46
- Abstract: DNA damage encountered during the cellular process of chromosomal replication can disrupt the replication machinery and result in mutagenesis or lethality. The RecA protein of Escherichia coli is essential for survival in this situation: It maintains the integrity of the arrested replication fork and signals the upregulation of over 40 gene products, of which most are required to restore the genomic template and to facilitate the resumption of processive replication. Although RecA was originally discovered as a gene product that was required to change the genetic information during sexual cell cycles, over three decades of research have revealed that it is also the key enzyme required to maintain the genetic information when DNA damage is encountered during replication in asexual cell cycles. In this review, we examine the significant experimental approaches that have led to our current understanding of the RecA-mediated processes that restore replication following encounters with DNA damage.
DNA damage-induced replication fork regression and processing in Escherichia coli
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Courcelle J; Donaldson JR; Chow KH; Courcelle CT
Science, 2003 299(5609):1064-7.
- Abstract: 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.
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- Highlight:
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- Rec'd and repaired
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- LeBrasseur N. Journal of Cell Biology (2003) 106:464-5
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Answering the Call: Coping with DNA Damage at
theMost Inopportune Time
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Crowley DJ; Courcelle J
Journal of Biomedicine and Biotechnology, (2002) 2(2): 66-74.
- Abstract: DNA damage incurred during the process of chromosomal replication has a particularly high possibility of resulting in mutagenesisor lethality for the cell. The SOS response of Escherichia coli appears to be well adapted for this particular situation and involves the coordinated up-regulation of genes whose products center upon the tasks of maintaining the integrity of the replication fork when it encounters DNA damage, delaying the replication process (a DNA damage checkpoint), repairing the DNA lesions or allowing replication to occur over these DNA lesions, and then restoring processive replication before the SOS response itself is turned off. Recent advances in the fields of genomics and biochemistry has given a much more comprehensive picture of the timing and coordination of events which allow cells to deal with potentially lethal or mutagenic DNA lesions at the time of chromosomal replication.
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Requirement for Uracil-DNA Glycosylase during
the Transition to Late-Phase Cytomegalovirus DNA Replication
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Courcelle CT; Courcelle J; Prichard MN; Mocarski ES
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Journal of Virology, 2001 Aug, 75(16): 7592-7601.
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- Abstract: Cytomegalovirus gene UL114, a homolog of
mammalian uracil-DNA glycosylase (UNG), is required for efficient viral
DNA replication. In quiescent fibroblasts, UNG mutant virus replication
is delayed for 48 h and follows the virus-induced expression of
cellular UNG. In contrast, mutant virus replication proceeds without
delay in actively growing fibroblasts that express host cell UNG. In
the absence of viral or host cell UNG expression, mutant virus fails to
proceed to late-phase DNA replication, characterized by rapid DNA
amplification. The data suggest that uracil incorporated early during
wild-type viral DNA replication must be removed by virus or host UNG
prior to late-phase amplification and encapsidation into progeny
virions. The process of uracil incorporation and excision may introduce
strand breaks to facilitate the transition from early-phase replication
to late-phase amplification.
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Participation of recombination proteins in rescue of arrested replication forks in UV-irradiated Escherichia coli need not involve recombination.
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Courcelle J; Hanawalt PC
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Proceedings of the National Academy of Sciences of the United States of
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America, 2001 Jul 17, 98(15): 8196-8202.
- Abstract: Alternative reproductive cycles make use of different strategies to generate different reproductive products. In Escherichia coli, recA and several other rec genes are required for the generation of recombinant genomes during Hfr conjugation. During normal asexual reproduction, many of these same genes are needed to generate clonal products from UV-irradiated cells. However, unlike conjugation, this latter process also requires the function of the nucleotide excision repair genes. Following UV irradiation, the recovery of DNA replication requires uvrA and uvrC, as well as recA, recF, and recR. The rec genes appear to be required to protect and maintain replication forks that are arrested at DNA lesions, based on the extensive degradation of the nascent DNA that occurs in their absence. The products of the recJ and recQ genes process the blocked replication forks before the resumption of replication and may affect the fidelity of the recovery process. We discuss a model in which several rec gene products process replication forks ar-rested by DNA damage to facilitate the repair of the blocking DNA lesions by nucleotide excision repair, thereby allowing processive replication to resume with no need for strand exchanges or recombination. The poor survival of cellular populations that depend on recombinational pathways (compared with that in their excision repair proficient counterparts) suggests that at least some of the rec genes may be designed to function together with nucleotide excision repair in a common and predominant pathway by which cells faithfully recover replication and survive following UV-induced DNA damage.
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Therefore, what are recombination proteins there for?
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Courcelle J; Ganesan AK; Hanawalt PC
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BioEssays 23:463-470, May 2001
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- Abstract: The order of discovery can have a profound effect upon the way in which we think about the function of a gene. In E. coli, recA is nearly essential for cell survival in the presence of DNA damage. However, recA was originally identified, as a gene required to obtain recombinant DNA molecules in conjugating bacteria. As a result, it has been frequently assumed that recA promotes the survival of bacteria containing DNA damage by recombination in which DNA strand exchanges occur. We now know that several of the processes that interact with or are controlled by recA, such as excision repair and translesion synthesis, operate to ensure that DNA replication occurs processively without strand exchanges. Yet the view persists in the literature that recA functions primarily to promote recombination during DNA repair. With the benefit of hindsight and more than three decades of additional research, we reexamine some of the classical experiments that established the concept of DNA repair by recombination, and we consider the possibilities that recombination is not an efficient mechanism for rescuing damaged cells, and that recA may be important for maintaining processive replication in a manner that does not generally promote recombination.
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- Editorial:
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- Gene names: the approaching end of a century-long dilemma
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- Wilkins A.S. BioEssays 23:377-378 May 2001
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Comparative gene expression profiles following UV exposure in wild type and SOS deficient Escherichia coli.
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Courcelle J; Khodursky A; Peter B; Brown PO; Hanawalt PC
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Genetics 158: 41-64 May 2001
- Abstract: The SOS response in UV-irradiated Escherichia coli includes the upregulation of several dozen genes that are negatively regulated by the LexA repressor. Using DNA microarrays containing amplified DNA fragments from 95.5% of all open reading frames identified on the E. coli chromosome, we have examined the changes in gene expression following UV exposure in both wild-type cells and lexA1 mutants, which are unable to induce genes under LexA control. We report here the time courses of expression of the genes surrounding the 26 documented lexA-regulated regions on the E. coli chromosome. We observed 17 additional sites that responded in a lexA-dependent manner and a large number of genes that were upregulated in a lexA-independent manner although upregulation in this manner was generally not more than twofold. In addition, several transcripts were either downregulated or degraded following UV irradiation. These newly identified UV-responsive genes are discussed with respect to their possible roles in cellular recovery following exposure to UV irradiation.
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Supplemental
link to Raw Microarray data Genetics 158: 41-64 May 2001
RecQ and RecJ Process Blocked Replication Forks Prior to the Resumption of Replication in UV-Irradiated Escherichia coli.
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Courcelle J; Hanawalt PC
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Molecular and General Genetics, 1999 Nov 262(3):543-51
- Abstract: The accurate recovery of replication following DNA damage and repair is critical to maintaining genomic integrity. In Escherichia coli, the recovery of replication following UV-induced DNA damage is dependent upon several proteins in the recF pathway, including RecF, RecO, and RecR. Two other recF pathway proteins, the RecQ helicase and the RecJ exonuclease, have been shown to affect the sites and frequencies at which illegitimate rearrangements occur following UV-induced DNA damage, suggesting that they also may function during the recovery of replication. We show here that RecQ and RecJ process the nascent DNA at blocked replication forks prior to the resumption of DNA synthesis. The processing involves selective degradation of the nascent lagging DNA strand and it requires both RecQ and RecJ. We suggest that this processing may serve to lengthen the substrate that can be recognized and stabilized by the RecA protein at the replication fork, thereby helping to assure the accurate recovery of replication after the obstructing lesion has been repaired.
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