Biomedical subjects

Justin Courcelle

Publications and source records attributed to Justin Courcelle.

11 recordsLinked to original sources

PubMed2006

RuvABC is required to resolve holliday junctions that accumulate following replication on damaged templates in Escherichia coli.

RuvABC is a complex that promotes branch migration and resolution of Holliday junctions. Although ruv mutants are hypersensitive to UV irradiation, the molecular event(s) that necessitate RuvABC processing in vivo are not known. Here, we used a combination of two-dimensional gel analysis and electron microscopy to reveal that although ruvAB and ruvC mutants are able to resume replication following arrest at UV-induced lesions, molecules that replicate in the presence of DNA damage accumulate unresolved Holliday junctions. The failure to resolve the Holliday junctions on the fully replicated molecules correlates with a delayed loss of genomic integrity that is likely to account for the loss of viability in these cells. The strand exchange intermediates that accumulate in ruv mutants are distinct from those observed at arrested replication forks and are not subject to resolution by RecG. These results indicate that the Holliday junctions observed in ruv mutants are intermediates of a repair pathway that is distinct from that of the recovery of arrested replication forks. A model is proposed in which RuvABC is required to resolve junctions that arise during the repair of a subset of nonarresting lesions after replication has passed through the template.

Bacterial Proteins↗

PubMed2006

Nascent DNA processing by RecJ favors lesion repair over translesion synthesis at arrested replication forks in Escherichia coli.

DNA lesions that arrest replication can lead to rearrangements, mutations, or lethality when not processed accurately. After UV-induced DNA damage in Escherichia coli, RecA and several recF pathway proteins are thought to process arrested replication forks and ensure that replication resumes accurately. Here, we show that the RecJ nuclease and RecQ helicase, which partially degrade the nascent DNA at blocked replication forks, are required for the rapid recovery of DNA synthesis and prevent the potentially mutagenic bypass of UV lesions. In the absence of RecJ, or to a lesser extent RecQ, the recovery of replication is significantly delayed, and both the recovery and cell survival become dependent on translesion synthesis by polymerase V. The RecJ-mediated processing is proposed to restore the region containing the lesion to a form that allows repair enzymes to remove the blocking lesion and DNA synthesis to resume. In the absence of nascent DNA processing, polymerase V can synthesize past the lesion to prevent lethality, although this occurs with slower kinetics and a higher frequency of mutagenesis.

Adenosine Triphosphatases↗

PubMed2006

Monitoring DNA replication following UV-induced damage in Escherichia coli.

The question of how the replication machinery accurately copies the genomic template in the presence of DNA damage has been intensely studied for more than forty years. A large number of genes has been characterized that, when mutated, are known to impair the ability of the cell to replicate in the presence of DNA damage. This chapter describes three techniques that can be used to monitor the progression, degradation, and structural properties of replication forks following UV-induced DNA damage in Escherichia coli.

DNA Damage↗

PubMed2005

Recs preventing wrecks.

The asexual cell cycle of E. coli produces two genetically identical clones of the parental cell through processive, semiconservative replication of the chromosome. When this process is prematurely disrupted by DNA damage, several recF pathway gene products play critical roles processing the arrested replication fork, allowing it to resume and complete its task. In contrast, when E. coli cultures are starved for thymine, these same gene products play a detrimental role, allowing replication to become unregulated and highly recombinagenic, resulting in lethality after prolonged starvation. Here, I briefly review the experimental observations that suggest how RecF maintains replication in the presence of DNA damage and discuss how this function may relate to the events that lead to a loss of viability during thymine starvation.

Chromosomes, Bacterial↗

PubMed2005

Nucleotide excision repair or polymerase V-mediated lesion bypass can act to restore UV-arrested replication forks in Escherichia coli.

Nucleotide excision repair and translesion DNA synthesis are two processes that operate at arrested replication forks to reduce the frequency of recombination and promote cell survival following UV-induced DNA damage. While nucleotide excision repair is generally considered to be error free, translesion synthesis can result in mutations, making it important to identify the order and conditions that determine when each process is recruited to the arrested fork. We show here that at early times following UV irradiation, the recovery of DNA synthesis occurs through nucleotide excision repair of the lesion. In the absence of repair or when the repair capacity of the cell has been exceeded, translesion synthesis by polymerase V (Pol V) allows DNA synthesis to resume and is required to protect the arrested replication fork from degradation. Pol II and Pol IV do not contribute detectably to survival, mutagenesis, or restoration of DNA synthesis, suggesting that, in vivo, these polymerases are not functionally redundant with Pol V at UV-induced lesions. We discuss a model in which cells first use DNA repair to process replication-arresting UV lesions before resorting to mutagenic pathways such as translesion DNA synthesis to bypass these impediments to replication progression.

DNA Repair↗

PubMed2004

When replication travels on damaged templates: bumps and blocks in the road.

Escherichia coli can accurately replicate their genome even when it contains hundreds of damaged bases. In this situation, processes such as DNA repair, translesion DNA synthesis, and recombination all contribute to the cell's ability to successfully complete this task. However, under conditions when these reactions go awry, these same processes can result in cell lethality, mutagenesis, or genetic instability. In order to understand the molecular events that can lead this normally faithful duplication of the genome to become less than perfect, it is essential to define the substrates and conditions when each of these processes are recruited to the replication fork.

DNA Damage↗

PubMed2004

RuvAB and RecG are not essential for the recovery of DNA synthesis following UV-induced DNA damage in Escherichia coli.

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 found that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis is resumed with kinetics that are similar to those 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.

Bacterial Proteins↗

PubMed2003

RecO acts with RecF and RecR to protect and maintain replication forks blocked by UV-induced DNA damage in Escherichia coli.

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.

Bacterial Proteins↗

PubMed2003

DNA damage-induced replication fork regression and processing in Escherichia coli.

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.

Adenosine Triphosphatases↗

PubMed2003

RecA-dependent recovery of arrested DNA replication forks.

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↗

PubMed2002

Answering the Call: Coping with DNA Damage at the Most Inopportune Time.

DNA damage incurred during the process of chromosomal replication has a particularly high possibility of resulting in mutagenesis or 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.

Journal Article↗