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Manganese Is Required for the Rapid Recovery of DNA Synthesis following Oxidative Challenge in Escherichia coli

Hutfilz CR; Wang NE; Hoff CA; Lee JA; Hackert BJ; Courcelle J; Courcelle CT

J Bacteriol (2019) 201:e00426-19

Abstract: Divalent metals such as iron and manganese play an important role in the cellular response to oxidative challenges and are required as cofactors by many enzymes. However, how these metals affect replication after oxidative challenge is not known. Here, we show that replication in Escherichia coli is inhibited following a challenge with hydrogen peroxide and requires manganese for the rapid recovery of DNA synthesis. We show that the manganese-dependent recovery of DNA synthesis occurs independent of lesion repair, modestly improves cell survival, and is associated with elevated rates of mutagenesis. The Mn-dependent mutagenesis involves both replicative and translesion polymerases and requires prior disruption by H2O2 to occur. Taking these findings together, we propose that replication in E. coli is likely to utilize an iron-dependent enzyme(s) that becomes oxidized and inactivated during oxidative challenges. The data suggest that manganese remetallates these or alternative enzymes to allow genomic DNA replication to resume, although with reduced fidelity.
Importance: Iron and manganese play important roles in how cell's cope with oxygen stress. However, how these metals affect the ability of cells to replicate after oxidative challenges is not known. Here, we show that replication in Escherichia coli is inhibited following a challenge with hydrogen peroxide and requires manganese for the rapid recovery of DNA synthesis. The manganese-dependent recovery of DNA synthesis occurs independently of lesion repair and modestly improves survival, but it also increases the mutation rate in cells. The results imply that replication in E. coli is likely to utilize an iron-dependent enzyme(s) that becomes oxidized and inactivated during oxidative challenges. We propose that manganese remetallates these or alternative enzymes to allow genomic DNA replication to resume, although with reduced fidelity. 

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RecBCD, SbcCD and ExoI process a substrate created by convergent replisomes to complete DNA replication

Hamilton NA;  Wendel BM; Weber EA; Courcelle CT; Courcelle J

Mol Microbiol (2019) 111:1638-1651

Abstract: The accurate completion of DNA replication on the chromosome requires RecBCD and structure specific SbcCD and ExoI nucleases. However, the substrates and mechanism by which this reaction occurs remains unknown. Here we show that these completion enzymes operate on plasmid substrates containing two replisomes, but are not required for plasmids containing one replisome. Completion on the two-replisome plasmids requires RecBCD, but does not require RecA and no broken intermediates accumulate in its absence, indicating that the completion reaction occurs normally in the absence of any double-strand breaks. Further, similar to the chromosome, we show that when the normal completion reaction is prevented, an aberrant RecA-mediated recombination process leads to amplifications that drive most of the instabilities associated with the two-replisome substrates. The observations imply that the substrate SbcCD, ExoI and RecBCD act upon in vivo is created specifically by two convergent replisomes, and demonstrate that the function of RecBCD in completing replication is independent of double-strand break repair, and likely promotes joining of the strands of the convergent replication forks.

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Limited Capacity or Involvement of Excision Repair, Double-Strand Breaks, or Translesion Synthesis for

Psoralen Cross-Link Repair in Escherichia coli

Cole JM; Acott JD; Courcelle CT; Courcelle J

Genetics (2018) 210:99-112

Abstract: DNA interstrand cross-links are complex lesions that covalently bind complementary strands of DNA and whose mechanism of repair remains poorly understood. In Escherichia coli, several gene products have been proposed to be involved in cross-link repair based on the hypersensitivity of mutants to cross-linking agents. However, cross-linking agents induce several forms of DNA damage, making it challenging to attribute mutant hypersensitivity specifically to interstrand cross-links. To address this, we compared the survival of UVA-irradiated repair mutants in the presence of 8-methoxypsoralen-which forms interstrand cross-links and monoadducts-to that of angelicin-a congener forming only monoadducts. We show that incision by nucleotide excision repair is not required for resistance to interstrand cross-links. In addition, neither RecN nor DNA polymerases II, IV, or V is required for interstrand cross-link survival, arguing against models that involve critical roles for double-strand break repair or translesion synthesis in the repair process. Finally, estimates based on Southern analysis of DNA fragments in alkali agarose gels indicate that lethality occurs in wild-type cells at doses producing as few as one to two interstrand cross-links per genome. These observations suggest that E. coli may lack an efficient repair mechanism for this form of damage.

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SbcC-SbcD and ExoI process convergent forks to complete chromosome replication

Wendel BM; Cole JM; Courcelle CT; Courcelle J

Proc Natl Acad Sci U S A. (2018) 115:349-354

Abstract: SbcC-SbcD are the bacterial orthologs of Mre11-Rad50, a nuclease complex essential for genome stability, normal development, and viability in mammals. In vitro, these enzymes degrade long DNA palindromic structures. When inactivated along with ExoI in Escherichia coli, or Sae2 in eukaryotes, palindromic amplifications arise and propagate in cells. However, long DNA palindromes are not normally found in bacterial or human genomes, leaving the cellular substrates and function of these enzymes unknown. Here, we show that during the completion of DNA replication, convergent replication forks form a palindrome-like structural intermediate that requires nucleolytic processing by SbcC-SbcD and ExoI before chromosome replication can be completed. Inactivation of these nucleases prevents completion from occurring, and under these conditions, cells maintain viability by shunting the reaction through an aberrant recombinational pathway that leads to amplifications and instability in this region. The results identify replication completion as an event critical to maintain genome integrity and cell viability, demonstrate SbcC-SbcD-ExoI acts before RecBCD and is required to initiate the completion reaction, and reveal how defects in completion result in genomic instability.

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Replication rapidly recovers and continues in the presence of hydroxyurea in Escherichia coli

Nazaretyan SA; Savic N; Sadek M; Hackert BJ; Courcelle J; Courcelle CT

J Bacteriol (2017) pii: JB.00713-17

Abstract: In both prokaryotes and eukaryotes, hydroxyurea is suggested to inhibit DNA replication by inactivating ribonucleotide reductase and depleting deoxyribonucleoside triphosphate pools. In this study, we show that the inhibition of replication in Escherichia coli is transient even at concentrations of 0.1 M hydroxyurea and that replication rapidly recovers and continues in its presence. The recovery of replication does not require the alternative ribonucleotide reductases, NrdEF and NrdDG, or translesion DNA polymerases, Pol II, Pol IV, or Pol V. Ribonucleotides are incorporated at higher frequencies during replication in the presence of hydroxyurea. However, these do not contribute significantly to the observed synthesis or toxicity. Hydroxyurea toxicity was only observed under conditions where the stability of hydroxyurea was compromised and byproducts, known to damage DNA directly, were allowed to accumulate. The results demonstrate that hydroxyurea is not a direct or specific inhibitor of DNA synthesis in vivo, and that the transient inhibition observed is most likely due to a general depletion of iron cofactors from enzymes when 0.1 M hydroxyurea is initially applied. Finally, the results support previous studies suggesting that hydroxyurea toxicity is mediated primarily through direct DNA damage induced by the breakdown products of hydroxyurea, rather than by inhibition of replication or depletion of deoxyribonucleotide levels in the cell.  
Importance: Hydroxyurea is commonly suggested to function by inhibiting DNA replication through the inactivation of ribonucleotide reductase and depleting deoxyribonucleoside triphosphate pools. Here, we show that hydroxyurea only transiently inhibits replication in Escherichia coli before it rapidly recovers and continues in the presence of this drug. The recovery of replication does not depend on alternative ribonucleotide reductases, translesion synthesis, or RecA. Further we show that hydroxyurea toxicity is only observed after toxic intermediates that accumulate when hydroxyurea breaks down, damage DNA and induce lethality. The results demonstrate that hydroxyurea toxicity is mediated indirectly by the formation of DNA damage, rather than by an inhibition of replication or depletion of deoxyribonucleotide levels in the cell.

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Cho Endonuclease Functions during DNA Interstrand Cross-Link Repair in Escherichia coli

Perera AV; Mendenhall JB; Courcelle CT; Courcelle J

J Bacteriol (2016) 198:3099-3108

Abstract: DNA interstrand cross-links are complex lesions that covalently link both strands of the duplex DNA. Lesion removal is proposed to be initiated via the UvrABC nucleotide excision repair complex; however, less is known about the subsequent steps of this complex repair pathway. In this study, we characterized the contribution of nucleotide excision repair mutants to survival in the presence of psoralen-induced damage. Unexpectedly, we observed that the nucleotide excision repair mutants exhibit differential sensitivity to psoralen-induced damage, with uvrC mutants being less sensitive than either uvrA or uvrB We show that Cho, an alternative endonuclease, acts with UvrAB and is responsible for the reduced hypersensitivity of uvrC mutants. We find that Cho's contribution to survival correlates with the presence of DNA interstrand cross-links, rather than monoadducts, and operates at a step after, or independently from, the initial incision during the global repair of psoralen DNA adducts from the genome.

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RecBCD is required to complete chromosomal replication: Implications for double-strand break frequencies and repair mechanisms

Courcelle J; Wendel BM; Livingstone DD; Courcelle CT

DNA Repair (2015) 32:86-95

Abstract: Several aspects of the mechanism of homologous double-strand break repair remain unclear. Although intensive efforts have focused on how recombination reactions initiate, far less is known about the molecular events that follow. Based upon biochemical studies, current models propose that RecBCD processes double-strand ends and loads RecA to initiate recombinational repair. However, recent studies have shown that RecBCD plays a critical role in completing replication events on the chromosome through a mechanism that does not involve RecA or recombination. Here, we examine several studies, both early and recent, that suggest RecBCD also operates late in the recombination process- after initiation, strand invasion, and crossover resolution have occurred. Similar to its role in completing replication, we propose a model in which RecBCD is required to resect and resolve the DNA synthesis associated with homologous recombination at the point where the missing sequences on the broken molecule have been restored. We explain how the impaired ability to complete chromosome replication in recBC and recD mutants is likely to account for the loss of viability and genome instability in these mutants, and conclude that spontaneous double-strand breaks and replication fork collapse occur far less frequently than previously speculated.

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Completion of DNA replication in Escherichia coli

Wendel BM; Courcelle CT; Courcelle J

Proc Natl Acad Sci U S A (2014) 111:16454-9

Abstract: The mechanism by which cells recognize and complete replicated regions at their precise doubling point must be remarkably efficient, occurring thousands of times per cell division along the chromosomes of humans. However, this process remains poorly understood. Here we show that, in Escherichia coli, the completion of replication involves an enzymatic system that effectively counts pairs and limits cellular replication to its doubling point by allowing converging replication forks to transiently continue through the doubling point before the excess, over-replicated regions are incised, resected, and joined. Completion requires RecBCD and involves several proteins associated with repairing double-strand breaks including, ExoI, SbcDC, and RecG. However, unlike double-strand break repair, completion occurs independently of homologous recombination and RecA. In some bacterial viruses, the completion mechanism is specifically targeted for inactivation to allow over-replication to occur during lytic replication. The results suggest that a primary cause of genomic instabilities in many double-strand-break-repair mutants arises from an impaired ability to complete replication, independent from DNA damage.

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Fate of the replisome following arrest by UV-induced DNA damage in Escherichia coli

Jeiranian HA; Schalow BJ; Courcelle CT; Courcelle J

Proc Natl Acad Sci U S A (2013) 110:11421-6

Abstract: Accurate replication in the presence of DNA damage is essential to genome stability and viability in all cells. In Escherichia coli, DNA replication forks blocked by UV-induced damage undergo a partial resection and RecF-catalyzed regression before synthesis resumes. These processing events generate distinct structural intermediates on the DNA that can be visualized in vivo using 2D agarose gels. However, the fate and behavior of the stalled replisome remains a central uncharacterized question. Here, we use thermosensitive mutants to show that the replisome's polymerases uncouple and transiently dissociate from the DNA in vivo. Inactivation of α, β, or τ subunits within the replisome is sufficient to signal and induce the RecF-mediated processing events observed following UV damage. By contrast, the helicase-primase complex (DnaB and DnaG) remains critically associated with the fork, leading to a loss of fork integrity, degradation, and aberrant intermediates when disrupted. The results reveal a dynamic replisome, capable of partial disassembly to allow access to the obstruction, while retaining subunits that maintain fork licensing and direct reassembly to the appropriate location after processing has occurred

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UvrD Participation in Nucleotide Excision Repair Is Required for the Recovery of DNA Synthesis following UV-Induced Damage in Escherichia coli

Newton KN, Courcelle CT, Courcelle J

J Nucleic Acids (2012) 2012:271453

Abstract: UvrD is a DNA helicase that participates in nucleotide excision repair and several replication-associated processes, including methyl-directed mismatch repair and recombination. UvrD is capable of displacing oligonucleotides from synthetic forked DNA structures in vitro and is essential for viability in the absence of Rep, a helicase associated with processing replication forks. These observations have led others to propose that UvrD may promote fork regression and facilitate resetting of the replication fork following arrest. However, the molecular activity of UvrD at replication forks in vivo has not been directly examined. In this study, we characterized the role UvrD has in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that UvrD is required for DNA synthesis to recover. However, in the absence of UvrD, the displacement and partial degradation of the nascent DNA at the arrested fork occur normally. In addition, damage-induced replication intermediates persist and accumulate in uvrD mutants in a manner that is similar to that observed in other nucleotide excision repair mutants. These data indicate that, following arrest by DNA damage, UvrD is not required to catalyze fork regression in vivo and suggest that the failure of uvrD mutants to restore DNA synthesis following UV-induced arrest relates to its role in nucleotide excision repair.

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Cellular characterization of the primosome and rep helicase in processing and restoration of replication following arrest by UV-induced DNA damage in Escherichia coli

Courcelle CT, Landstrom AJ, Anderson B, Courcelle J

J Bacteriol (2012) 194:3977-86

Abstract: Following arrest by UV-induced DNA damage, replication is restored through a sequence of steps that involve partial resection of the nascent DNA by RecJ and RecQ, branch migration and processing of the fork DNA surrounding the lesion by RecA and RecF-O-R, and resumption of DNA synthesis once the blocking lesion has been repaired or bypassed. In vitro, the primosomal proteins (PriA, PriB, and PriC) and Rep are capable of initiating replication from synthetic DNA fork structures, and they have been proposed to catalyze these events when replication is disrupted by certain impediments in vivo. Here, we characterized the role that PriA, PriB, PriC, and Rep have in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that the partial degradation and processing of the arrested replication fork occurs normally in both rep and primosome mutants. In each mutant, the nascent degradation ceases and DNA synthesis initially resumes in a timely manner, but the recovery then stalls in the absence of PriA, PriB, or Rep. The results demonstrate a role for the primosome and Rep helicase in overcoming replication forks arrested by UV-induced damage in vivo and suggest that these proteins are required for the stability and efficiency of the replisome when DNA synthesis resumes but not to initiate de novo replication downstream of the lesion.

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Inefficient replication reduces RecA-mediated repair of UV-damaged plasmids introduced into competent Escherichia coli.

Jeiranian HA, Courcelle CT, Courcelle J 

Plasmid (2012) 68:113-24.

Abstract: Transformation of Escherichia coli with purified plasmids containing DNA damage is frequently used as a tool to characterize repair pathways that operate on chromosomes. In this study, we used an assay that allowed us to quantify plasmid survival and to compare how efficiently various repair pathways operate on plasmid DNA introduced into cells relative to their efficiency on chromosomal DNA. We observed distinct differences between the mechanisms operating on the transforming plasmid DNA and the chromosome. An average of one UV-induced lesion was sufficient to inactivate ColE1-based plasmids introduced into nucleotide excision repair mutants, suggesting an essential role for repair on newly introduced plasmid DNA. By contrast, the absence of RecA, RecF, RecBC, RecG, or RuvAB had a minimal effect on the survival of the transforming plasmid DNA containing UV-induced damage. Neither the presence of an endogenous homologous plasmid nor the induction of the SOS response enhanced the survival of transforming plasmids. Using two-dimensional agarose-gel analysis, both replication- and RecA-dependent structures that were observed on established, endogenous plasmids following UV-irradiation, failed to form on UV-irradiated plasmids introduced into E. coli. We interpret these observations to suggest that the lack of RecA-mediated survival is likely to be due to inefficient replication that occurs when plasmids are initially introduced into cells, rather than to the plasmid's size, the absence of homologous sequences, or levels of recA expression.

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Mfd is required for rapid recovery of transcription following UV-induced DNA damage but not oxidative DNA damage in Escherichia coli

Schalow BJ, Courcelle CT, Courcelle J 

J Bacteriol (2012) 194:2637-45

Abstract: Transcription-coupled repair (TCR) is a cellular process by which some forms of DNA damage are repaired more rapidly from transcribed strands of active genes than from nontranscribed strands or the overall genome. In humans, the TCR coupling factor, CSB, plays a critical role in restoring transcription following both UV-induced and oxidative DNA damage. It also contributes indirectly to the global repair of some forms of oxidative DNA damage. The Escherichia coli homolog, Mfd, is similarly required for TCR of UV-induced lesions. However, its contribution to the restoration of transcription and to global repair of oxidative damage has not been examined. Here, we report the first direct study of transcriptional recovery following UV-induced and oxidative DNA damage in E. coli. We observed that mutations in mfd or uvrA reduced the rate that transcription recovered following UV-induced damage. In contrast, no difference was detected in the rate of transcription recovery in mfd, uvrA, fpg, nth, or polB dinB umuDC mutants relative to wild-type cells following oxidative damage. mfd mutants were also fully resistant to hydrogen peroxide (H(2)O(2)) and removed oxidative lesions from the genome at rates comparable to wild-type cells. The results demonstrate that Mfd promotes the rapid recovery of gene expression following UV-induced damage in E. coli. In addition, these findings imply that Mfd may be functionally distinct from its human CSB homolog in that it does not detectably contribute to the recovery of gene expression or global repair following oxidative damage.

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Escherichia coli Fpg glycosylase is nonrendundant and required for the rapid global repair of oxidized purine and pyrimidine damage in vivo

Schalow BJ, Courcelle CT, Courcelle J

J Mol Biol. (2011) J410(:183-93

Abstract: Endonuclease (Endo) III and formamidopyrimidine-N-glycosylase (Fpg) are two of the predominant DNA glycosylases in Escherichia coli that remove oxidative base damage. In cell extracts and purified form, Endo III is generally more active toward oxidized pyrimidines, while Fpg is more active towards oxidized purines. However, the substrate specificities of these enzymes partially overlap in vitro. Less is known about the relative contribution of these enzymes in restoring the genomic template following oxidative damage. In this study, we examined how efficiently Endo III and Fpg repair their oxidative substrates in vivo following treatment with hydrogen peroxide. We found that Fpg was nonredundant and required to rapidly remove its substrate lesions on the chromosome. In addition, Fpg also repaired a significant portion of the lesions recognized by Endo III, suggesting that it plays a prominent role in the global repair of both purine damage and pyrimidine damage in vivo. By comparison, Endo III did not affect the repair rate of Fpg substrates and was only responsible for repairing a subset of its own substrate lesions in vivo. The absence of Endo VIII or nucleotide excision repair did not significantly affect the global repair of either Fpg or Endo III substrates in vivo. Surprisingly, replication recovered after oxidative DNA damage in all mutants examined, even when lesions persisted in the DNA, suggesting the presence of an efficient mechanism to process or overcome oxidative damage encountered during replication.

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Visualization of UV-induced replication intermediates in E. coli using two-dimensional agarose-gel analysis

Jeiranian HA, Schalow BJ, Courcelle J.

J Vis Exp. (2010) 46:2220

Abstract: Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.


ATP binding, ATP hydrolysis, and protein dimerization are required for RecF to catalyze an early step in the processing and recovery of replication forks disrupted by DNA damage

Michel-Marks E, Courcelle CT, Korolev S, Courcelle J

J Mol Biol (2010) 401:579-89

Abstract: In Escherichia coli, the recovery of replication following disruption by UV-induced DNA damage requires the RecF protein and occurs through a process that involves stabilization of replication fork DNA, resection of nascent DNA to allow the offending lesion to be repaired, and reestablishment of a productive replisome on the DNA. RecF forms a homodimer and contains an ATP binding cassette ATPase domain that is conserved among eukaryotic SMC (structural maintenance of chromosome) proteins, including cohesin, condensin, and Rad50. Here, we investigated the functions of RecF dimerization, ATP binding, and ATP hydrolysis in the progressive steps involved in recovering DNA synthesis following disruption by DNA damage. RecF point mutations with altered biochemical properties were constructed in the chromosome. We observed that protein dimerization, ATP binding, and ATP hydrolysis were essential for maintaining and processing the arrested replication fork, as well as for restoring DNA synthesis. In contrast, stabilization of the RecF protein dimer partially protected the DNA at the arrested fork from degradation, although overall processing and recovery remained severely impaired.

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Nucleotide excision repair is a predominant mechanism for processing nitrofurazone-induced DNA damage in Escherichia coli

Ona KR, Courcelle CT, Courcelle J

J Bacteriol. (2009) 191:4959-65

Abstract: Nitrofurazone is reduced by cellular nitroreductases to form N(2)-deoxyguanine (N(2)-dG) adducts that are associated with mutagenesis and lethality. Much attention recently has been given to the role that the highly conserved polymerase IV (Pol IV) family of polymerases plays in tolerating adducts induced by nitrofurazone and other N(2)-dG-generating agents, yet little is known about how nitrofurazone-induced DNA damage is processed by the cell. In this study, we characterized the genetic repair pathways that contribute to survival and mutagenesis in Escherichia coli cultures grown in the presence of nitrofurazone. We find that nucleotide excision repair is a primary mechanism for processing damage induced by nitrofurazone. The contribution of translesion synthesis to survival was minor compared to that of nucleotide excision repair and depended upon Pol IV. In addition, survival also depended on both the RecF and RecBCD pathways. We also found that nitrofurazone acts as a direct inhibitor of DNA replication at higher concentrations. We show that the direct inhibition of replication by nitrofurazone occurs independently of DNA damage and is reversible once the nitrofurazone is removed. Previous studies that reported nucleotide excision repair mutants that were fully resistant to nitrofurazone used high concentrations of the drug (200 microM) and short exposure times. We demonstrate here that these conditions inhibit replication but are insufficient in duration to induce significant levels of DNA damage.

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Shifting replication between IInd, IIIrd, and IVth gears

Courcelle J

Proc Natl Acad Sci U S A. (2009) 106:6027-8

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RecA-dependent recovery of arrested DNA replication forks

Al-Hadid Q, Ona K, Courcelle CT, Courcelle J.

Mutat Res. (2008) 645:19-26

Abstract: RecA is required for recombinational processes and cell survival following UV-induced DNA damage. recA433 is a historically important mutant allele that contains a single amino acid substitution (R243H). This mutation separates the recombination and survival functions of RecA. recA433 mutants remain proficient in recombination as measured by conjugation or transduction, but are hypersensitive to UV-induced DNA damage. The cellular functions carried out by RecA require either recF pathway proteins or recBC pathway proteins to initiate RecA-loading onto the appropriate DNA substrates. In this study, we characterized the ability of recA433 to carry out functions associated with either the recF pathway or recBC pathway. We show that several phenotypic deficiencies exhibited by recA433 mutants are similar to recF mutants but distinct from recBC mutants. In contrast to recBC mutants, recA433 and recF mutants fail to process or resume replication following disruption by UV-induced DNA damage. However, recA433 and recF mutants remain proficient in conjugational recombination and are resistant to formaldehyde-induced protein-DNA crosslinks, functions that are impaired in recBC mutants. The results are consistent with a model in which the recA433 mutation selectively impairs RecA functions associated with the RecF pathway, while retaining the ability to carry out RecBCD pathway-mediated functions. These results are discussed in the context of the recF and recBC pathways and the potential substrates utilized in each case.

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RecBCD and RecJ/RecQ Initiate DNA Degradation on Distinct Substrates in UV-Irradiated Escherichia coli

Chow KH; Courcelle J

Rad Research (2007) 168:499-506

Abstract: After UV irradiation, recA mutants fail to recover replication, and a dramatic and nearly complete degradation of the genomic DNA occurs. Although the RecBCD helicase/nuclease complex is known to mediate this catastrophic DNA degradation, it is not known how or where this degradation is initiated. Previous studies have speculated that RecBCD targets and initiates degradation from the nascent DNA at replication forks arrested by DNA damage. To test this question, we examined which enzymes were responsible for the degradation of genomic DNA and the nascent DNA in UV-irradiated recA cells. We show here that, although RecBCD degrades the genomic DNA after UV irradiation, it does not target the nascent DNA at arrested replication forks. Instead, we observed that the nascent DNA at arrested replication forks in recA cultures is degraded by RecJ/RecQ, similar to what occurs in wild-type cultures. These findings indicate that the genomic DNA degradation and nascent DNA degradation in UV-irradiated recA mutants are mediated separately through RecBCD and RecJ/RecQ, respectively. In addition, they demonstrate that RecBCD initiates degradation at a site(s) other than the arrested replication fork directly.

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Inactivation of the DnaB helicase leads to the collapse and degradation of the replication fork: a comparison to UV-induced arrest

Belle JJ; Casey A; Courcelle CT; Courcelle J

J Bacteriol (2007) 189:5452-62

Abstract: Replication forks face a variety of structurally diverse impediments that can prevent them from completing their task. The mechanism by which cells overcome these hurdles is likely to vary depending on the nature of the obstacle and the strand in which the impediment is encountered. Both UV-induced DNA damage and thermosensitive replication proteins have been used in model systems to inhibit DNA replication and characterize the mechanism by which it recovers. In this study, we examined the molecular events that occur at replication forks following inactivation of a thermosensitive DnaB helicase and found that they are distinct from those that occur following arrest at UV-induced DNA damage. Following UV-induced DNA damage, the integrity of replication forks is maintained and protected from extensive degradation by RecA, RecF, RecO, and RecR until replication can resume. By contrast, inactivation of DnaB results in extensive degradation of the nascent and leading-strand template DNA and a loss of replication fork integrity as monitored by two-dimensional agarose gel analysis. The degradation that occurs following DnaB inactivation partially depends on several genes, including recF, recO, recR, recJ, recG, and xonA. Furthermore, the thermosensitive DnaB allele prevents UV-induced DNA degradation from occurring following arrest even at the permissive temperature, suggesting a role for DnaB prior to loading of the RecFOR proteins. We discuss these observations in relation to potential models for both UV-induced and DnaB(Ts)-mediated replication inhibition.

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Structural conservation of RecF and Rad50: implications for DNA recognition and RecF function

Koroleva O; Makharashvili N; Courcelle CT; Courcelle J; Korolev S

EMBO J (2007) 26:867-77

Abstract: RecF, together with RecO and RecR, belongs to a ubiquitous group of recombination mediators (RMs) that includes eukaryotic proteins such as Rad52 and BRCA2. RMs help maintain genome stability in the presence of DNA damage by loading RecA-like recombinases and displacing single-stranded DNA-binding proteins. Here, we present the crystal structure of RecF from Deinococcus radiodurans. RecF exhibits a high degree of structural similarity with the head domain of Rad50, but lacks its long coiled-coil region. The structural homology between RecF and Rad50 is extensive, encompassing the ATPase subdomain and the so-called 'Lobe II' subdomain of Rad50. The pronounced structural conservation between bacterial RecF and evolutionarily diverged eukaryotic Rad50 implies a conserved mechanism of DNA binding and recognition of the boundaries of double-stranded DNA regions. The RecF structure, mutagenesis of conserved motifs and ATP-dependent dimerization of RecF are discussed with respect to its role in promoting presynaptic complex formation at DNA damage sites.

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

Donaldson JR; Courcelle CT; Courcelle J 

J Biol Chem (2006) 281:28811-21.

Abstract: RuvABC is a complex that promotes branch migration and resolution of Holliday junctions. While 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 non-arresting lesions after replication has passed through the template.

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

Courcelle CT; Chow KH; Casey A; Courcelle J 

Proc Natl Acad Sci U S A (2006) 103:9154-9

Abstract: 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.

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Monitoring DNA replication following UV-induced damage in Escherichia coli

Courcelle CT; Courcelle J 

Methods Enz (2006) 409:425-41

Abstract: The question of how replication 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 have been characterized that, when mutated, are known to impair the ability of the cell to replicate in the presence of DNA damage.  This article 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.

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

Courcelle CT; Belle JJ; Courcelle J

J Bact (2005)  187:6953-61

Abstract: 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 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.

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Recs preventing wrecks

Courcelle J

Mutat Res (2005) 577:217-27
Abstract: 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.

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

Courcelle CT; Belle JJ; Courcelle J 

Res Microbiol (2004) 55:231-7

Abstract: 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.

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RecG or RuvAB is not required for the resumption of replication following UV irradiation Escherichia coli

Donaldson JR; Courcelle CT; Courcelle J 

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.

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

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.

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RecA-dependent recovery of arrested DNA replication forks

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.

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

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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Rec'd and repaired

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

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

Courcelle CT; Courcelle J; Prichard MN; Mocarski ES

Journal of Virology, 2001 Aug, 75(16): 7592-7601. 

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.

Courcelle J; Hanawalt PC 

Proceedings of the National Academy of Sciences of the United States of

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?

Courcelle J; Ganesan AK; Hanawalt PC 

BioEssays 23:463-470, May 2001 

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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Gene names: the approaching end of a century-long dilemma

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.

Courcelle J; Khodursky A; Peter B; Brown PO; Hanawalt PC

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.

Courcelle J; Hanawalt PC

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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Recovery of DNA replication in UV-irradiated Escherichia coli requires both excision repair and recF protein function. 

Courcelle J; Crowley DJ; Hanawalt PC.  

Journal of Bacteriology, 1999 Feb, 181(3):916-22. 

Abstract: After UV doses that disrupt DNA replication, the recovery of replication at replication forks in Escherichia coli requires a functional copy of the recF gene. In recF mutants, replication fails to recover and extensive degradation of the nascent DNA occurs, suggesting that recF function is needed to stabilize the disrupted replication forks and facilitate the process of recovery. We show here that the ability of recF to promote the recovery of replication requires that the disrupting lesions be removed. In the absence of excision repair, recF+ cells protect the nascent DNA at replication forks, but replication does not resume. The classical view is that recombination proteins operate in pathways that are independent from DNA repair, and therefore the functions of Rec proteins have been studied in repair-deficient cells. However, mutations in either uvr or recF result in failure to recover replication at UV doses from which wild-type cells recover efficiently, suggesting that recF and excision repair contribute to a common pathway in the recovery of replication.
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recF and recR are required for the resumption of replication at DNA replication forks in Escherichia coli.

Courcelle J; Carswell-Crumpton C; Hanawalt PC.

Proceedings of the National Academy of Sciences of the United States of

America, 1997 Apr 15, 94(8):3714-9.

Abstract: Escherichia coli containing a mutation in recF are hypersensitive to UV. However, they exhibit normal levels of conjugational or transductional recombination unless the major pathway (recBC) is defective. This implies that the UV sensitivity of recF mutants is not due to a defect in recombination such as occurs during conjugation or transduction. Here, we show that when replication is disrupted, at least two genes in the recF pathway, recF and recR, are required for the resumption of replication at DNA replication forks, and that in their absence, localized degradation occurs at the replication forks. Our observations support a model in which recF and recR are required to reassemble a replication holoenzyme at the site of a DNA replication fork. These results, when taken together with previous literature, suggest that the UV hypersensitivity of recF cells is due to an inability to resume replication at disrupted replication forks rather than to a defect in recombination. Current biochemical and genetic data on the conditions under which recF-mediated recombination occurs suggest that the recombinational intermediate also may mimic the structure of a disrupted replication fork.
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Is recF a DNA replication protein?

Kogoma T. Proceedings of the National Academy of Sciences of the United States of America, 1997 Apr 15, 94(8):3483-4.
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Kinetics of pyrimidine(6-4)pyrimidone photoproduct repair in Escherichia coli. 

Koehler DR; Courcelle J; Hanawalt PC.

Journal of Bacteriology, 1996 Mar, 178(5):1347-50.

Abstract: We compared the removal of pyrimidine(6-4)pyrimidone photoproducts [(6-4) photoproducts] and cyclobutane pyrimidine dimers (CPDs) from the genome of repair-proficient Escherichia coli, using monoclonal antibodies specific for each type of lesion. We found that (6-4) photoproducts were removed at a higher rate than CPDs in the first 30 min following a moderate UV dose (40 J/m2). The difference in rates was less than that typically reported for cultured mammalian cells, in which the removal of (6-4) photoproducts is far more rapid than that of CPDs.
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Analysis of a feline immunodeficiency virus provirus reveals patterns of gene sequence conservation distinct from human immunodeficiency virus type1. 

Sodora DL; Courcelle J; Brojatsch J; Berson A; Wang YC; Dow SW; Hoover EA;

Mullins JI.

Aids Research and Human Retroviruses, 1995 Apr, 11(4):531-3.

Thymine ring saturation and fragmentation products: lesion bypass, misinsertion and implications for mutagenesis. 

Evans J; Maccabee M; Hatahet Z; Courcelle J; Bockrath R; Ide H; Wallace S.  

Mutation Research, 1993 May, 299(3-4):147-56.

Abstract: We have used thymine glycol and dihydrothymine as representative ring saturation products resulting from free-radical interaction with DNA pyrimidines, and urea glycosides and beta-ureidoisobutyric acid (UBA) as models for pyrimidine-ring fragmentation products. We have shown that thymine glycol and the ring-fragmentation products urea and beta-ureidoisobutyric acid, as well as abasic sites, are strong blocks to DNA polymerases in vitro. In contrast, dihydrothymine is not a block to any of the polymerases tested. For thymine glycol, termination sites were observed opposite the putative lesions, whereas for the ring-fragmentation products, the termination sites were primarily one base prior to the lesion. These and other data have suggested that thymine glycol codes for an A, and that a base is stably inserted opposite the damage, whereas when a base is inserted opposite the non-coding lesions, it is removed by the 3-->5 exonuclease activity of DNA polymerase I. Despite their efficiency as blocking lesions, thymine glycol, urea and UBA can be bypassed at low frequency in certain specific sequence contexts. When the model lesions were introduced individually into single-stranded biologically active DNA, we found that thymine glycol, urea, beta-ureidoisobutyric acid, and abasic sites were all lethal lesions having an activation efficiency of 1, whereas dihydrothymine was not. Thus the in vitro studies predicted the in vivo results. When the survival of biologically active single-stranded DNA was examined in UV-induced Escherichia coli cells where the block to replication was released, no increase in survival was observed for DNA containing urea or abasic sites, suggesting inefficient bypass of these lesions. In contrast, beta-ureidoisobutyric acid survival was slightly enhanced, and transfecting DNA containing thymine glycols was significantly reactivated. When mutation induction by unique lesions was measured using f1-K12 hybrid DNA containing an E. coli target gene, thymine glycols and dihydrothymine were found to be inefficient as premutagenic lesions, suggesting that in vivo, as in vitro, they primarily code for A. In contrast, urea and beta-ureidoisobutyric acid were efficient premutagenic lesions, with beta-ureidoisobutyric acid being about 4-5-fold more effective than urea glycosides, which have approximately the same rate of mutation induction as abasic sites from purines. Sequence analysis of the mutations resulting from these ring-fragmentation products shows that the mutations produced are both lesion and sequence context dependent. The possible roles that bypass efficiency and lesion-directed misinsertion might play in mutagenesis are discussed.

Phil Hanawalt, Justin Courcelle, and Susan Wallace

1996 Gordon Conference on Mutagenesis, Plymouth NH

J Courcelle 1999 (PhD)

CS Tan 2000 (PhD)

KH Chow 2004 (Masters)

JR Donaldson 2005 (PhD)

JJ Belle 2007 (PhD)

KR Ona 2009 (Masters)

E Michel-Marks 2011 (Masters)

N Savic 2012 (Undergrad Honors)

K Newton 2012 (Masters)

A Jeiranian 2012 (PhD)

B Anderson 2013 (Undergrad Honors)

M Sadek 2014 (Undergrad McNair Scholar)

AV Perera 2015 (Masters)

DD Divingstone 2015 (Undergrad Honors)

C Hutfilz 2017 (Undergrad Honors)

JM Cole 2018 (Masters)

BM Wendel 2018 (PhD)

J Acott 2018 (Undergrad Honors)

J Acott 2018 (McNair Scholar)

E Weber 2019 (Undergrad Honors)

N Hamilton 2019 (Masters)