Determination of DNA lesion bypass using a ChIP-based assay

DNA lesion bypass is mediated by DNA damage tolerance (DDT) pathways and homologous recombination (HR). The DDT pathways, which involve translesion synthesis and template switching (TS), are activated by the ubiquitylation (ub) of PCNA through components of the RAD6-RAD18 pathway, whereas the HR pathway is independent of RAD18. However, it is unclear how these processes are coordinated within the context of chromatin. Here we show that Bre1, an ubiquitin ligase specific for histone H2B, is recruited to chromatin in a manner coupled to replication of damaged DNA. In the absence of Bre1 or H2Bub, cells exhibit accumulation of unrepaired DNA lesions. Consequently, the damaged forks become unstable and resistant to repair. We provide physical, genetic, and cytological evidence that H2Bub contributes toward both Rad18-dependent TS and replication fork repair by HR. Using an inducible system of DNA damage bypass, we further show that H2Bub is required for the regulation of DDT after genome duplication. We propose that Bre1-H2Bub facilitates fork recovery and gap-filling repair by controlling chromatin dynamics in response to replicative DNA damage.

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An underlying mechanism for the increased mutagenesis of lagging-strand genes inBacillus subtilis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1416651112 ◽

2015 ◽

Vol 112 (10) ◽

pp. E1096-E1105 ◽

Cited By ~ 46

Author(s):

Samuel Million-Weaver ◽

Ariana N. Samadpour ◽

Daniela A. Moreno-Habel ◽

Patrick Nugent ◽

Mitchell J. Brittnacher ◽

...

Keyword(s):

Excision Repair ◽

Underlying Mechanism ◽

Mutation Rates ◽

Dependent Manner ◽

Lesion Bypass ◽

Dna Lesion ◽

Accelerated Evolution ◽

Recombination Protein ◽

Y Family ◽

Lagging Strand

We previously reported that lagging-strand genes accumulate mutations faster than those encoded on the leading strand inBacillus subtilis. Although we proposed that orientation-specific encounters between replication and transcription underlie this phenomenon, the mechanism leading to the increased mutagenesis of lagging-strand genes remained unknown. Here, we report that the transcription-dependent and orientation-specific differences in mutation rates of genes require theB. subtilisY-family polymerase, PolY1 (yqjH). We find that without PolY1, association of the replicative helicase, DnaC, and the recombination protein, RecA, with lagging-strand genes increases in a transcription-dependent manner. These data suggest that PolY1 promotes efficient replisome progression through lagging-strand genes, thereby reducing potentially detrimental breaks and single-stranded DNA at these loci. Y-family polymerases can alleviate potential obstacles to replisome progression by facilitating DNA lesion bypass, extension of D-loops, or excision repair. We find that the nucleotide excision repair (NER) proteins UvrA, UvrB, and UvrC, but not RecA, are required for transcription-dependent asymmetry in mutation rates of genes in the two orientations. Furthermore, we find that the transcription-coupling repair factor Mfd functions in the same pathway as PolY1 and is also required for increased mutagenesis of lagging-strand genes. Experimental and SNP analyses ofB. subtilisgenomes show mutational footprints consistent with these findings. We propose that the interplay between replication and transcription increases lesion susceptibility of, specifically, lagging-strand genes, activating an Mfd-dependent error-prone NER mechanism. We propose that this process, at least partially, underlies the accelerated evolution of lagging-strand genes.

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Complex Formation with Rev1 Enhances the Proficiency of Saccharomyces cerevisiae DNA Polymerase ζ for Mismatch Extension and for Extension Opposite from DNA Lesions

Molecular and Cellular Biology ◽

10.1128/mcb.01671-06 ◽

2006 ◽

Vol 26 (24) ◽

pp. 9555-9563 ◽

Cited By ~ 88

Author(s):

Narottam Acharya ◽

Robert E. Johnson ◽

Satya Prakash ◽

Louise Prakash

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Polymerase ◽

Dna Lesions ◽

Synthetic Activity ◽

Lesion Bypass ◽

Dna Lesion ◽

C Terminus ◽

Accessory Subunit ◽

Induced Mutagenesis ◽

Y Family

ABSTRACT Rev1, a Y family DNA polymerase (Pol) functions together with Polζ, a B family Pol comprised of the Rev3 catalytic subunit and Rev7 accessory subunit, in promoting translesion DNA synthesis (TLS). Extensive genetic studies with Saccharomyces cerevisiae have indicated a requirement of both Polζ and Rev1 for damage-induced mutagenesis, implicating their involvement in mutagenic TLS. Polζ is specifically adapted to promote the extension step of lesion bypass, as it proficiently extends primer termini opposite DNA lesions, and it is also a proficient extender of mismatched primer termini on undamaged DNAs. Since TLS through UV-induced lesions and various other DNA lesions does not depend upon the DNA-synthetic activity of Rev1, Rev1 must contribute to Polζ-dependent TLS in a nonenzymatic way. Here, we provide evidence for the physical association of Rev1 with Polζ and show that this binding is mediated through the C terminus of Rev1 and the polymerase domain of Rev3. Importantly, a rev1 mutant that lacks the C-terminal 72 residues which inactivate interaction with Rev3 exhibits the same high degree of UV sensitivity and defectiveness in UV-induced mutagenesis as that conferred by the rev1Δ mutation. We propose that Rev1 binding to Polζ is indispensable for the targeting of Polζ to the replication fork stalled at a DNA lesion. In addition to this structural role, Rev1 binding enhances the proficiency of Polζ for the extension of mismatched primer termini on undamaged DNAs and for the extension of primer termini opposite DNA lesions.

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