Physical interactions between MCM and Rad51 facilitate replication fork lesion bypass and ssDNA gap filling by non-recombinogenic functions

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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DNA transposons mediate duplications via transposition-independent and -dependent mechanisms in metazoans

Nature Communications ◽

10.1038/s41467-021-24585-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Shengjun Tan ◽

Huijing Ma ◽

Jinbo Wang ◽

Man Wang ◽

Mengxia Wang ◽

...

Keyword(s):

Inverted Repeat ◽

Replication Fork ◽

Single Copy ◽

Terminal Inverted Repeat ◽

Gap Filling ◽

New Genes ◽

Repeat Dna ◽

New Gene ◽

Gene Structures ◽

Reference Genomes

AbstractDespite long being considered as “junk”, transposable elements (TEs) are now accepted as catalysts of evolution. One example is Mutator-like elements (MULEs, one type of terminal inverted repeat DNA TEs, or TIR TEs) capturing sequences as Pack-MULEs in plants. However, their origination mechanism remains perplexing, and whether TIR TEs mediate duplication in animals is almost unexplored. Here we identify 370 Pack-TIRs in 100 animal reference genomes and one Pack-TIR (Ssk-FB4) family in fly populations. We find that single-copy Pack-TIRs are mostly generated via transposition-independent gap filling, and multicopy Pack-TIRs are likely generated by transposition after replication fork switching. We show that a proportion of Pack-TIRs are transcribed and often form chimeras with hosts. We also find that Ssk-FB4s represent a young protein family, as supported by proteomics and signatures of positive selection. Thus, TIR TEs catalyze new gene structures and new genes in animals via both transposition-independent and -dependent mechanisms.

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PARP10 promotes cellular proliferation and tumorigenesis by alleviating replication stress

10.1101/337220 ◽

2018 ◽

Author(s):

Emily M. Schleicher ◽

Adri M. Galvan ◽

George-Lucian Moldovan ◽

Claudia M. Nicolae

Keyword(s):

Replication Fork ◽

Cellular Proliferation ◽

Gene Knockout ◽

Replication Stress ◽

Cellular Transformation ◽

Tumor Formation ◽

Dna Lesions ◽

Lesion Bypass ◽

Replication Fork Arrest ◽

Adp Ribosyltransferase

ABSTRACTDuring carcinogenesis, cells are exposed to increased replication stress due to replication fork arrest at sites of DNA lesions and other difficult to replicate regions. Efficient fork restart and DNA repair are important for cancer cell proliferation. We previously showed that the ADP-ribosyltransferase PARP10 interacts with the replication protein PCNA and promotes lesion bypass by recruiting specialized, non-replicative DNA polymerases. Here, we show that PARP10 is overexpressed in a large proportion of human tumors. To understand the role of PARP10 in cellular transformation, we inactivated PARP10 in HeLa cancer cells by CRISPR/Cas9-mediated gene knockout, and overexpressed it in non-transformed RPE-1 cells. We found that PARP10 promotes cellular proliferation and replication fork elongation. Mechanistically, PARP10 overexpression alleviated cellular sensitivity to replication stress by fostering the restart of stalled replication forks. Importantly, mouse xenograft studies indicated that loss of PARP10 reduces the tumorigenesis activity of HeLa cells, while its overexpression results in tumor formation by non-transformed RPE-1 cells. Our findings indicate that PARP10 promotes cellular transformation by alleviating replication stress, and suggest that targeting PARP10 may represent a novel therapeutic approach.

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A Comprehensive View of Translesion Synthesis in Escherichia coli

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.00002-20 ◽

2020 ◽

Vol 84 (3) ◽

Author(s):

Shingo Fujii ◽

Robert P. Fuchs

Keyword(s):

Escherichia Coli ◽

Genetic Study ◽

Translesion Synthesis ◽

Gap Filling ◽

Lesion Bypass ◽

Content Type ◽

Comprehensive View ◽

Pol V ◽

Whole Process ◽

Per Se

The lesion bypass pathway, translesion synthesis (TLS), exists in essentially all organisms and is considered a pathway for postreplicative gap repair and, at the same time, for lesion tolerance. As with the saying “a trip is not over until you get back home,” studying TLS only at the site of the lesion is not enough to understand the whole process of TLS. Recently, a genetic study uncovered that polymerase V (Pol V), a poorly expressed Escherichia coli TLS polymerase, is not only involved in the TLS step per se but also participates in the gap-filling reaction over several hundred nucleotides.

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Requirement of Replication Checkpoint Protein Kinases Mec1/Rad53 for Postreplication Repair in Yeast

mBio ◽

10.1128/mbio.00079-11 ◽

2011 ◽

Vol 2 (3) ◽

Cited By ~ 9

Author(s):

Venkateswarlu Gangavarapu ◽

Sergio R. Santa Maria ◽

Satya Prakash ◽

Louise Prakash

Keyword(s):

Replication Fork ◽

Dna Lesions ◽

Yeast Cells ◽

Template Switching ◽

Replication Forks ◽

Lesion Site ◽

Lesion Bypass ◽

Content Type ◽

Replication Checkpoint ◽

Template Strand

ABSTRACT DNA lesions in the template strand block the replication fork. In Saccharomyces cerevisiae, replication through DNA lesions occurs via a Rad6/Rad18-dependent pathway where lesions can be bypassed by the action of translesion synthesis (TLS) DNA polymerases η and ζ or by Rad5-mediated template switching. An alternative Rad6/Rad18-independent but Rad52-dependent template switching pathway can also restore the continuity of the replication fork. The Mec1/Rad53-dependent replication checkpoint plays a crucial role in the maintenance of stable and functional replication forks in yeast cells with DNA damage; however, it has remained unclear which of the lesion bypass processes requires the activation of replication checkpoint-mediated fork stabilization. Here we show that postreplication repair (PRR) of newly synthesized DNA in UV-damaged yeast cells is inhibited in the absence of Mec1 and Rad53 proteins. Since TLS remains functional in cells lacking these checkpoint kinases and since template switching by the Rad5 and Rad52 pathways provides the alternative means of lesion bypass and requires Mec1/Rad53, we infer that lesion bypass by the template switching pathways occurs in conjunction with the replication fork that has been stabilized at the lesion site by the action of Mec1/Rad53-mediated replication checkpoint. IMPORTANCE Eukaryotic cells possess mechanisms called checkpoints that act to stop the cell cycle when DNA replication is halted by lesions in the template strand. Upon stalling of the ongoing replication at the lesion site, the recruitment of Mec1 and Rad53 kinases to the replication ensemble initiates the checkpoint wherein Mec1-mediated phosphorylation of Rad53 activates the pathway. A crucial role of replication checkpoint is to stabilize the replication fork by maintaining the association of DNA polymerases with the other replication components at the stall site. Our observations that Mec1 and Rad53 are required for lesion bypass by template switching have important implications for whether lesion bypass occurs in conjunction with the stalled replication ensemble or in gaps that could have been left behind the newly restarted forks. We discuss this important issue and suggest that lesion bypass in Saccharomyces cerevisiae cells occurs in conjunction with the stalled replication forks and not in gaps.

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Coordination between nucleotide excision repair and specialized polymerase DnaE2 action enables DNA damage survival in non-replicating bacteria

10.1101/2021.02.15.431208 ◽

2021 ◽

Author(s):

Asha Mary Joseph ◽

Saheli Daw ◽

Ismath Sadhir ◽

Anjana Badrinarayanan

Keyword(s):

Dna Damage ◽

Nucleotide Excision Repair ◽

Replication Fork ◽

Excision Repair ◽

Genome Integrity ◽

Replication Machinery ◽

Gap Filling ◽

Independent Manner ◽

Dna Lesion ◽

Nucleotide Excision

AbstractTranslesion synthesis (TLS) is a highly conserved mutagenic DNA lesion tolerance pathway, which employs specialized, low-fidelity DNA polymerases to synthesize across lesions. Current models suggest that activity of these polymerases is predominantly associated with ongoing replication, functioning either at or behind the replication fork. Here we provide evidence for DNA damage-dependent function of a specialized polymerase, DnaE2, in replication-independent conditions. We develop an assay to follow lesion repair in non-replicating Caulobacter and observe that components of the replication machinery localize on DNA in response to damage. These localizations persist in the absence of DnaE2 or if catalytic activity of the polymerase is mutated. Single-stranded DNA gaps for SSB binding and low-fidelity polymerase-mediated synthesis are generated by nucleotide excision repair, as replisome components fail to localize in its absence. This mechanism of gap-filling facilitates cell cycle restoration when cells are released into replication-permissive conditions. Thus, such cross-talk (between activity of NER and specialized polymerases in subsequent gap-filling) helps preserve genome integrity and enhances survival in a replication-independent manner.

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Coordination between nucleotide excision repair and specialized polymerase DnaE2 action enables DNA damage survival in non-replicating bacteria

eLife ◽

10.7554/elife.67552 ◽

2021 ◽

Vol 10 ◽

Author(s):

Asha Mary Joseph ◽

Saheli Daw ◽

Ismath Sadhir ◽

Anjana Badrinarayanan

Keyword(s):

Dna Damage ◽

Nucleotide Excision Repair ◽

Replication Fork ◽

Excision Repair ◽

Genome Integrity ◽

Replication Machinery ◽

Gap Filling ◽

Independent Manner ◽

Dna Lesion ◽

Nucleotide Excision

Translesion synthesis (TLS) is a highly conserved mutagenic DNA lesion tolerance pathway, which employs specialized, low-fidelity DNA polymerases to synthesize across lesions. Current models suggest that activity of these polymerases is predominantly associated with ongoing replication, functioning either at or behind the replication fork. Here we provide evidence for DNA damage-dependent function of a specialized polymerase, DnaE2, in replication-independent conditions. We develop an assay to follow lesion repair in non-replicating Caulobacter and observe that components of the replication machinery localize on DNA in response to damage. These localizations persist in the absence of DnaE2 or if catalytic activity of this polymerase is mutated. Single-stranded DNA gaps for SSB binding and low-fidelity polymerase-mediated synthesis are generated by nucleotide excision repair, as replisome components fail to localize in the absence of NER. This mechanism of gap-filling facilitates cell cycle restoration when cells are released into replication-permissive conditions. Thus, such cross-talk (between activity of NER and specialized polymerases in subsequent gap-filling) helps preserve genome integrity and enhances survival in a replication-independent manner.

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Homologous Recombination: To Fork and Beyond

Genes ◽

10.3390/genes9120603 ◽

2018 ◽

Vol 9 (12) ◽

pp. 603 ◽

Cited By ~ 17

Author(s):

Félix Prado

Keyword(s):

Homologous Recombination ◽

Chromosome Segregation ◽

Replication Fork ◽

Genome Duplication ◽

Current Knowledge ◽

Replication Forks ◽

Lesion Bypass ◽

Dna Damage Tolerance ◽

Multiple Factors ◽

Dynamic Replication

Accurate completion of genome duplication is threatened by multiple factors that hamper the advance and stability of the replication forks. Cells need to tolerate many of these blocking lesions to timely complete DNA replication, postponing their repair for later. This process of lesion bypass during DNA damage tolerance can lead to the accumulation of single-strand DNA (ssDNA) fragments behind the fork, which have to be filled in before chromosome segregation. Homologous recombination plays essential roles both at and behind the fork, through fork protection/lesion bypass and post-replicative ssDNA filling processes, respectively. I review here our current knowledge about the recombination mechanisms that operate at and behind the fork in eukaryotes, and how these mechanisms are controlled to prevent unscheduled and toxic recombination intermediates. A unifying model to integrate these mechanisms in a dynamic, replication fork-associated process is proposed from yeast results.

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Interpretation and Gap Filling in International Commercial Contracts

10.1017/9781780689760 ◽

2019 ◽

Author(s):

Ayse Nihan Karadayi Yalim

Keyword(s):

Gap Filling

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