Mechanistic Insights From Single-Molecule Studies of Repair of Double Strand Breaks

DNA double strand breaks (DSBs) are among some of the most deleterious forms of DNA damage. Left unrepaired, they are detrimental to genome stability, leading to high risk of cancer. Two major mechanisms are responsible for the repair of DSBs, homologous recombination (HR) and nonhomologous end joining (NHEJ). The complex nature of both pathways, involving a myriad of protein factors functioning in a highly coordinated manner at distinct stages of repair, lend themselves to detailed mechanistic studies using the latest single-molecule techniques. In avoiding ensemble averaging effects inherent to traditional biochemical or genetic methods, single-molecule studies have painted an increasingly detailed picture for every step of the DSB repair processes.

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Structural mechanism of DNA-end synapsis in the non-homologous end joining pathway for repairing double-strand breaks: bridge over troubled ends

Biochemical Society Transactions ◽

10.1042/bst20180518 ◽

2019 ◽

Vol 47 (6) ◽

pp. 1609-1619 ◽

Cited By ~ 5

Author(s):

Qian Wu

Keyword(s):

Single Molecule ◽

Genome Instability ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Structural Mechanism ◽

Strand Breaks ◽

Single Molecule Studies ◽

Non Homologous End Joining ◽

Dna Ends

Non-homologous end joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), which is the most toxic DNA damage in cells. Unrepaired DSBs can cause genome instability, tumorigenesis or cell death. DNA end synapsis is the first and probably the most important step of the NHEJ pathway, aiming to bring two broken DNA ends close together and provide structural stability for end processing and ligation. This process is mediated through a group of NHEJ proteins forming higher-order complexes, to recognise and bridge two DNA ends. Spatial and temporal understanding of the structural mechanism of DNA-end synapsis has been largely advanced through recent structural and single-molecule studies of NHEJ proteins. This review focuses on core NHEJ proteins that mediate DNA end synapsis through their unique structures and interaction properties, as well as how they play roles as anchor and linker proteins during the process of ‘bridge over troubled ends'.

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DNA polymerases δ and λ cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1507833112 ◽

2015 ◽

Vol 112 (50) ◽

pp. E6907-E6916 ◽

Cited By ~ 16

Author(s):

Damon Meyer ◽

Becky Xu Hua Fu ◽

Wolf-Dietrich Heyer

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Polymerase ◽

Genome Stability ◽

Double Strand Breaks ◽

Dna Polymerase Delta ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Repair Efficiency ◽

Repair Pathways

Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol δ) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol δ proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol δ and DNA polymerase 4 (Pol λ), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol λ showed faster kinetics associating with MMEJ substrates following DSB induction than Pol δ. The association of Pol δ depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol δ recruitment was diminished in cells lacking Pol λ. These data suggest cooperative involvement of both polymerases in MMEJ.

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Repair of DNA Double-Strand Breaks by the Nonhomologous End Joining Pathway

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-080320-110356 ◽

2021 ◽

Vol 90 (1) ◽

Author(s):

Benjamin M. Stinson ◽

Joseph J. Loparo

Keyword(s):

Genome Stability ◽

Double Strand Breaks ◽

Nonhomologous End Joining ◽

Annual Review ◽

Publication Date ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Break Site ◽

Dna Ends

DNA double-strand breaks pose a serious threat to genome stability. In vertebrates, these breaks are predominantly repaired by nonhomologous end joining (NHEJ), which pairs DNA ends in a multiprotein synaptic complex to promote their direct ligation. NHEJ is a highly versatile pathway that uses an array of processing enzymes to modify damaged DNA ends and enable their ligation. The mechanisms of end synapsis and end processing have important implications for genome stability. Rapid and stable synapsis is necessary to limit chromosome translocations that result from the mispairing of DNA ends. Furthermore, end processing must be tightly regulated to minimize mutations at the break site. Here, we review our current mechanistic understanding of vertebrate NHEJ, with a particular focus on end synapsis and processing. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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Evolutionary and Comparative Analysis of Bacterial Nonhomologous End Joining Repair

Genome Biology and Evolution ◽

10.1093/gbe/evaa223 ◽

2020 ◽

Vol 12 (12) ◽

pp. 2450-2466

Author(s):

Mohak Sharda ◽

Anjana Badrinarayanan ◽

Aswin Sai Narain Seshasayee

Keyword(s):

Genome Stability ◽

Double Strand Breaks ◽

Nonhomologous End Joining ◽

Ancestral Reconstruction ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Domains Of Life ◽

History Of ◽

Two Kingdoms

Abstract DNA double-strand breaks (DSBs) are a threat to genome stability. In all domains of life, DSBs are faithfully fixed via homologous recombination. Recombination requires the presence of an uncut copy of duplex DNA which is used as a template for repair. Alternatively, in the absence of a template, cells utilize error-prone nonhomologous end joining (NHEJ). Although ubiquitously found in eukaryotes, NHEJ is not universally present in bacteria. It is unclear as to why many prokaryotes lack this pathway. Toward understanding what could have led to the current distribution of bacterial NHEJ, we carried out comparative genomics and phylogenetic analysis across ∼6,000 genomes. Our results show that this pathway is sporadically distributed across the phylogeny. Ancestral reconstruction further suggests that NHEJ was absent in the eubacterial ancestor and can be acquired via specific routes. Integrating NHEJ occurrence data for archaea, we also find evidence for extensive horizontal exchange of NHEJ genes between the two kingdoms as well as across bacterial clades. The pattern of occurrence in bacteria is consistent with correlated evolution of NHEJ with key genome characteristics of genome size and growth rate; NHEJ presence is associated with large genome sizes and/or slow growth rates, with the former being the dominant correlate. Given the central role these traits play in determining the ability to carry out recombination, it is possible that the evolutionary history of bacterial NHEJ may have been shaped by requirement for efficient DSB repair.

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Faculty Opinions recommendation of Polq-Mediated End Joining Is Essential for Surviving DNA Double-Strand Breaks during Early Zebrafish Development.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726318081.793518723 ◽

2016 ◽

Author(s):

Cecilia Moens

Keyword(s):

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Zebrafish Development

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Analysis of chromatid-break-repair detects a homologous recombination to non-homologous end-joining switch with increasing load of DNA double-strand breaks

Mutation Research/Genetic Toxicology and Environmental Mutagenesis ◽

10.1016/j.mrgentox.2021.503372 ◽

2021 ◽

pp. 503372

Author(s):

Tamara Murmann-Konda ◽

Aashish Soni ◽

Martin Stuschke ◽

George Iliakis

Keyword(s):

Homologous Recombination ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Chromatid Break ◽

Break Repair ◽

Non Homologous End Joining ◽

Increasing Load

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The Maize Transposable Element Ac Induces Recombination Between the Donor Site and an Homologous Ectopic Sequence

Genetics ◽

10.1093/genetics/146.3.1143 ◽

1997 ◽

Vol 146 (3) ◽

pp. 1143-1151

Author(s):

Gil Shalev ◽

Avraham A Levy

Keyword(s):

Transposable Element ◽

Donor Site ◽

Double Strand Breaks ◽

Plant Genome ◽

Blue Staining ◽

Dna Double Strand Breaks ◽

End Joining ◽

Reading Frame ◽

Strand Breaks ◽

Ectopic Recombination

The prominent repair mechanism of DNA double-strand breaks formed upon excision of the maize Ac transposable element is via nonhomologous end joining. In this work we have studied the role of homologous recombination as an additional repair pathway. To this end, we developed an assay whereby β-Glucuronidase (GUS) activity is restored upon recombination between two homologous ectopic (nonallelic) sequences in transgenic tobacco plants. One of the recombination partners carried a deletion at the 5′ end of GUS and an Ac or a Ds element inserted at the deletion site. The other partner carried an intact 5′ end of the GUS open reading frame and had a deletion at the 3′ end of the gene. Based on GUS reactivation data, we found that the excision of Ac induced recombination between ectopic sequences by at least two orders of magnitude. Recombination events, visualized by blue staining, were detected in seedlings, in pollen and in protoplasts. DNA fragments corresponding to recombination events were recovered exclusively in crosses with Ac-carrying plants, providing physical evidence for Ac-induced ectopic recombination. The occurrence of ectopic recombination following double-strand breaks is a potentially important factor in plant genome evolution.

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