This Special Issue (SI) of Environmental and Molecular Mutagenesis (EMM), entitled “Inspiring Basic and Applied Research in Genome Integrity Mechanisms,” is to update the community on recent findings and advances on genome integrity mechanisms with emphasis on their importance for basic and environmental health sciences. This SI includes two research articles, one brief research communication, and four reviews that highlight cutting edge research findings and perspectives, from both established leaders and junior trainees, on DNA repair mechanisms. In particular, the authors provided an updated understanding on several distinct enzymes (e.g., DNA polymerase beta, DNA polymerase theta, DNA glycosylase NEIL2) and the associated molecular mechanisms in base excision repair, nucleotide excision repair, and microhomology‐mediated end joining of double‐strand breaks. In addition, genome‐wide sequencing analysis or site‐specific mutational signature analysis of DNA lesions from environmental mutagens (e.g., UV light and aflatoxin) provide further characterization and sequence context impact of DNA damage and mutations. This SI is dedicated to the legacy of Dr. Samuel H. Wilson from the U.S. National Institute of Environmental Health Sciences at the National Institutes of Health.
more » « less- PAR ID:
- 10500462
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Environmental and Molecular Mutagenesis
- ISSN:
- 0893-6692
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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The plant-specific RNA Polymerase V (Pol V) plays a key role in gene silencing, but its role in repair of double stranded DNA breaks is unclear. Excision of the transposable element mPing creates double stranded breaks that are repaired by NHEJ. We measured mPing excision site repair in multiple DNA methylation mutants including pol V using an mPing : GFP reporter. Two independent mutant alleles of pol V showed less GFP expression, indicating that the Pol V protein plays a role in excision site repair. Sequence analysis of the pol V excision sites indicated an elevated rate of large deletions consistent with less efficient repair. These results clarify the role of Pol V, but not other RNA-directed DNA methylation proteins (Pol IV) or maintenance DNA methylation pathways ( MET1 ), in the repair of double-strand DNA breaks.more » « less
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The plant-specific RNA Polymerase V (Pol V) plays a key role in gene silencing, but its role in repair of double stranded DNA breaks is unclear. Excision of the transposable element mPing creates double stranded breaks that are repaired by NHEJ. We measured mPing excision site repair in multiple DNA methylation mutants including pol V using an mPing:GFP reporter. Two independent mutant alleles of pol V showed less GFP expression, indicating that the Pol V protein plays a role in excision site repair. Sequence analysis of the pol V excision sites indicated an elevated rate of large deletions consistent with less efficient repair. These results clarify the role of Pol V, but not other RNA-directed DNA methylation proteins (Pol IV) or maintenance DNA methylation pathways (MET1), in the repair of double-strand DNA breaks.more » « less
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Abstract Background Nucleotide excision repair is the primary DNA repair mechanism that removes bulky DNA adducts such as UV-induced pyrimidine dimers. Correspondingly, genome-wide mapping of nucleotide excision repair with eXcision Repair sequencing (XR-seq), provides comprehensive profiling of DNA damage repair. A number of XR-seq experiments at a variety of conditions for different damage types revealed heterogenous repair in the human genome. Although human repair profiles were extensively studied, how repair maps vary between primates is yet to be investigated. Here, we characterized the genome-wide UV-induced damage repair in gray mouse lemur,
Microcebus murinus , in comparison to human.Results We derived fibroblast cell lines from mouse lemur, exposed them to UV irradiation, and analyzed the repair events genome-wide using the XR-seq protocol. Mouse lemur repair profiles were analyzed in comparison to the equivalent human fibroblast datasets. We found that overall UV sensitivity, repair efficiency, and transcription-coupled repair levels differ between the two primates. Despite this, comparative analysis of human and mouse lemur fibroblasts revealed that genome-wide repair profiles of the homologous regions are highly correlated, and this correlation is stronger for highly expressed genes. With the inclusion of an additional XR-seq sample derived from another human cell line in the analysis, we found that fibroblasts of the two primates repair UV-induced DNA lesions in a more similar pattern than two distinct human cell lines do.
Conclusion Our results suggest that mouse lemurs and humans, and possibly primates in general, share a homologous repair mechanism as well as genomic variance distribution, albeit with their variable repair efficiency. This result also emphasizes the deep homologies of individual tissue types across the eukaryotic phylogeny.
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Abstract Transcription-coupled repair is essential for the removal of DNA lesions from the transcribed genome. The pathway is initiated by CSB protein binding to stalled RNA polymerase II. Mutations impairing CSB function cause severe genetic disease. Yet, the ATP-dependent mechanism by which CSB powers RNA polymerase to bypass certain lesions while triggering excision of others is incompletely understood. Here we build structural models of RNA polymerase II bound to the yeast CSB ortholog Rad26 in nucleotide-free and bound states. This enables simulations and graph-theoretical analyses to define partitioning of this complex into dynamic communities and delineate how its structural elements function together to remodel DNA. We identify an allosteric pathway coupling motions of the Rad26 ATPase modules to changes in RNA polymerase and DNA to unveil a structural mechanism for CSB-assisted progression past less bulky lesions. Our models allow functional interpretation of the effects of Cockayne syndrome disease mutations.
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Trakselis, Michael A. (Ed.)The genome of prokaryotes can be damaged by a variety of endogenous and exogenous factors, including reactive oxygen species, UV exposure, and antibiotics. To better understand these repair processes and the impact they may have on DNA replication, normal genome maintenance processes can be perturbed by removing or editing associated genes and monitoring DNA repair outcomes. In particular, the replisome activities of DNA unwinding by the helicase and DNA synthesis by the polymerase must be tightly coupled to prevent any appreciable single strand DNA (ssDNA) from accumulating and amplifying genomic stress. If decoupled, vulnerable ssDNA would persist, likely leading to double strand breaks (DSBs) or requiring replication restart mechanisms downstream of a stall. In either case, free 3'-OH strands would exist, resulting from ssDNA gaps in the leading strand or complete DSBs. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) can enzymatically label ssDNA ends with bromo-deoxy uridine triphosphate (BrdU) to detect free 3'-OH DNA ends in the E. coli genome. Labeled DNA ends can be detected and quantified using fluorescence microscopy or flow cytometry. This methodology is useful in applications where in situ investigation of DNA damage and repair are of interest, including effects from enzyme mutations or deletions and exposure to various environmental conditions.more » « less