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Pathways and signatures of mutagenesis at targeted DNA nicks
Yinbo Zhang1, Luther Davis1, Nancy Maizels1,2
1Department of Immunology, University of Washington Medical School, Seattle, Washington, United States of America.
Abstract:
Nicks are the most frequent form of DNA damage and a potential source of mutagenesis in human cells. By deep sequencing, we have identified factors and pathways that promote and limit mutagenic repair at a targeted nick in human cells. Mutations were distributed asymmetrically around the nick site. BRCA2 inhibited all categories of mutational events, including indels, SNVs and HDR. DNA2 and RPA promoted resection. DNA2 inhibited 1 bp deletions but contributed to longer deletions, as did REV7. POLQ stimulated SNVs. Parallel analysis of DSBs targeted to the same site identified similar roles for DNA2 and POLQ (but not REV7) in promoting deletions and for POLQ in stimulating SNVs. Insertions were infrequent at nicks, and most were 1 bp in length, as at DSBs. The translesion polymerase REV1 stimulated +1 insertions at one nick site but not another, illustrating the potential importance of sequence context in determining the outcome of mutagenic repair. These results highlight the potential for nicks to promote mutagenesis, especially in BRCA-deficient cells, and identify mutagenic signatures of DNA2, REV1, REV3, REV7 and POLQ.
Insights
DNA nicks, a common DNA damage, can lead to mutations. This study identifies key proteins like BRCA2, DNA2, and POLQ that influence mutagenic repair pathways at nicks, revealing specific mutation signatures.
Area of Science:
- Molecular Biology
- Genetics
- DNA Repair Mechanisms
Background:
- DNA nicks represent the most frequent DNA damage in human cells.
- These nicks are a potential source of mutagenesis, impacting genomic stability.
- Understanding repair pathways is crucial for preventing genetic alterations.
Purpose of the Study:
- To identify factors and pathways governing mutagenic repair at targeted DNA nicks.
- To elucidate the roles of specific proteins in nick repair outcomes.
- To compare nick repair mutagenesis with double-strand break (DSB) repair.
Main Methods:
- Deep sequencing of targeted nicks in human cells.
- Analysis of mutation distribution and types (indels, SNVs).
- Comparative analysis with targeted double-strand breaks (DSBs).
Main Results:
- Mutations showed asymmetric distribution around nick sites.
- BRCA2 inhibited all mutation types; DNA2 and RPA promoted DNA resection.
- POLQ stimulated single nucleotide variants (SNVs); REV7 contributed to deletions. DNA2 also contributed to longer deletions.
- REV1's role in insertions was sequence-context dependent.
Conclusions:
- DNA nicks can promote mutagenesis, particularly in BRCA-deficient cells.
- Specific proteins (DNA2, REV1, REV7, POLQ) exhibit distinct mutagenic signatures at nicks.
- Sequence context influences the mutagenic outcome of nick repair.
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