An official website of the United States government
Abstract DNA double-strand breaks require repair or risk corrupting the language of life. To ensure genome integrity and viability, multiple DNA double-strand break repair pathways function in eukaryotes. Two such repair pathways, canonical non-homologous end joining and homologous recombination, have been extensively studied, while other pathways such as microhomology-mediated end joint and single-strand annealing, once thought to serve as back-ups, now appear to play a fundamental role in DNA repair. Here, we review the molecular details and hierarchy of these four DNA repair pathways, and where possible, a comparison for what is known between animal and fungal models. We address the factors contributing to break repair pathway choice, and aim to explore our understanding and knowledge gaps regarding mechanisms and regulation in filamentous pathogens. We additionally discuss how DNA double-strand break repair pathways influence genome engineering results, including unexpected mutation outcomes. Finally, we review the concept of biased genome evolution in filamentous pathogens, and provide a model, termed Biased Variation, that links DNA double-strand break repair pathways with properties of genome evolution. Despite our extensive knowledge for this universal process, there remain many unanswered questions, for which the answers may improve genome engineering and our understanding of genome evolution.
more »
« less
Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice
Abstract The Mre11-Rad50-Nbs1 (MRN) complex recognizes and processes DNA double-strand breaks for homologous recombination by performing short-range removal of 5ʹ strands. Endonucleolytic processing by MRN requires a stably bound protein at the break site—a role we postulate is played by DNA-dependent protein kinase (DNA-PK) in mammals. Here we interrogate sites of MRN-dependent processing by identifying sites of CtIP association and by sequencing DNA-PK-bound DNA fragments that are products of MRN cleavage. These intermediates are generated most efficiently when DNA-PK is catalytically blocked, yielding products within 200 bp of the break site, whereas DNA-PK products in the absence of kinase inhibition show greater dispersal. Use of light-activated Cas9 to induce breaks facilitates temporal resolution of DNA-PK and Mre11 binding, showing that both complexes bind to DNA ends before release of DNA-PK-bound products. These results support a sequential model of double-strand break repair involving collaborative interactions between homologous and non-homologous repair complexes.
more »
« less
- Award ID(s):
- 1933303
- PAR ID:
- 10463267
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 14
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract Alternative end joining (alt-EJ) mechanisms, such as polymerase theta-mediated end joining, are increasingly recognized as important contributors to inaccurate double-strand break repair. We previously proposed an alt-EJ model whereby short DNA repeats near a double-strand break anneal to form secondary structures that prime limited DNA synthesis. The nascent DNA then pairs with microhomologous sequences on the other break end. This synthesis-dependent microhomology-mediated end joining (SD-MMEJ) explains many of the alt-EJ repair products recovered following I-SceI nuclease cutting in Drosophila. However, sequence-specific factors that influence SD-MMEJ repair remain to be fully characterized. Here, we expand the utility of the SD-MMEJ model through computational analysis of repair products at Cas9-induced double-strand breaks for 1100 different sequence contexts. We find evidence at single nucleotide resolution for sequence characteristics that drive successful SD-MMEJ repair. These include optimal primer repeat length, distance of repeats from the break, flexibility of DNA sequence between primer repeats, and positioning of microhomology templates relative to preferred primer repeats. In addition, we show that DNA polymerase theta is necessary for most SD-MMEJ repair at Cas9 breaks. The analysis described here includes a computational pipeline that can be utilized to characterize preferred mechanisms of alt-EJ repair in any sequence context.more » « less
-
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
-
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
-
Telomeres cap chromosome ends with specialized chromatin composed of DNA repeats bound by a multiprotein complex called shelterin. Fission yeast telomeres can be formed by cleaving a “proto-telomere” bearing 48 bp of telomere repeats to form a new stable chromosomal end that prevents the rapid degradation seen at similar DNA double-strand breaks (DSBs). This end-protection was investigated in viable mutants lacking telomere-associated proteins. Telomerase, the shelterin components Taz1, Rap1, or Poz1 or the telomere-associated protein Rif1 were not required to form a stable chromosome end after cleavage of the proto-telomere. However, cells lacking the fission yeast shelterin component Ccq1 converted the cleaved telomere repeat-capped end to a rapidly degraded DSB. Degradation was greatly reduced by eliminating the nuclease activity of Mre11, a component of the Mre11-Rad50-Nbs1/Xrs2 complex that processes DSBs. These results demonstrate a novel function for Ccq1 to effect end-protection by restraining Mre11-dependent degradation.more » « less
