Abstract
DNA double-strand breaks (DSBs) are toxic lesions, which if improperly repaired can result in cell death or genomic instability. DSB repair is usually facilitated by the classical non-homologous end joining (C-NHEJ), or homologous recombination (HR) pathways. However, a mutagenic alternative NHEJ pathway, microhomology-mediated end joining (MMEJ), can also be deployed. While MMEJ is suppressed by C-NHEJ, the relationship between HR and MMEJ is less clear. Here, we describe a role for HR genes in suppressing MMEJ in human cells. By monitoring DSB mis-repair using a sensitive HPRT assay, we found that depletion of HR proteins, including BRCA2, BRCA1 or RPA, resulted in a distinct mutational signature associated with significant increases in break-induced mutation frequencies, deletion lengths and the annealing of short regions of microhomology (2–6 bp) across the break-site. This signature was dependent on CtIP, MRE11, POLQ and PARP, and thus indicative of MMEJ. In contrast to CtIP or MRE11, depletion of BRCA1 resulted in increased partial resection and MMEJ, thus revealing a functional distinction between these early acting HR factors. Together these findings indicate that HR factors suppress mutagenic MMEJ following DSB resection.
Highlights
DNA double strand breaks (DSBs) are deleterious lesions that if left unrepaired can lead to cell death, while if misrepaired can give rise to genomic instability, leading to tumorigenesis [1]
We have previously shown that siRNA-mediated depletions of SETD2 or RAD51 had similar effects on I-SceI-induced DSBs in the HPRT gene, namely an increased overall frequency of HPRT mutagenesis associated with increases in both deletion lengths and use of microhomologies on either side of the deletions [51]
Similar to the depletion of RAD51, SETD2, BRCA2 and BRCA1, depletion of either Cterminal binding protein interacting protein (CtIP) or MRE11 resulted in a significant increase in the I-SceI-induced mutation frequency (3.5%; P = 0.0007; and 3.6%; P < 0.0001, respectively) compared to 1.1% in NT controls (Figure 4A)
Summary
DNA double strand breaks (DSBs) are deleterious lesions that if left unrepaired can lead to cell death, while if misrepaired can give rise to genomic instability, leading to tumorigenesis [1]. Resection is initiated by the endonucleolytic activity of the MRE11-RAD50-NBS1 (MRN) complex and the Cterminal binding protein interacting protein (CtIP), which exposes short ssDNA tails [9,10]. These become substrates for the extensive resection mediators, Exo, DNA2 and BLM [11,12]. During synthesis-dependent strand annealing (SDSA) [22], the invading and extended strand is expelled from the D-loop to anneal to the second end which, following gap filling and ligation, results in error-free repair [23]
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