Microhomology-mediated end joining (MMEJ) is a Ku and Ligase IV independent mechanism for repair of DNA double-strand breaks, which contributes to chromosome rearrangements. Here we used a chromosomal end-joining assay to determine the genetic requirements for MMEJ in Saccharomyces cerevisiae. We found that end resection influences the ability to expose microhomologies; however, it is not rate limiting for MMEJ in wild-type cells. The frequency of MMEJ increased by up to 350-fold in rfa1 hypomorphic mutants, suggesting that replication protein A (RPA) bound to the ssDNA overhangs formed by resection prevents spontaneous annealing between microhomologies. In vitro, the mutant RPA complexes were unable to fully extend ssDNA and were compromised in their ability to prevent spontaneous annealing. We propose the helix-destabilizing activity of RPA channels ssDNA intermediates from mutagenic MMEJ to error-free homologous recombination, thus preserving genome integrity.

Mammalian polymerase theta (Polθ) is a multifunctional enzyme that promotes error-prone DNA repair by alternative-NHEJ (alt-NHEJ). Here we perform structure-function analyses and report that, in addition to the polymerase domain, the helicase activity plays a central role during Polθ-mediated double-strand break (DSB) repair. Our results show that Polθ-helicase promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells. The helicase activity also suppresses CRISPR/Cas9 mediated gene targeting by homologous recombination (HR). In vitro experiments reveal that Polθ–helicase facilitates the removal of RPA from resected DSBs to allow their annealing and subsequent joining by alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, we show that the inhibition of RPA1 enhances end-joining and suppresses recombination. Taken together, our results reveal that the balance between HR and alt-NHEJ is controlled by opposing activities of Polθ and RPA, providing further insight into the regulation of repair pathway choice in mammalian cells.

Foldback priming at DNA double-stranded breaks is one mechanism proposed to initiate palindromic gene amplification, a common feature of cancer cells. Here we show small (5–9 bp) inverted repeats drive the formation of large palindromic duplications, the major class of chromosomal rearrangements recovered from yeast cells lacking Sae2 or the Mre11 nuclease. RPA dysfunction increased the frequency of palindromic duplications in Sae2 or Mre11 nuclease-deficient cells by ~1000-fold, consistent with intra-strand annealing to create a hairpin-capped chromosome that is subsequently replicated to form a dicentric isochromosome. The palindromic duplications were frequently associated with duplication of a second chromosome region bounded by a repeated sequence and a telomere, suggesting the dicentric chromosome breaks and repairs by recombination between dispersed repeats to acquire a telomere. We propose secondary structures within single-stranded DNA are potent instigators of genome instability, and RPA and Mre11-Sae2 play important roles in preventing their formation and propagation, respectively.

The Mre11-Rad50-Xrs2/NBS1 (MRX/N) nuclease/ATPase complex plays structural and catalytic roles in the repair of DNA doublestrand breaks (DSBs) and is the DNA damage sensor for Tel1/ATM kinase activation. Saccharomyces cerevisiae Sae2 can function with MRX to initiate 5′-3′ end resection and also plays an important role in attenuation of DNA damage signaling. Here we describe a class of mre11 alleles that suppresses the DNA damage sensitivity of sae2Δ cells by accelerating turnover of Mre11 at DNA ends, shutting off the DNA damage checkpoint and allowing cell cycle progression. The mre11 alleles do not suppress the end resection or hairpinopening defects of the sae2Δ mutant, indicating that these functions of Sae2 are not responsible for DNA damage resistance. The purified M P110L RX complex shows reduced binding to single-and double-stranded DNA in vitro relative to wild-type MRX, consistent with the increased turnover of Mre11 from damaged sites in vivo. Furthermore, overproduction of Mre11 causes DNA damage sensitivity only in the absence of Sae2. Together, these data suggest that it is the failure to remove Mre11 from DNA ends and attenuate Rad53 kinase signaling that causes hypersensitivity of sae2Δ cells to clastogens.DNA repair | Mre11 | Sae2 | DNA damage checkpoint

Replication protein A (RPA) is the main eukaryotic single-stranded DNA (ssDNA) binding protein, having essential roles in all DNA metabolic reactions involving ssDNA. RPA binds ssDNA with high affinity, thereby preventing the formation of secondary structures and protecting ssDNA from the action of nucleases, and directly interacts with other DNA processing proteins. Here we discuss recent results supporting the idea that one function of RPA is to prevent annealing between short repeats that can lead to chromosome rearrangements by microhomology-mediated end joining or the formation of hairpin structures that are substrates for structure-selective nucleases. We suggest that replication fork catastrophe caused by depletion of RPA could result from cleavage of secondary structures by nucleases, and that failure to cleave hairpin structures formed at DNA ends could lead to gene amplification. These studies highlight the important role RPA plays in maintaining genome integrity.