Microhomology-mediated end joining (MMEJ) is a major pathway for Ku-independent alternative nonhomologous end joining, which contributes to chromosomal translocations and telomere fusions, but the underlying mechanism of MMEJ in mammalian cells is not well understood. In this study, we demonstrated that, distinct from Ku-dependent classical nonhomologous end joining, MMEJ-even with very limited end resection-requires cyclin-dependent kinase activities and increases significantly when cells enter S phase. We also showed that MMEJ shares the initial end resection step with homologous recombination (HR) by requiring meiotic recombination 11 homolog A (Mre11) nuclease activity, which is needed for subsequent recruitment of Bloom syndrome protein (BLM) and exonuclease 1 (Exo1) to DNA double-strand breaks (DSBs) to promote extended end resection and HR. MMEJ does not require S139-phosphorylated histone H2AX (γ-H2AX), suggesting that initial end resection likely occurs at DSB ends. Using a MMEJ and HR competition repair substrate, we demonstrated that MMEJ with short end resection is used in mammalian cells at the level of 10-20% of HR when both HR and nonhomologous end joining are available. Furthermore, MMEJ is used to repair DSBs generated at collapsed replication forks. These studies suggest that MMEJ not only is a backup repair pathway in mammalian cells, but also has important physiological roles in repairing DSBs to maintain cell viability, especially under genomic stress.BLM/Exo1 | CtIP | DNA repair pathway | DNA damage | genome stability D NA double-strand breaks (DSBs) can be repaired by multiple pathways. The classical nonhomologous end joining (C-NHEJ) pathway relies on Ku70/Ku80 and ligates DSB ends without a template (1). Homologous recombination (HR), an error-free pathway, uses a homologous template to repair DSBs (2) and is initiated by end resection from DSB ends to generate a long stretch of singlestrand DNA (ssDNA) for strand invasion. Although C-NHEJ is active throughout the cell cycle, HR is used when cells enter S and G2 because cyclin-dependent kinases (CDKs) are needed for promoting end resection to activate HR (3-5).In the absence of C-NHEJ factors such as Ku70, Ku80, or DNA ligase IV, robust alternative nonhomologous end joining (alt-NHEJ) activity is observed in various organisms including yeast and mammals (6, 7). Many alt-NHEJ events, classified as microhomology-mediated end joining (MMEJ), require end resection and join the ends by base pairing at microhomology sequences (5-25 nucleotides), resulting in deletions at the junctions (6). However, other alt-NHEJ pathways without using microhomology regions also exist.Genetic analyses in yeast reveal that MMEJ is Rad52-independent, distinguishing it from HR and single-strand annealing (SSA) repair pathways, whereas the Mre11-Rad50-Xrs2 (MRX) complex and DNA ligase IV are needed for MMEJ (8-10). Further studies suggest that in yeast, Srs2 helicase, Sae2 nuclease [CtBP-interacting protein (CtIP) homologue], Tel1 [ataxia telangiectasia mutated (A...

Summary Chromosomal rearrangements often occur at genomic loci with DNA secondary structures, such as common fragile sites (CFSs) and palindromic repeats. We developed assays in mammalian cells that revealed CFS-derived AT-rich sequences and Alu inverted repeats (Alu-IRs) are mitotic recombination hotspots, requiring the repair functions of CtIP and the Mre11/Rad50/Nbs1 complex (MRN). We also identified an endonuclease activity of CtIP which is dispensable for end resection and homologous recombination (HR) at I-SceI-generated "clean" double-strand breaks (DSBs), but is required for repair of DSBs occurring at CFS-derived AT-rich sequences. In addition, CtIP nuclease defective mutants are impaired in Alu-IRs-induced mitotic recombination. These studies suggest that an end resection-independent CtIP function is important for processing DSB ends with secondary structures to promote HR. Furthermore, our studies uncover an important role of MRN, CtIP and their associated nuclease activities in protecting CFSs in mammalian cells.

Stabilizing high-efficiency perovskite solar cells (PSCs) at operating conditions remains an unresolved issue hampering its large-scale commercial deployment. Here, we report a star-shaped polymer to improve charge transport and inhibit ion migration at the perovskite interface. The incorporation of multiple chemical anchor sites in the star-shaped polymer branches strongly controls the crystallization of perovskite film with lower trap density and higher carrier mobility and thus inhibits the nonradiative recombination and reduces the charge-transport loss. Consequently, the modified inverted PSCs show an optimal power conversion efficiency of 22.1% and a very high fill factor (FF) of 0.862, corresponding to 95.4% of the Shockley-Queisser limited FF (0.904) of PSCs with a 1.59-eV bandgap. The modified devices exhibit excellent long-term operational and thermal stability at the maximum power point for 1000 hours at 45°C under continuous one-sun illumination without any significant loss of efficiency.

hSSB1 (human single strand DNA-binding protein 1) has been shown to participate in homologous recombination (HR)-dependent repair of DNA double strand breaks (DSBs) and ataxia telangiectasia-mutated (ATM)-mediated checkpoint pathways. Here we present evidence that hSSB2, a homolog of hSSB1, plays a role similar to hSSB1 in DNA damage-response pathways. This was evidenced by findings that hSSB2-depleted cells resemble hSSB1-depleted cells in hypersensitivity to DNA-damaging reagents, reduced efficiency in HR-dependent repair of DSBs, and defective ATM-dependent phosphorylation. Notably, hSSB1 and hSSB2 form separate complexes with two identical proteins, INTS3 and hSSBIP1 (C9ORF80). Cells depleted of INTS3 and hSSBIP1 also exhibited hypersensitivity to DNA damage reagents, chromosomal instability, and reduced ATM-dependent phosphorylation. hSSBIP1 was rapidly recruited to laser-induced DSBs, a feature also similar to that reported for hSSB1. Depletion of INTS3 decreased the stability of hSSB1 and hSSBIP1, suggesting that INTS3 may provide a scaffold to allow proper assembly of the hSSB complexes. Thus, our data demonstrate that hSSB1 and hSSB2 form two separate complexes with similar structures, and both are required for efficient HR-dependent repair of DSBs and ATM-dependent signaling pathways.

In response to DNA damage, the Fanconi anemia (FA) core complex functions as a signaling machine for monoubiquitination of FANCD2 and FANCI. It remains unclear whether this complex can also participate in subsequent DNA repair. We have shown previously that the FANCM constituent of the complex contains a highly conserved helicase domain and an associated ATP-dependent DNA translocase activity. Here we show that FANCM also possesses an ATP-independent binding activity and an ATP-dependent bi-directional branch-point translocation activity on a synthetic four-way junction DNA, which mimics intermediates generated during homologous recombination or at stalled replication forks. Using an siRNA-based complementation system, we found that the ATP-dependent activities of FANCM are required for cellular resistance to a DNA-crosslinking drug, mitomycin C, but not for the monoubiquitination of FANCD2 and FANCI. In contrast, monoubiquitination requires the entire helicase domain of FANCM, which has both ATP dependent and independent activities. These data are consistent with participation of FANCM and its associated FA core complex in the FA pathway at both signaling through monoubiquitination and the ensuing DNA repair.