Understanding The Roles Of SOSS1and ZNHIT1/SRCAP In Homologous Recombination Repair

Endogenous and exogenous genotoxic agents, such as ionizing radiation and free radicals generated from cellular metabolism can cause DNA damage, which will lead to genomic instability and tumorigenesis if unrepaired or repaired incorrectly. DNA damage can be divided into multiple types, including single-strand breaks, double-strand breaks (DSBs), intrastrand cross-links, interstrand cross-links, pyrimidine dimer formation and base mismatch. DSBs are one of the most dangerous types of DNA damage, causing gene rearrangement, chromosome loss, gene fusion or frame shift, cell death and genetic diseases including cancer. Two major pathways responsible for DSB repair in eukaryotic cells are non-homologous end joining (NHEJ) repair and homologous recombination (HR) repair. It is generally believed that NHEJ is an error-prone pathway, which directly joins the two ends of DSB and leads to the deletion of partial DNA. HR is an error-free repair pathway for DSBs, which is active mainly in S and G2phases. HR requires a sequence of sister chromatid similar or identical to the broken DNA to synthesize DNA, and therefore maintains genomic integrity as well as stability. DNA end resection in the DSB ends is critical for the choice between NHEJ and HR, which is the initial step of HR pathway. Firstly, the MRN (MRE11, RAD50, and NBS1) complex and CtIP collaborately process the DSB ends to generate3’ overhang ssDNA tails. RPA complex then binds to the ssDNA to form RPA-ssDNA structures, and recruits ATRIP-ATR to DNA damage sites to activate ATR kinase. ATR activation on RPA-ssDNA elicits cell cycle checkpoints and promotes the repair of DNA damage. Secondly, with the assistance of HR mediators (such as BRCA1, BRCA2, PALB2and RAD51paralogs), RAD51displaces RPA to form RAD51-ssDNA filaments. RAD51-ssDNA filament catalyzes strand invasion to match homolog DNA template sequence and forms D-loop structure. Finally, RAD51dissociates from DNA to expose the3’end which is required for DNA synthesis. Accompanied by DNA synthesis, the cross between newly synthesised DNA and templated DNA leads to the form of double Holliday Junction (dHJ). As a key recombination intermediate in HR, dHJ can be dissolved by the BLM-TopoIIIa-RMI1-RMI2(BTR) complex to produce non-crossover products. After this process, cells complete high-fidelity HR repair.SSBs are essential for a series of DNA metabolic processes such as DNA replication, repair and recombination pathways. These SSBs protect ssDNA from nucleolytic damage, prevent hairpin formation and block DNA reannealing until the processing pathway is successfully completed. In eukaryocytes, SOSS1(sensor of ssDNA1) complex and RPA complex are the main SSBs involved in the repair of damaged DNA. We focus on the molecular mechanism and the regulation of SOSS1complex as well as the function of RPA complex in HR repair.SOSS1complex is composed of SOSSA, SOSSB1and SOSSC. Through recognising and binding ssDNA, SOSS1complex promotes HR repair. However, compared to RPA complex, the study of SOSS1complex initiates later and the molecular mechanism remains unclear. Therefore, we take advantage of the technology of structural biology to study the crystal structure of the N-terminal half of SOSSA (SOSSAn) in complex with SOSSB1and SOSSC. Meanwhile, from the viewpoint of molecular biology, we study the molecular mechanism of how the SOS S1complex assembles. We show that SOSSA serves as a scaffold to bind both SOSSB1and SOSSC for the assembly of the SOSS1complex. SOSSA promotes the stability of other subunits in SOSS1complex and their precise location in the nucleus. SOSSB1is the key partner involved in ssDNA binding. SOSSB1interacts with both SOSSAN and ssDNA via two distinct surfaces. The recognition of ssDNA with a length of up to nine nucleotides is mediated solely by SOSSB1. Furthermore, we identify the critical amino acid sites involved in mediating the assembly of SOSS1complex and the recognition of ssDNA. The point mutants of these sites display defects in DSB repair, indicating the importance of SOSS1in HR pathway.RPA complex plays crucial roles in virtually all aspects of eukaryotic DNA metabolism. RPA complex is a sensor of DNA damage to activate DNA damage checkpoint, which efficiently maintains genomic integrity. In HR repair,3’ssDNA generated by end resection is coated by RPA complex, which promotes strand invasion catalyzed by RAD51-ssDNA nucleoprotein filament. But the regulatory mechanism of the recruitment for RPA complex in DNA damage sites remains elusive. Due to the highly condensed and tightly wrapped structure of chromosome, the MRN complex as well as CtIP required for DNA end resection can’t be recruited to DNA damage sites effectively. SRCAP is an ATP-dependent chromatin-remodeling enzyme. Mutations in SRCAP are responsible for a genetic disorder known as Floating-Harbor syndrome. We find that the human SRCAP chromatin remodeling enzyme and ZNHIT1, a small subunit of the SRCAP complex, participate in the relaxation of chromatin structure after DNA damage. We further demonstrate that SRCAP/ZNHIT1promotes the recruitment of CtIP to DNA damage sites and the subsequent CtIP-dependent DNA-end resection. Our findings establish SRCAP/ZNHIT1as a newly identified regulator of DNA damage response pathways and highlight the importance of ATP-dependent chromatin remodeling in maintaining genomic integrity.In conclusion, both SOSS1and RPA complex are recruited to DSBs, functioning independently and cooperatively in the HR repair pathway.