Structural Basis And Functional Importance Of INTS3/INTS6 Assembly In DNA Double-strand Break Repair Pathway

Genome is often subjected to environmental and metabolic DNA damaging agents.It is estimated that each cell encounters 104-105 lesions per day.In eukaryotes,two distinct pathways,homologous recombination(HR)and non-homologous end joining(NHEJ)have evolved to repair double strand breaks(DSBs).As a more accurate mechanism,HR requires a sister chromatid as the template to ensure genome integrity,only occurs in the S and G2 phases of the cell cycle.One of the initial steps in the process of HR is the resection of DSBs to generate a 3' single-stranded DNA(ssDNA)overhang,which is essential for RAD51-mediated strand exchange.SOSS1 is a single-stranded DNA(ssDNA)-binding protein complex that plays a critical role in DSB repair.SOSS1 consists of three subunits:INTS3,SOSSC,and hSSB1,with INTS3 serving as a scaffold to stabilize this complex.SOSS1 can bind to ssDNA with a minimal length of-35 nucleotides(nt)and its DNA binding affinity is-30-fold stronger than that of SSB1 alone.Structural studies of INTS3N/hSSBl/SOSSC showed that hSSB1 solely contributes to the binding of ssDNA while neither SOSSC nor INTS3N is critical for ssDNA binding,implying that the C-terminal of INTS3(INTS3c)may be involved in the recognition of longer ssDNA.Recent studies have further identified INTS6 as an additional Integrator component involved in the DSB repair pathway by regulating the INTS3/hSSB1/INTS6 complex,but how INTS3 interacts with INTS6,hence impacting DBS repair is not clear.In the present study,we determined the crystal structure of INTS3c in complex with the C-terminal of INTS6(INTS6c)at a resolution of 2.4 A.Structure analysis revealed that two INTS3c subunits dimerized and interacted with INTS6c via conserved residues.Subsequent biochemical analyses further confirmed that INTS3c formed a stable dimer both in vitro and in vivo,and electrophoretic mobility shift assay showed that INTS3 dimerization was important for recognizing the longer ssDNA.Functional studies revealed that perturbation of INTS3c dimerization or INTS3c/INTS6c interaction inhibited the DSB repair process Altogether,these results unravel the underappreciated role of INTS3 dimerization and the molecular basis of INTS3/INTS6 interaction in DSB repair.