Crystal structure of the INTS3/INTS6 complex reveals the functional importance of INTS3 dimerization in DSB repair

Yu Jia,Nannan Su,Jun Huang,Haiwei Song,Qiangzu Sun,Zixiu Cheng,Sakshibeedu R Bharath

doi:10.1038/s41421-021-00283-0

Abstract

SOSS1 is a single-stranded DNA (ssDNA)-binding protein complex that plays a critical role in double-strand DNA break (DSB) repair. SOSS1 consists of three subunits: INTS3, SOSSC, and hSSB1, with INTS3 serving as a scaffold to stabilize this complex. Moreover, the integrator complex subunit 6 (INTS6) participates in the DNA damage response through direct binding to INTS3, but how INTS3 interacts with INTS6, thereby impacting DSB repair, is not clear. Here, we determined the crystal structure of the C-terminus of INTS3 (INTS3c) in complex with the C-terminus of INTS6 (INTS6c) at a resolution of 2.4 Å. Structural analysis revealed that two INTS3c subunits dimerize and interact with INTS6c via conserved residues. Subsequent biochemical analyses confirmed that INTS3c forms a stable dimer and INTS3 dimerization is important for recognizing the longer ssDNA. Perturbation of INTS3c dimerization and disruption of the INTS3c/INTS6c interaction impair 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.

Highlights

DNA double-strand breaks (DSBs) arise when the integrity of genomic DNA is challenged by a variety of endogenous and exogenous DNA-damaging agents such as replication fork collapse, oxidative stress, and ionizing radiation (IR)[1]
We showed that INTS3 uses its C-terminal region to mediate its interaction with integrator complex subunit 6 (INTS6)
Consistent with the previous observations that the INTS3/INTS6 interaction is required for the recruitment of INTS6 to the DNA damage sites in response to IR21, our structure-based functional assays showed that the disruption of the INTS3c/INTS6 fragment consisting of residues 800−887 (INTS6c) interaction inhibits the DSB repair process

Summary

Introduction

DNA double-strand breaks (DSBs) arise when the integrity of genomic DNA is challenged by a variety of endogenous and exogenous DNA-damaging agents such as replication fork collapse, oxidative stress, and ionizing radiation (IR)[1]. DSBs are highly cytotoxic lesions that disrupt the continuity of a chromosome[2]. Defective DSB repair leads to genome instability and is associated with developmental disorders, premature aging, and tumorigenesis[3,4]. Two main pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ), have evolved to repair DSBs5–7. As a more accurate single-stranded DNA (ssDNA) overhang, which is essential for Rad51-mediated strand exchange[9,10,11]. Following the resection of DSBs, the replication protein A (RPA)

Methods

Results

Conclusion