Crystal Structure of INTS3/INTS6 complex reveals Functional Importance of INTS3 dimerization in DSB Repair

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 DBS 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. Structure 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.

A biophysical and structural analysis of DNA binding by oligomeric hSSB1 (NABP2/OBFC2B)

“…Notably, our structural model differs from a model described in a recently deposited preprint ( Figure 6 in reference (45)) incorporating hSSB1, ssDNA, INT3, and INTS6. However, in contrast to our model and published data (18), this model (45) reveals direct contacts between the N-terminus of INTS3 and ssDNA. Further structural and biophysical studies will be required to shed light on the exact composition and structural details of the entire SOSS1 complex, comprising hSSB1 (SOSSB1), ssDNA, INTS3 (SOSSA), C9orf80 (SOSSC) and INTS6.…”

“…Interestingly, our structural data differs from a model described in a recently deposited preprint (Figure 6 in Jia et al 32 ) incorporating hSSB1, ssDNA, INT3, and INTS6. In contrast to our and published data 18 , this model 32 reveals direct contacts between the N-terminus of INTS3 and ssDNA.…”

Structure of the catalytic core of the Integrator complex

“…Although this approach successfully uncovered factors involved in complex formation, it could not determine its stoichiometry or inter-subunit contacts, and such architectural information remained missing. Other biochemical approaches revealed the association of INTS4/9/11 ( Albrecht et al., 2018 ) as well as INTS3/6 ( Jia et al., 2020 ; Skaar et al., 2009 ; Zhang et al., 2013 ). Building on these findings, we developed a method that allowed us to identify two previously unknown sub-complexes, INTS5/8 and INTS10/13/14.…”