Two Interaction Surfaces Between Xpa and Rpa Organize the Preincision Complex in Nucleotide Excision Repair

Authors

Mihyun Kim
Hyun-Suk Kim
Areetha D'Souza
Kaitlyn Gallagher
Eunwoo Jeong
Agnieszka Topolska-Wós
Kateryna Ogorodnik Le Meur
Chi-Lin Tsai
Miaw-Sheue Tsai
Minyong Kee
John A Tainer
Jung-Eun Yeo
Walter J Chazin
Orlando D Schärer

Publication Date

8-23-2022

Journal

Proceedings of the National Academy of Sciences of the United States of America

Abstract

The xeroderma pigmentosum protein A (XPA) and replication protein A (RPA) proteins fulfill essential roles in the assembly of the preincision complex in the nucleotide excision repair (NER) pathway. We have previously characterized the two interaction sites, one between the XPA N-terminal (XPA-N) disordered domain and the RPA32 C-terminal domain (RPA32C), and the other with the XPA DNA binding domain (DBD) and the RPA70AB DBDs. Here, we show that XPA mutations that inhibit the physical interaction in either site reduce NER activity in biochemical and cellular systems. Combining mutations in the two sites leads to an additive inhibition of NER, implying that they fulfill distinct roles. Our data suggest a model in which the interaction between XPA-N and RPA32C is important for the initial association of XPA with NER complexes, while the interaction between XPA DBD and RPA70AB is needed for structural organization of the complex to license the dual incision reaction. Integrative structural models of complexes of XPA and RPA bound to single-stranded/double-stranded DNA (ss/dsDNA) junction substrates that mimic the NER bubble reveal key features of the architecture of XPA and RPA in the preincision complex. Most critical among these is that the shape of the NER bubble is far from colinear as depicted in current models, but rather the two strands of unwound DNA must assume a U-shape with the two ss/dsDNA junctions localized in close proximity. Our data suggest that the interaction between XPA and RPA70 is key for the organization of the NER preincision complex.

Keywords

DNA, DNA Damage, DNA Repair, Protein Binding, Protein Domains, Replication Protein A, Xeroderma Pigmentosum Group A Protein, DNA repair, nucleotide excision repair, replication protein A, xeroderma pigmentosum protein A

DOI

10.1073/pnas.2207408119

PMID

35969784

PMCID

PMC9407234

PubMedCentral® Posted Date

8-15-2022

PubMedCentral® Full Text Version

Post-print

Published Open-Access

yes