TY - JOUR

T1 - RPA and XPA interaction with DNA structures mimicking intermediates of the late stages in nucleotide excision repair

AU - Krasikova, Yuliya S.

AU - Rechkunova, Nadejda I.

AU - Maltseva, Ekaterina A.

AU - Lavrik, Olga I.

N1 - Publisher Copyright: © 2018 Krasikova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

PY - 2018/1/10

Y1 - 2018/1/10

N2 - Replication protein A (RPA) and the xeroderma pigmentosum group A (XPA) protein are indispensable for both pathways of nucleotide excision repair (NER). Here we analyze the interaction of RPA and XPA with DNA containing a flap and different size gaps that imitate intermediates of the late NER stages. Using gel mobility shift assays, we found that RPA affinity for DNA decreased when DNA contained both extended gap and similar sized flap in comparison with gapped-DNA structure. Moreover, crosslinking experiments with the flap-gap DNA revealed that RPA interacts mainly with the ssDNA platform within the long gap and contacts flap in DNA with a short gap. XPA exhibits higher affinity for bubble-DNA structures than to flap-gap-containing DNA. Protein titration analysis showed that formation of the RPA-XPA-DNA ternary complex depends on the protein concentration ratio and these proteins can function as independent players or in tandem. Using fluorescently-labelled RPA, direct interaction of this protein with XPA was detected and characterized quantitatively. The data obtained allow us to suggest that XPA can be involved in the post-incision NER stages via its interaction with RPA.

AB - Replication protein A (RPA) and the xeroderma pigmentosum group A (XPA) protein are indispensable for both pathways of nucleotide excision repair (NER). Here we analyze the interaction of RPA and XPA with DNA containing a flap and different size gaps that imitate intermediates of the late NER stages. Using gel mobility shift assays, we found that RPA affinity for DNA decreased when DNA contained both extended gap and similar sized flap in comparison with gapped-DNA structure. Moreover, crosslinking experiments with the flap-gap DNA revealed that RPA interacts mainly with the ssDNA platform within the long gap and contacts flap in DNA with a short gap. XPA exhibits higher affinity for bubble-DNA structures than to flap-gap-containing DNA. Protein titration analysis showed that formation of the RPA-XPA-DNA ternary complex depends on the protein concentration ratio and these proteins can function as independent players or in tandem. Using fluorescently-labelled RPA, direct interaction of this protein with XPA was detected and characterized quantitatively. The data obtained allow us to suggest that XPA can be involved in the post-incision NER stages via its interaction with RPA.

KW - DNA Repair

KW - DNA/chemistry

KW - Electrophoretic Mobility Shift Assay

KW - Humans

KW - Photoaffinity Labels

KW - Protein Binding

KW - Recombinant Proteins/metabolism

KW - Replication Protein A/metabolism

KW - Xeroderma Pigmentosum Group A Protein/metabolism

KW - DAMAGED DNA

KW - COMPLEX

KW - RECOGNITION

KW - REPLICATION PROTEIN-A

KW - SINGLE-STRANDED-DNA

KW - BINDING PROTEIN

KW - OLIGONUCLEOTIDES

KW - DUAL INCISION

KW - TFIIH

KW - MOLECULAR-MECHANISMS

UR - http://www.scopus.com/inward/record.url?scp=85040321000&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0190782

DO - 10.1371/journal.pone.0190782

M3 - Article

C2 - 29320546

AN - SCOPUS:85040321000

VL - 13

SP - e0190782

JO - PLoS ONE

JF - PLoS ONE

SN - 1932-6203

IS - 1

M1 - 0190782

ER -