TY - JOUR

T1 - Stalling of Eukaryotic Translesion DNA Polymerases at DNA-Protein Cross-Links

AU - Yudkina, Anna V.

AU - Shilkin, Evgeniy S.

AU - Makarova, Alena V.

AU - Zharkov, Dmitry O.

N1 - Funding: This research was funded by the Russian Science Foundation (grant No. 21-74-00061). A.V.Y. is a recipient of the Russian Presidential Fellowship (SP-174.2021.4). Partial salary support from the Russian Ministry of Science and Higher Education (State funded budget projects 121031300056-8 and FSUS-2020-0035). This study was also partially supported by the financing of research of the Institute of Molecular Genetics of the National Research Centre “Kurchatov Institute” (No. 121030200227-6) (A.V.M. and E.S.S.). Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

PY - 2022/2

Y1 - 2022/2

N2 - DNA-protein cross-links (DPCs) are extremely bulky adducts that interfere with replication. In human cells, they are processed by SPRTN, a protease activated by DNA polymerases stuck at DPCs. We have recently proposed the mechanism of the interaction of DNA polymerases with DPCs, involving a clash of protein surfaces followed by the distortion of the cross-linked protein. Here, we used a model DPC, located in the single-stranded template, the template strand of double-stranded DNA, or the displaced strand, to study the eukaryotic translesion DNA polymerases ζ (POLζ), ι (POLι) and η (POLη). POLι demonstrated poor synthesis on the DPC-containing substrates. POLζ and POLη paused at sites dictated by the footprints of the polymerase and the cross-linked protein. Beyond that, POLζ was able to elongate the primer to the cross-link site when a DPC was in the template. Surprisingly, POLη was not only able to reach the cross-link site but also incorporated 1-2 nucleotides past it, which makes POLη the most efficient DNA polymerase on DPC-containing substrates. However, a DPC in the displaced strand was an insurmountable obstacle for all polymerases, which stalled several nucleotides before the cross-link site. Overall, the behavior of translesion polymerases agrees with the model of protein clash and distortion described above.

AB - DNA-protein cross-links (DPCs) are extremely bulky adducts that interfere with replication. In human cells, they are processed by SPRTN, a protease activated by DNA polymerases stuck at DPCs. We have recently proposed the mechanism of the interaction of DNA polymerases with DPCs, involving a clash of protein surfaces followed by the distortion of the cross-linked protein. Here, we used a model DPC, located in the single-stranded template, the template strand of double-stranded DNA, or the displaced strand, to study the eukaryotic translesion DNA polymerases ζ (POLζ), ι (POLι) and η (POLη). POLι demonstrated poor synthesis on the DPC-containing substrates. POLζ and POLη paused at sites dictated by the footprints of the polymerase and the cross-linked protein. Beyond that, POLζ was able to elongate the primer to the cross-link site when a DPC was in the template. Surprisingly, POLη was not only able to reach the cross-link site but also incorporated 1-2 nucleotides past it, which makes POLη the most efficient DNA polymerase on DPC-containing substrates. However, a DPC in the displaced strand was an insurmountable obstacle for all polymerases, which stalled several nucleotides before the cross-link site. Overall, the behavior of translesion polymerases agrees with the model of protein clash and distortion described above.

KW - DNA polymerases

KW - DNA replication

KW - DNA–protein cross-link

KW - translesion synthesis

KW - DNA Replication

KW - DNA-Directed DNA Polymerase/genetics

KW - Nucleotides

KW - Eukaryota/genetics

KW - Proteins/genetics

KW - DNA/genetics

KW - Translesion synthesis

UR - http://www.scopus.com/inward/record.url?scp=85125349182&partnerID=8YFLogxK

UR - https://www.mendeley.com/catalogue/7edae555-3fca-3e6d-82bb-dff31ef1768a/

U2 - 10.3390/genes13020166

DO - 10.3390/genes13020166

M3 - Article

C2 - 35205211

AN - SCOPUS:85125349182

VL - 13

JO - Genes

JF - Genes

SN - 2073-4425

IS - 2

M1 - 166

ER -