TY - JOUR

T1 - Variable termination sites of DNA polymerases encountering a DNA–protein cross-link

AU - Yudkina, Anna V.

AU - Dvornikova, Antonina P.

AU - Zharkov, Dmitry O.

N1 - Publisher Copyright: © 2018 Yudkina 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 - 2018/6/1

Y1 - 2018/6/1

N2 - DNA-protein cross-links (DPCs) are important DNA lesions induced by endogenous cross-linking agents such as formaldehyde or acetaldehyde, as well as ionizing radiation, cancer chemotherapeutic drugs, and abortive action of some enzymes. Due to their very bulky nature, they are expected to interfere with DNA and RNA synthesis and DNA repair. DPCs are highly genotoxic and the ability of cells to deal with them is relevant for many chemotherapeutic interventions. However, interactions of DNA polymerases with DPCs have been poorly studied due to the lack of a convenient experimental model. We have used NaBH4-induced trapping of E. coli formamidopyrimidine-DNA glycosylase with DNA to construct model DNA polymerase substrates containing a DPC in single-stranded template, or in the template strand of double-stranded DNA, or in the non-template (displaced) strand of double-stranded DNA. Nine DNA polymerases belonging to families A, B, X, and Y were studied with respect to their behavior upon encountering a DPC: Klenow fragment of E. coli DNA polymerase I, Thermus aquaticus DNA polymerase I, Pyrococcus furiosus DNA polymerase, Sulfolobus solfataricus DNA polymerase IV, human DNA polymerases β, κ and λ, and DNA polymerases from bacteriophages T4 and RB69. Although none were able to fully bypass DPCs in any context, Family B DNA polymerases (T4, RB69) and Family Y DNA polymerase IV were able to elongate the primer up to the site of the cross-link if a DPC was located in single-stranded template or in the displaced strand. In other cases, DNA synthesis stopped 4–5 nucleotides before the site of the cross-link in single-stranded template or in double-stranded DNA if the polymerases could displace the downstream strand. We suggest that termination of DNA polymerases on a DPC is mostly due to the unrelieved conformational strain experienced by the enzyme when pressing against the cross-linked protein molecule.

AB - DNA-protein cross-links (DPCs) are important DNA lesions induced by endogenous cross-linking agents such as formaldehyde or acetaldehyde, as well as ionizing radiation, cancer chemotherapeutic drugs, and abortive action of some enzymes. Due to their very bulky nature, they are expected to interfere with DNA and RNA synthesis and DNA repair. DPCs are highly genotoxic and the ability of cells to deal with them is relevant for many chemotherapeutic interventions. However, interactions of DNA polymerases with DPCs have been poorly studied due to the lack of a convenient experimental model. We have used NaBH4-induced trapping of E. coli formamidopyrimidine-DNA glycosylase with DNA to construct model DNA polymerase substrates containing a DPC in single-stranded template, or in the template strand of double-stranded DNA, or in the non-template (displaced) strand of double-stranded DNA. Nine DNA polymerases belonging to families A, B, X, and Y were studied with respect to their behavior upon encountering a DPC: Klenow fragment of E. coli DNA polymerase I, Thermus aquaticus DNA polymerase I, Pyrococcus furiosus DNA polymerase, Sulfolobus solfataricus DNA polymerase IV, human DNA polymerases β, κ and λ, and DNA polymerases from bacteriophages T4 and RB69. Although none were able to fully bypass DPCs in any context, Family B DNA polymerases (T4, RB69) and Family Y DNA polymerase IV were able to elongate the primer up to the site of the cross-link if a DPC was located in single-stranded template or in the displaced strand. In other cases, DNA synthesis stopped 4–5 nucleotides before the site of the cross-link in single-stranded template or in double-stranded DNA if the polymerases could displace the downstream strand. We suggest that termination of DNA polymerases on a DPC is mostly due to the unrelieved conformational strain experienced by the enzyme when pressing against the cross-linked protein molecule.

KW - Bacteriophage T4/enzymology

KW - Borohydrides/chemistry

KW - DNA Adducts/chemistry

KW - DNA Replication

KW - DNA, Single-Stranded/biosynthesis

KW - DNA-Directed DNA Polymerase/metabolism

KW - DNA-Formamidopyrimidine Glycosylase/metabolism

KW - DNA/biosynthesis

KW - Escherichia coli Proteins/metabolism

KW - Escherichia coli/enzymology

KW - Guanine/analogs & derivatives

KW - Humans

KW - Oligonucleotides/chemistry

KW - Pyrococcus furiosus/enzymology

KW - Sulfolobus solfataricus/enzymology

KW - Transcription Termination, Genetic

KW - BASE-EXCISION-REPAIR

KW - HUMAN-CELLS

KW - ESCHERICHIA-COLI

KW - REPLICATION BYPASS

KW - LESION-BYPASS

KW - MECHANISM

KW - BETA

KW - GENOMIC INSTABILITY

KW - MAJOR GROOVE

KW - STRAND-DISPLACEMENT

UR - http://www.scopus.com/inward/record.url?scp=85048039410&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0198480

DO - 10.1371/journal.pone.0198480

M3 - Article

C2 - 29856874

AN - SCOPUS:85048039410

VL - 13

SP - e0198480

JO - PLoS ONE

JF - PLoS ONE

SN - 1932-6203

IS - 6

M1 - 0198480

ER -