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

Anna V. Yudkina, Antonina P. Dvornikova, Dmitry O. Zharkov

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)

Abstract

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.

Original languageEnglish
Article number0198480
Pages (from-to)e0198480
Number of pages17
JournalPLoS ONE
Volume13
Issue number6
DOIs
Publication statusPublished - 1 Jun 2018

Keywords

  • Bacteriophage T4/enzymology
  • Borohydrides/chemistry
  • DNA Adducts/chemistry
  • DNA Replication
  • DNA, Single-Stranded/biosynthesis
  • DNA-Directed DNA Polymerase/metabolism
  • DNA-Formamidopyrimidine Glycosylase/metabolism
  • DNA/biosynthesis
  • Escherichia coli Proteins/metabolism
  • Escherichia coli/enzymology
  • Guanine/analogs & derivatives
  • Humans
  • Oligonucleotides/chemistry
  • Pyrococcus furiosus/enzymology
  • Sulfolobus solfataricus/enzymology
  • Transcription Termination, Genetic
  • BASE-EXCISION-REPAIR
  • HUMAN-CELLS
  • ESCHERICHIA-COLI
  • REPLICATION BYPASS
  • LESION-BYPASS
  • MECHANISM
  • BETA
  • GENOMIC INSTABILITY
  • MAJOR GROOVE
  • STRAND-DISPLACEMENT

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