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

Date Published: June 1, 2018

Publisher: Public Library of Science

Author(s): Anna V. Yudkina, Antonina P. Dvornikova, Dmitry O. Zharkov, Giovanni Maga.

http://doi.org/10.1371/journal.pone.0198480

Abstract

DNA-protein cross-links (DPCs) are important DNA lesions induced by endogenous crosslinking 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.

Partial Text

DNA of all living organisms is perpetually exposed to various exogenous and endogenous genotoxic agents, and is subject to damage [1]. DNA lesions can induce mutations, which lead to cancer and contribute to aging. Since cellular DNA is tightly bound to a variety of structural, regulatory and catalytic proteins, DNA-protein cross-links (DPCs) are among the common types of DNA damage. They can be generated by genotoxic agents such as aldehydes, ionizing and UV radiation, oxidative stress, and some chemotherapy drugs, or through covalent capture of some DNA-dependent enzymes (methyltransferases, topoisomerases) in abortive reaction events [2–4]. Different analytical methods produce estimates of 0.5–70 DPCs per 107 DNA bases as a background level in human cells [5–7].

Relative to a typical size of DNA lesions, DNA–protein cross-links are very bulky adducts. As a DPC model, we have used Fpg protein trapped on DNA by NaBH4 treatment; according to its crystal structure, Fpg fully or partially covers ~7 nt on the damaged DNA strand, and ~9 nt on the complementary strand [20] (S1 Fig). In our substrates, 8-oxoguanine, the lesion that Fpg excises and gets trapped to this position, was 13 nucleotides from the 3′-terminus of the primer. In turn, DNA polymerases, even though their size varies, are also large molecules (38–114 kDa for polymerases used in our study; cf. 30 kDa for Fpg). Thus, from the steric considerations, DNA polymerase should run into the protein part of the DPC soon after the start of synthesis. The available X-ray structures [47,48] allow us to estimate the distance at the start between the polymerase and Fpg for Pol β and RB69pol. When Fpg is cross-linked to the displaced strand, the surfaces of Pol β and RB69pol are, respectively, 6 nt and 5 nt away from the surface of Fpg. Thus, the termination of Pol β 4–5 nt before the site of the cross-link, correspond to an overlap of 2–3 nt between its footprint and the footprint of Fpg. When Fpg is cross-linked to the template strand, the surfaces of Pol β and Fpg are separated by 4 nt, due to the asymmetry of the Fpg footprint, and the overlap at the termination site is 0–1 nt. Even more strikingly, stalled RB69pol overlaps over at least 4 nt with Fpg in the template strand and nearly with the full footprint of Fpg cross-linked to the displaced strand.

 

Source:

http://doi.org/10.1371/journal.pone.0198480

 

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