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

Anna V Yudkina,Alena V Makarova,Dmitry O Zharkov,Evgeniy S Shilkin

doi:10.3390/genes13020166

Abstract

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.

Highlights

translesion DNA synthesis (TLS) involves a set of specific DNA polymerases that have a wide and flexible active site with no substrate conformational selection and lack proofreading, which helps them to bypass the lesion at the expense of low synthesis accuracy
We have investigated whether DNA polymerases ι (POLι), η (POLη), and ζ (POLζ), which are involved in TLS in eukaryotic cells, are able to bypass a DNA-protein cross-links (DPCs) or reach the cross-link site when the DPC is located in the template strand of single-stranded or double-stranded DNA, or in the non-template displaced strand of a DNA duplex (Figure 1)
To investigate the ability of hPOLι to approach a DPC, we performed primer extension reactions on the model DPC-containing substrates in the presence of dNTPs and Mg2+ or Mn2+ ions (Figure 2a). hPOLι is a low-processivity DNA polymerase, typically incorporating 1–3 nucleotides per association [46]. It is more active on gapped DNA substrates incorporating 7–10 nucleotides and possesses limited strand displacement activity [47,48]

Summary

Introduction

DNA is always associated with a variety of proteins. this tight association increases the risk of haphazard covalent attachment of proteins to DNA with the formation of DNA-protein cross-links (DPCs). The polymerases show widely varying ability to elongate the primer in the presence of a DPC, with some stopping almost immediately after the collision with the surface of the cross-linked protein and others extending almost to the point of the cross-link, which apparently requires major deformation of the DPC’s protein part This could possibly facilitate DPC recognition by downstream repair proteases. TLS involves a set of specific DNA polymerases that have a wide and flexible active site with no substrate conformational selection and lack proofreading, which helps them to bypass the lesion at the expense of low synthesis accuracy. Most of these polymerases belong to the structural family Y, which, in human cells, encompasses DNA polymerases ι, κ, η, and Rev.

Primer Extension Reactions

Substrate Design

Inefficient Synthesis by POLι on DPC-Containing Substrates

POLζ Is Able to Reach the Cross-Link Site

POLη Able to Reach the Cross-Link Site and Beyond