Translesion activity of PrimPol on DNA with cisplatin and DNA\u2013protein cross-links

Tahir H Tahirov,Alena V Makarova,Elizaveta O Boldinova,Dmitry O Zharkov,Evgeniy S Shilkin,Anna V Yudkina,Diana I Gagarinskaya,Andrey G Baranovskiy

doi:10.1038/s41598-021-96692-y

Abstract

Human PrimPol belongs to the archaeo-eukaryotic primase superfamily of primases and is involved in de novo DNA synthesis downstream of blocking DNA lesions and non-B DNA structures. PrimPol possesses both DNA/RNA primase and DNA polymerase activities, and also bypasses a number of DNA lesions in vitro. In this work, we have analyzed translesion synthesis activity of PrimPol in vitro on DNA with an 1,2-intrastrand cisplatin cross-link (1,2-GG CisPt CL) or a model DNA–protein cross-link (DpCL). PrimPol was capable of the 1,2-GG CisPt CL bypass in the presence of Mn2+ ions and preferentially incorporated two complementary dCMPs opposite the lesion. Nucleotide incorporation was stimulated by PolDIP2, and yeast Pol ζ efficiently extended from the nucleotides inserted opposite the 1,2-GG CisPt CL in vitro. DpCLs significantly blocked the DNA polymerase activity and strand displacement synthesis of PrimPol. However, PrimPol was able to reach the DpCL site in single strand template DNA in the presence of both Mg2+ and Mn2+ ions despite the presence of the bulky protein obstacle.

Highlights

Human PrimPol belongs to the archaeo-eukaryotic primase superfamily of primases and is involved in de novo DNA synthesis downstream of blocking DNA lesions and non-B DNA structures
PrimPol binding to DNA and its activity can be regulated by accessory proteins: replication protein A (RPA)[13,14,15] and DNA polymerase δ-interacting protein 2 (PolDIP2)[16]
Avian and human PrimPol-deficient cells demonstrate elevated sensitivity to cisplatin treatment that indicates the role of PrimPol in the tolerance of cisplatin-induced DNA d amage[3,4,9]

Summary

Introduction

Human PrimPol belongs to the archaeo-eukaryotic primase superfamily of primases and is involved in de novo DNA synthesis downstream of blocking DNA lesions and non-B DNA structures. PrimPol possesses both DNA/RNA primase and DNA polymerase activities, and bypasses a number of DNA lesions in vitro. We have analyzed translesion synthesis activity of PrimPol in vitro on DNA with an 1,2-intrastrand cisplatin cross-link (1,2-GG CisPt CL) or a model DNA–protein crosslink (DpCL). PrimPol is capable of bypassing some DNA lesions in vitro suggesting that PrimPol may perform DNA translesion synthesis (TLS). DNA cross-links (CL) represent an abundant class of DNA lesions in a genome They may be formed between bases of one DNA strand (intrastrand CL), bases of different DNA strands (interstrand CL) and between DNA and a protein molecule (DNA–protein CL, DpCL). Efficient repair, TLS and replication restart might contribute to tumor resistance to cisplatin (reviewed in[23])

Methods

Results

Conclusion