Strand Displacement Activity of PrimPol.

Elizaveta O Boldinova,Ekaterina A Belousova,Alena V Makarova,Ekaterina A Maltseva,Olga I Lavrik,Svetlana N Khodyreva,Diana I Gagarinskaya

doi:10.3390/ijms21239027

Abstract

Human PrimPol is a unique enzyme possessing DNA/RNA primase and DNA polymerase activities. In this work, we demonstrated that PrimPol efficiently fills a 5-nt gap and possesses the conditional strand displacement activity stimulated by Mn2+ ions and accessory replicative proteins RPA and PolDIP2. The DNA displacement activity of PrimPol was found to be more efficient than the RNA displacement activity and FEN1 processed the 5′-DNA flaps generated by PrimPol in vitro.

Highlights

Human PrimPol is an archaeo-eukaryotic primase with DNA polymerase activity found in nuclei and mitochondria [1,2]
It has been suggested that the main function of PrimPol is re-initiation of stalled replication forks using de novo DNA synthesis and re-priming downstream DNA lesions and non-B DNA structures such as G-quadruplexes [4,5,6,7]
We investigated how PrimPol responds to a gapped DNA structure and analyzed its ability to displace downstream DNA or RNA strands

Summary

Introduction

Human PrimPol is an archaeo-eukaryotic primase with DNA polymerase activity found in nuclei and mitochondria [1,2]. We demonstrated that PrimPol possesses the DNA strand displacement activity, which is stimulated by Mn2+ ions, replication protein A (RPA) and polymerase delta-interacting protein 2 (PolDIP2), and that the resulting flap can be subsequently removed by FEN1. PPrriimmPPooll eefffificciieennttllyy ffiilllleedd tthhee ggaapp ccoonnttaaiinniinngg uunnddaammaaggeedd GG oorr 88--ooxxoo--GG ((FFiigguurree 11AA)). OOnnllyy ttrraaccee DDNNAA ssttrraanndd ddiissppllaacceemmeenntt aaccttiivviittyy ooff PPrriimmPPooll wwaass oobbsseerrvveedd iinn tthhee pprreesseennccee ooff MMgg22++ iioonnss ((FFiigguurree 11AA,, llaanneess 55––88)). Endonuclease FEN1 excises 5 -flap structures in DNA during the long-patch BER [13,24,25] and stimulates the DNA strand displacement activity of Pol β and Pol λ [11,12,13]. The process of strand displacement synthesis is involved in many DNA transactions in vivo, such as d-loop invasion in homologous recombination and break-induced replication, non-homologous end-joining and microhomology-mediated end-joining, long-patch base excision repair, and Okazaki fragment maturation. The RNA displacement activity of PrimPol was not efficient, suggesting that PrimPol is blocked after encountering an Okazaki fragment

Proteins

DNA Substrates

Primer Extension Reactions