PrimPol‐mediated repriming facilitates replication traverse of DNA interstrand crosslinks

Abstract

DNA interstrand crosslinks (ICLs) induced by endogenous aldehydes or chemotherapeutic agents interfere with essential processes such as replication and transcription. ICL recognition and repair by the Fanconi Anemia pathway require the formation of an X-shaped DNA structure that may arise from convergence of two replication forks at the crosslink or traversing of the lesion by a single replication fork. Here, we report that ICL traverse strictly requires DNA repriming events downstream of the lesion, which are carried out by PrimPol, the second primase-polymerase identified in mammalian cells after Pola/Primase. The recruitment of PrimPol to the vicinity of ICLs depends on its interaction with RPA, but not on FANCM translocase or the BLM/TOP3A/RMI1-2 (BTR) complex that also participate in ICL traverse. Genetic ablation of PRIMPOL makes cells more dependent on the fork convergence mechanism to initiate ICL repair, and PRIMPOL KO cells and mice display hypersensitivity to ICL-inducing drugs. These results open the possibility of targeting PrimPol activity to enhance the efficacy of chemotherapy based on DNA crosslinking agents.

Replication of the Mammalian Genome by Replisomes Specific for Euchromatin and Heterochromatin

“…While replication restart past monoadduct blocks has been known for many years ( Rupp and Howard-Flanders, 1968 ; Heller and Marians, 2006 ; Lehmann and Fuchs, 2006 ; Taylor and Yeeles, 2018 ; Guilliam and Yeeles, 2020 ), our observations were contrary to over 50 years of conventional wisdom ( Marmur and Grossman, 1961 ). However, ICL traverse has been confirmed by recent work from other laboratories ( Mutreja et al, 2018 ; González-Acosta et al, 2021 ).…”

“…Restart of replication would require priming downstream of the ICL. Recently the Mendez lab described the requirement of the PrimPol primase for about 50% of traverse events ( González-Acosta et al, 2021 ). While these results identify PrimPol as important for traverse they also argue that there are other factors that support repriming downstream of an ICL.…”

Arabidopsis thaliana PrimPol is a primase and lesion bypass DNA polymerase with the biochemical characteristics to cope with DNA damage in the nucleus, mitochondria, and chloroplast

“…PrimPol is the most recent DNA polymerase identified in metazoans. Its evolutionary relationships with the AEP superfamily of primases agrees with its unique role in repriming stalled replication forks by synthesizing “de novo” DNA primers 21 , 21 , 28 – 32 , 59 . According to the postulate “ PrimPol is an ancient eukaryotic gene ” 21 , herein we show that his enzyme was present in eukaryotes before the divergence between plants and animals.…”

“…Human PrimPol (HsPrimPol) in complex with a template/primer DNA and an incoming deoxynucleotide illustrates that the AEP domain of PrimPol makes limited contacts with the primer-strand allowing the active site to bind two nucleotides at the same time 26 and biochemical studies demonstrate that the Zn-finger domain interacts with the initial nucleotide and the cryptic G. HsPrimPol is localized in the nucleus and mitochondria 21 , 24 . Although HsPrimPol is a non-essential protein, this enzyme is involved in repriming, even in the absence of damaged DNA, at lesions that block nuclear DNA or structures that halt the replication fork 22 , 29 – 32 .…”

Human PrimPol Discrimination against Dideoxynucleotides during Primer Synthesis

“…In the present work, we questioned how the PrimPol present in mitochondria [7] assists Polγ to cope with this blocking agent. In 2017, Torregrosa-Muñumer et al [11] demonstrated that PrimPol can reinitiate ddC-and UV-stalled mtDNA replication by priming mtDNA replication from non-conventional origins, mirroring the repriming function of PrimPol in the nucleus [10,12,27]. Whereas PrimPol was required during mtDNA replication under stress conditions, no apparent differences were detected in mitochondrial replication intermediates for both unstressed WT or PrimPol −/− MEF cells.…”