Genetic interactions of repriming and translesion synthesis
PRIMPOL is the only known eukaryotic primase outside of the core replisome. It has been previously shown to restart DNA synthesis past replication impediments such as DNA damage or non-B-DNA secondary structures. To probe the genetic interactions of PRIMPOL, genome-wide CRISPR/Cas9 knockout screens were undertaken in the human TK6 cell line. The most interesting group of hits were RAD18, UBE2A and REV1 - three genes with key roles in DNA damage tolerance through translesion synthesis which appeared as hits depleted in PRIMPOL-/- cells in screens undertaken with cisplatin treatment. To further probe this interaction, REV1-/- PRIMPOL-/- cells were generated - consistent with the results of the screen, these cells showed increased sensitivity to DNA damaging agents. Following cisplatin treatment, the double knockouts also showed greater DNA damage checkpoint activation consistent with increased replication stress, increased cell cycle perturbations (particularly in p53-deficient backgrounds) and greater genetic instability as assessed by micronucleus formation. The roles of REV1 and PRIMPOL were also probed in the context of replication of non-B-DNA secondary structures. REV1 appeared to be of greater importance in the replication of G-quadruplex structures than PRIMPOL when assessed through inhibition of growth upon treatment with the G-quadruplex-stabilising ligand PhenDC3, a result in contrast with previously observed data regarding the replication of G-quadruplex structures in the avian DT40 cell line. The growth inhibition of REV1-/- cells upon PhenDC3 treatment occurred in the apparent absence of DNA damage signalling pathway activation and was relieved upon knockout of p53, highlighting key differences in the response of REV1-deficient cells to replication impediments formed by DNA damage compared to non-B-DNA secondary structures. Overall, this work has furthered our knowledge of the effect of combined or individual deficiencies in repriming and translesion synthesis in response to diverse replication impediments.
PRIMPOL, REV1, DNA damage tolerance, Translesion synthesis, DNA damage, Non-B-DNA secondary structures, G-quadruplex
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