Abstract
Human PrimPol is a primase belonging to the AEP superfamily with the unique ability to synthesize DNA primers de novo, and a non-processive DNA polymerase able to bypass certain DNA lesions. PrimPol facilitates both mitochondrial and nuclear replication fork progression either acting as a conventional TLS polymerase, or repriming downstream of blocking lesions. In vivo assays have shown that PrimPol is rapidly recruited to sites of DNA damage by interaction with the human replication protein A (RPA). In agreement with previous findings, we show here that the higher affinity of RPA for ssDNA inhibits PrimPol activities in short ssDNA templates. In contrast, once the amount of ssDNA increases up to a length in which both proteins can simultaneously bind ssDNA, as expected during replicative stress conditions, PrimPol and RPA functionally interact, and their binding capacities are mutually enhanced. When using M13 ssDNA as template, RPA stimulated both the primase and polymerase activities of PrimPol, either alone or in synergy with Polε. These new findings supports the existence of a functional PrimPol/RPA association that allows repriming at the exposed ssDNA regions formed in the leading strand upon replicase stalling.
Highlights
DNA polymerases are the enzymes responsible for making and repairing the DNA to ensure cell viability
Co-immunoprecipitation assays have demonstrated that the most carboxy-terminal region of human PrimPol mediates the interaction with the large subunit of replication protein A (RPA), the human single-stranded DNA binding protein[6]
When there is a lesion in the DNA template or any other condition impeding elongation of the leading strand, the polymerase responsible could be blocked, becoming uncoupled to the unwinding helicase, promoting an accumulation of a long stretch of uncopied ssDNA template that could not be readily covered by RPA
Summary
Human PrimPol is a primase belonging to the AEP superfamily with the unique ability to synthesize DNA primers de novo, and a non-processive DNA polymerase able to bypass certain DNA lesions. In vivo assays have shown that PrimPol is rapidly recruited to sites of DNA damage by interaction with the human replication protein A (RPA). Compelling evidence demonstrates that the DNA primase activity of human PrimPol facilitates DNA replication by repriming DNA synthesis beyond lesions, bulky structures or any circumstance that block elongation, leaving behind an unreplicated gap to be repaired post-replicatively[9,10,11]. Those DNA primers synthesized by human PrimPol can be efficiently elongated by the replicative polymerases Polγ and Polε[7]. Such a paradox is explored here in this paper, by studying the functional interaction of these two proteins, and how the length of the template defines either a stimulatory or inhibitory effect of RPA on PrimPol catalytic activities
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