Abstract
The intolerance of DNA polymerase δ (Polδ) to incorrect base pairing contributes to its extremely high accuracy during replication, but is believed to inhibit translesion synthesis (TLS). However, chicken DT40 cells lacking the POLD3 subunit of Polδ are deficient in TLS. Previous genetic and biochemical analysis showed that POLD3 may promote lesion bypass by Polδ itself independently of the translesion polymerase Polζ of which POLD3 is also a subunit. To test this hypothesis, we have inactivated Polδ proofreading in pold3 cells. This significantly restored TLS in pold3 mutants, enhancing dA incorporation opposite abasic sites. Purified proofreading-deficient human Polδ holoenzyme performs TLS of abasic sites in vitro much more efficiently than the wild type enzyme, with over 90% of TLS events resulting in dA incorporation. Furthermore, proofreading deficiency enhances the capability of Polδ to continue DNA synthesis over UV lesions both in vivo and in vitro. These data support Polδ contributing to TLS in vivo and suggest that the mutagenesis resulting from loss of Polδ proofreading activity may in part be explained by enhanced lesion bypass.
Highlights
Pol␦ synthesizes DNA with remarkably high fidelity making only a single error per 106 nucleotides synthesised in vivo [1]
Expression of proofreading deficient Pol␦ partially normalized the slow proliferation of pold3 cells, but not that of polζ cells
We have previously shown that cells lacking both POLD3 and two major translesion synthesis (TLS) polymerases, Pol and Pol, are inviable [19]
Summary
Pol␦ synthesizes DNA with remarkably high fidelity making only a single error per 106 nucleotides synthesised in vivo [1]. Pol , enzymes that have less spatially constrained active sites and that can accommodate the distorted base pairing created by damaged bases [6] While these characteristics allow TLS polymerases to bypass lesions, when coupled with the enzymes’ lack of proofreading activity, their deployment results in a reduction in fidelity of several orders of magnitude compared with Pol␦ and Pol⑀ (reviewed in [1]). Expression of proofreading-deficient POLD1 substantially changes the spectrum of mutagenesis arising from TLS past UV damage and abasic sites in POLD3+ cells These observations provide direct evidence that Pol␦ makes a substantial contribution to TLS in vivo and suggests that at least some of the mutagenesis in the absence of the proofreading activity of Pol␦, as observed for instance in a subset of cancers, is the result of more proficient lesion bypass by the enzyme
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
