Human DNA polymerase η has reverse transcriptase activity in cellular environments

Abstract

Edited by Patrick SungClassical DNA and RNA polymerase (pol) enzymes have defined roles with their respective substrates, but several pols have been found to have multiple functions. We reported previously that purified human DNA pol (hpol ) can incorporate both deoxyribonucleoside triphosphates (dNTPs) and ribonucleoside triphosphates (rNTPs) and can use both DNA and RNA as substrates. X-ray crystal structures revealed that two pol residues, Phe-18 and Tyr-92, behave as steric gates to influence sugar selectivity. However, the physiological relevance of these phenomena has not been established. Here, we show that purified hpol adds rNTPs to DNA primers at physiological rNTP concentrations and in the presence of competing dNTPs. When two rATPs were inserted opposite a cyclobutane pyrimidine dimer, the substrate was less efficiently cleaved by human RNase H2. Human XP-V fibroblast extracts, devoid of hpol , could not add rNTPs to a DNA primer, but the expression of transfected hpol in the cells restored this ability. XP-V cell extracts did not add dNTPs to DNA primers hybridized to RNA, but could when hpol was expressed in the cells. HEK293T cell extracts could add dNTPs to DNA primers hybridized to RNA, but lost this ability if hpol was deleted. Interestingly, a similar phenomenon was not observed when other translesion synthesis (TLS) DNA polymerases-hpol , , or -were individually deleted. These results suggest that hpol is one of the major reverse transcriptases involved in physiological processes in human cells.

Impact of 1,N6-ethenoadenosine, a damaged ribonucleotide in DNA, on translesion synthesis and repair

“…Our laboratory has previously shown that hpol η can act as a reverse transcriptase in human cell extracts (38), and here we examined this novel function of hpol η using modified RNA as a template, employing physiological concentrations of dNTPs and rNTPs ( Fig. 3) under in vitro conditions.…”

“…The TLS process has garnered considerable attention due to the tolerance of damage in DNA in an errorfree or error-prone manner (39,40). TLS also plays an important role in tolerance of ribonucleotides in DNA, and the main key player is hpol η (especially in dealing with lagging strands derived from Okazaki fragments) (16,24,38,(41)(42)(43)(44)(45)(46). Studies with 8-oxo-rG in DNA indicate that hpol η is required for the tolerance of this adduct (24).…”

“…Studies with 8-oxo-rG in DNA indicate that hpol η is required for the tolerance of this adduct (24). The versatile hpol η is now also known for its reverse transcriptase and transcription-associated roles (38,(47)(48)(49)(50). Damage in RNA has not been considered to disturb the cellular processes, possibly due to general lack of replication of RNA and its innately lower stability as well as the more rapid turnover (51)(52)(53)(54).…”

“…We now report TLS, RT, and RNA primer extension events and incision of the most abundant nucleotide, rATP, and its naturally-occurring damaged analog 1,N 6 -erA. We utilized hpol η for the TLS, RT, and RNA primer extension studies due to its recently discovered novel functions (37, 38,44,47,49,74,75) and RNase H2 for the incision studies. Our laboratory has recently shown that hpol η can act as a reverse transcriptase in human cells (38) and also that it utilizes RNA as a primer in in vitro replication assays, apart from its role in TLS (74).…”

“…We utilized hpol η for the TLS, RT, and RNA primer extension studies due to its recently discovered novel functions (37, 38,44,47,49,74,75) and RNase H2 for the incision studies. Our laboratory has recently shown that hpol η can act as a reverse transcriptase in human cells (38) and also that it utilizes RNA as a primer in in vitro replication assays, apart from its role in TLS (74). We examined the roles of hpol η as a TLS polymerase, reverse transcriptase, and RNA primer extension events in the presence of a damaged ribonucleotide in DNA as well as in RNA under physiological conditions (Fig.…”