Translesion DNA Synthesis Catalyzed by Human Pol η and Pol κ across 1,N 6-Ethenodeoxyadenosine

Abstract

1,N6 -Ethenodeoxyadenosine, a DNA adduct generated by exogenous and endogenous sources, severely blocks DNA synthesis and induces miscoding events in human cells. To probe the mechanism for in vivo translesion DNA synthesis across this adduct, in vitro primer extension studies were conducted using newly identified human DNA polymerases (pol) and , which have been shown to catalyze translesion DNA synthesis past several DNA lesions. Steady-state kinetic analyses and analysis of translesion products have revealed that the synthesis is >100-fold more efficient with pol than with pol and that both error-free and error-prone syntheses are observed with these enzymes. The miscoding events include both base substitution and frameshift mutations. These results suggest that both polymerases, particularly pol , may contribute to the translesion DNA synthesis events observed for 1,N 6 -ethenodeoxyadenosine in human cells.In the last few years, several new human DNA polymerases (pols), 1 which are likely to be involved in translesion DNA synthesis (TLS), were discovered. This list includes pol (1), pol (2, 3), pol (4), and pol (5). Pol , pol , and pol are encoded by the hRAD30A, hRAD30B, and DINB1 genes, respectively. These new pols form a Rad30/UmuC/DinB/REV1 superfamily (2, 3, 6). Pol consists of two gene products: hREV3 containing pol activity and hREV7 with an unknown function (5). In general, these pols catalyze TLS more efficiently than previously known pols. They synthesize DNA in a distributive manner and tend to show lower replication fidelity than other pols such as pol ␣, pol ␤, and pol ␦ when unmodified DNA is used as a template (7-10).There are several pieces of evidence for the involvement of human pol and pol in TLS in vivo (5,(11)(12)(13). Although the involvement of human pol or pol has not yet been established in vivo, the Escherichia coli homologue of pol , pol IV, has been shown to play a role in TLS in vivo (14). Pol , which is missing in xeroderma pigmentosum variant cells (13,15), is shown to catalyze efficient TLS across the cis-syn cyclobutane thymine-thymine dimer by inserting two dAMPs opposite the lesion (16). Therefore, the cancer proneness of xeroderma pigmentosum variant patients is thought to be caused by the lack of accurate TLS across this and/or other UV photo products. Pol also catalyzes TLS across other DNA lesions such as cisplatin G-G intrastrand cross-link (16), acetylaminofluorene-dG (16), and 8-oxodeoxyguanosine (17) Based on these findings, we are motivated to study the efficiency and fidelity of TLS catalyzed by these novel pols across 1,N 6 -ethenodeoxyadenosine (⑀dA) to probe the in vivo TLS mechanism. We have shown that ⑀dA is miscoding in simian and human cells by inducing ⑀dA3 T, ⑀dA3 G, and ⑀dA3 C (21, 22). This adduct is produced in animals exposed to vinyl compounds such as the human carcinogen vinyl chloride. Surprisingly, this adduct is also found in unexposed animals and humans with lipid peroxidation products being the suspected source of this adduct (23). Our in vitr...

Miscoding properties of 1,N6-ethanoadenine, a DNA adduct derived from reaction with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea

“…In search for enzymes which could be responsible for such misinsertion and lesion bypass, various translestional DNA polymerases were tested. Previous in vitro primer extension studies showed that both pol η and pol κ catalyze error-free and error-prone synthesis past εA with pol η being much more efficient than pol κ in such bypass [33]. Our in vitro results reported here also 13 support a possible role of pol η in cellular translesion synthesis across εA as based on the relatively efficient bypass and ~ 50% of A misinsertion opposite εA, which would lead to an εA→T transversion.…”

“…The position of the top bands in Figure 4A (center and right) also suggests the co-existence of full-length 9 and one base deletion, the latter of which remains to be determined. Such a deletion was frequently observed with pol η in replicating an εA-template as previously described [33].…”

“…Studies using in vitro replication systems have revealed that the three related Y family enzymes, pols η (Rad30A), ι (Rad30B) and κ (DinB1), are capable of bypassing various DNA lesions [e.g. [28][29][30][31][32][33][34][35][36] that are normally blockers of classical DNA polymerases such as pol α and pol β. However, such translesion syntheses can be error-free or error-prone.…”

RAD18 polymorphisms are associated with platinum-based chemotherapy toxicity in Chinese patients with non-small cell lung cancer

“…Platinum compounds react with DNA and lead to DNA lesions, including intrastrand crosslinks (Pt-d[CpG], Pt-d [ApG] and Pt-d [GpNgG]), interstrand crosslinks and single nucleotide damage involving guanine [43] . Hence, TLS that allows bypass of the intrastrand crosslinks and constitutes a critical initial step in interstrand crosslink repair [44] is advantageous to tumor cells' survival [45][46][47][48] . These crosslink bypasses can result in resistance against platinum-based chemotherapy [47,49] .…”

“…Hence, TLS that allows bypass of the intrastrand crosslinks and constitutes a critical initial step in interstrand crosslink repair [44] is advantageous to tumor cells' survival [45][46][47][48] . These crosslink bypasses can result in resistance against platinum-based chemotherapy [47,49] . TLS plays a similar role in normal cells in the face of damaging lesions and influences the side effects of platinum-based compounds.…”