AtREV1, a Y-Family DNA Polymerase in Arabidopsis, Has Deoxynucleotidyl Transferase Activity in Vitro

Abstract

To clarify the functions of the Arabidopsis thaliana REV1 (AtREV1) protein, we expressed it in Escherichia coli and purified it to near homogeneity. The deoxynucleotidyl transferase activity of the recombinant AtREV1 was examined in vitro using a primer extension assay. The recombinant AtREV1 transferred one or two nucleotides to the primer end. It efficiently inserted dCMP regardless of the opposite base. AtREV1 also inserted a dCMP opposite an apurinic/apyrimidinic site, which is physiologically generated or induced by various DNA-damaging agents. In contrast, AtREV1 had no insertion activities against UV-inducible DNA lesions as reported in yeast or mammalian system. Although the substrate specificity of AtREV1 was rather narrow in the presence of magnesium ion, it widened in the presence of manganese ion. These results suggest that AtREV1 serves as a deoxycytidyl transferase in plant cells.

Role of AtPolζ, AtRev1, and AtPolη in UV Light-Induced Mutagenesis in Arabidopsis

“…Reduction of the spontaneous mutation frequency in rev3-1 and rev1-1 indicates that AtPolz and AtRev1 are involved in spontaneous mutagenesis, as reported for yeast (Kalinowski et al, 1995;Harfe and Jinks-Robertson, 2000). In our previous report, we suggested that AtPolz and AtRev1 are involved in the bypass of apurinic/apyrimidinic sites that are generated under normal physiological conditions (Takahashi et al, 2007). Additionally, our preliminary results suggested the involvement of AtPolz and AtRev1 in g-rayinduced mutations (M. Nakagawa, S. Takahashi, A. Tanaka, I. Narumi, and A.N.…”

“…4A). It is conceivable that AtRev1 plays a role in recruiting other TLS-type polymerase(s) such as AtPolz, AtPolk, and AtPolh, thereby allowing these enzymes to perform the bypass of damaged DNA, as suggested in our previous report (Takahashi et al, 2007).…”

“…Recombinant AtRev1 showed no insertion activity against templates with this type of DNA damage (Takahashi et al, 2007). However, in vivo replication assays in other organisms revealed that although Polz and Rev1 are required for the bypass of 6-4TPs (Otsuka et al, 2005), they are dispensable for the bypass of CTDs (Nelson et al, 2000;Gibbs et al, 2005).…”

“…In a previous report, we showed the possibility that AtPolz and AtRev1 might cooperate to bypass certain types of DNA damage (Takahashi et al, 2007). When the rev3-1 and rev1-1 mutants were grown under longterm UV-B light exposure, their fresh weights were similarly inhibited (Takahashi et al, 2005).…”

“…AtREV3 and AtREV7 encode a catalytic subunit and a regulatory subunit of AtPolz, respectively. AtRev1, encoded by the AtREV1 gene, possesses deoxynucleotidyl transferase activity with low fidelity and inserts a nucleotide opposite apurinic/apyrimidinic sites in vitro (Takahashi et al, 2007). AtPolh, encoded by AtPOLH, bypasses cyclobutane pyrimidine dimers with comparable activity to human or yeast Polh (Anderson et al, 2008;Hoffman et al, 2008).…”