Mechanisms of dCMP Transferase Reactions Catalyzed by Mouse Rev1 Protein

Masaharu Sumii,Yuji Masuda,Mamoru Takahashi,Saburo Fukuda,Kenji Kamiya

doi:10.1074/jbc.m110149200

Abstract

The Rev1 protein, a member of a large family of translesion DNA polymerases, catalyzes a dCMP transfer reaction. Recombinant mouse Rev1 protein was found to insert a dCMP residue opposite guanine, adenine, thymine, cytosine, uracil, and an apurinic/apyrimidinic site and to have weak ability for transfer to a mismatched terminus. The mismatch-extension ability was strongly enhanced by a guanine residue on the template near the mismatched terminus; this was not the case with an apurinic/apyrimidinic site and the other template nucleotides. Kinetic analysis of the dCMP transferase reaction provided evidence for high affinity for dCTP with template G but not the other templates, whereas the template nucleotide did not much affect the V(max) value. Furthermore, it could be established that the mouse Rev1 protein inserts dGMP and dTMP residues opposite template guanine at a V(max) similar to that for dCMP.

Highlights

In yeast Saccharomyces cerevisiae, the REV1 gene is required for damage-induced and spontaneous mutagenesis [1,2,3,4,5,6,7]
The enzyme is capable of incorporating a dCMP residue opposite G and A, U, and AP sites [12, 17], it is not clear how the Rev1 protein plays a role in mutagenesis
We found that the mouse Rev1 protein transfers a dCMP residue opposite template G and A, T, C, U, and AP sites and extends a mismatched terminus by the addition of a dCMP residue

Summary

EXPERIMENTAL PROCEDURES

Animals—Mice of the C57BL/6N and C3H/He strains were purchased from Charles River Laboratories Inc., Atsugi, Japan. Northern Blot Analysis of Mouse Rev Gene Expression in Various Mouse Tissues—A Northern blot membrane with 2 ␮g of poly(A)ϩ mRNAs from tissues of C57BL/6N ϫ C3H/He mice was hybridized with a 32P-labeled probe generated by PCR using primers 5Ј-TCCCAGATTGACCAGTCTGTT-3Ј and 5Ј-TCAGGTCACTTTCAGTGTGCT-3Ј in ExpressHybTM Hybridization Solution (CLONTECH) at 65 °C and washed with 0.1 ϫ SSC and 0.1% SDS at the same temperature. Two-ml aliquots of lysate were applied at 0.1 ml/min to a 1-ml HiTrap chelating column (Amersham Biosciences, Inc.), which had been flushed with 2 ml of 0.1 M NiSO4 and equilibrated with buffer A (50 mM HEPES-NaOH, pH 7.5, 1 M NaCl, 10% glycerol, 10 mM ␤-mercaptoethanol) containing 10 mM imidazole, 5 mM ATP, and 10 mM MgCl2. The recombinant human REV1 protein and its mutant, D569A/E570A, were purified by nickel-chelating column and gel filtration chromatography as described [18]. The amount of DNA present in each band was quantified using a Bio-Imaging Analyzer BAS2000 (Fuji Photo Film Co., Ltd.)

RESULTS

Specific activityb

DISCUSSION

AP dGTP dTTP