Introduction

Formamidopyrimidine-DNA glycosylase (FPG) is one of several enzymes that can remove damaged or inappropriate bases from deoxyribose in DNA, starting a process of base excision repair. FPG is present in bacteria, from which it has been purified and well characterized as to specificity and mechanism of action (Bailly et al., 1989; Michaels et al., 1991; Boiteaux et al, 1992; Castaing et al., 1993; O'Connor et al., 1993; Karakaya, et al., 1997; Rabow and Kow, 1997; Zharkov et al., 1997). Although it was named for its ability to remove fapy-A and fapy-G, it also shows glycosylase activity on 8-oxo-G. All bacterial forms possess a seven-amino acid N-terminal sequence, which is an essential part of the active site. Like many other DNA glycosylases, bacterial FPG in vitro also cleaves DNA at abasic sites.

Cloning procedures have identified two mRNAs from Arabidopsis thaliana corresponding to two forms of FPG (Nakabeppu et al., 1998; Ohtsubo et al., 1998; Murphy and Gao, 1998). Sequence comparisons indicate that the mRNAs are produced by alternative splicing of the transcript from a single gene, AtMMH. Both forms of the protein have the conserved N-terminal amino-acid sequence and other sequence identities with the bacterial proteins. The C-terminal regions of the two forms are very different and unlike that of bacterial FPG . Both Arabidopsis FPGs have abasic lyase activity and cleave DNA oxidized by treatment with methylene blue and light, but only one form cleaves an oligonucleotide containing 8-oxo-G, demonstrating that the alternative splicing may affect the specificity of the enzyme (Gao and Murphy, Photochem. Photobiol., in press). One form has a nuclear localization sequence (Ohtsubo et al., 1998), and the other does not, suggesting that the two forms differ also in cellular localization.

Because other enzymes in the base excision repair pathway of human cells have multiple forms apparently produced by alternative splicing (Nilsen et al., 1997; Oda et al., 1999; Nishioka et al., 1999; Ohtsubo wt al., 2000), we have extended our search for alternative expression products of the AtMMH gene. We now report the discovery of three new cDNA clones and two distinct PCR fragments that together bring the total of potentialArabidopsis FPG protein variants to six.