Abstract
The Mag1 and Tpa1 proteins from budding yeast (Saccharomyces cerevisiae) have both been reported to repair alkylation damage in DNA. Mag1 initiates the base excision repair pathway by removing alkylated bases from DNA, and Tpa1 has been proposed to directly repair alkylated bases as does the prototypical oxidative dealkylase AlkB from Escherichia coli. However, we found that in vivo repair of methyl methanesulfonate (MMS)-induced alkylation damage in DNA involves Mag1 but not Tpa1. We observed that yeast strains without tpa1 are no more sensitive to MMS than WT yeast, whereas mag1-deficient yeast are ∼500-fold more sensitive to MMS. We therefore investigated the substrate specificity of Mag1 and found that it excises alkylated bases that are known AlkB substrates. In contrast, purified recombinant Tpa1 did not repair these alkylated DNA substrates, but it did exhibit the prolyl hydroxylase activity that has also been ascribed to it. A comparison of several of the kinetic parameters of Mag1 and its E. coli homolog AlkA revealed that Mag1 catalyzes base excision from known AlkB substrates with greater efficiency than does AlkA, consistent with an expanded role of yeast Mag1 in repair of alkylation damage. Our results challenge the proposal that Tpa1 directly functions in DNA repair and suggest that Mag1-initiated base excision repair compensates for the absence of oxidative dealkylation of alkylated nucleobases in budding yeast. This expanded role of Mag1, as compared with alkylation repair glycosylases in other organisms, could explain the extreme sensitivity of Mag1-deficient S. cerevisiae toward alkylation damage.
Highlights
The Mag1 and Tpa1 proteins from budding yeast (Saccharomyces cerevisiae) have both been reported to repair alkylation damage in DNA
Repair of the alkylated base 1,N6ethenoadenine (⑀A) in E. coli can be initiated by the DNA glycosylase AlkA and completed by other enzymes in the base excision repair (BER) pathway, or direct reversal repair (DRR) can be catalyzed by the DNA dealkylase AlkB in a reaction requiring ferrous iron, oxygen, and 2-oxoglutarate (2OG) (Fig. 1B)
We constructed yeast strains lacking mag1, tpa1, or both genes using standard methods. These strains were exposed to methyl methanesulfonate (MMS) to compare the contributions of Mag1 and Tpa1 to the repair of alkylation damage
Summary
The Mag and Tpa proteins from budding yeast (Saccharomyces cerevisiae) have both been reported to repair alkylation damage in DNA. Our results challenge the proposal that Tpa directly functions in DNA repair and suggest that Mag1-initiated base excision repair compensates for the absence of oxidative dealkylation of alkylated nucleobases in budding yeast This expanded role of Mag, as compared with alkylation repair glycosylases in other organisms, could explain the extreme sensitivity of Mag1-deficient S. cerevisiae toward alkylation damage. It has been suggested that some bacteria and archaea that appear to lack an AlkB-family oxidative dealkylase instead use BER with an expanded substrate repertoire to repair alkylation damage [1,2,3] In support of this idea, DNA glycosylases from these organisms have been shown to excise alkylated bases that have more commonly been thought to be substrates for direct repair by AlkB and related DNA dealkylases. We set out to clarify the roles of Mag and Tpa in the repair of alkylation damage to DNA in S. cerevisiae
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