Expansion of base excision repair compensates for a lack of DNA repair by oxidative dealkylation in budding yeast

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.

The Shu Complex Prevents Mutagenesis and Cytotoxicity of Single-Strand Specific Alkylation Lesions

“…A previous study claimed that yeast TPA1 is an AlKB homolog (Shivange, Kodipelli et al, 2014). However, in our hands, and consistent with a recent report (Admiraal et al, 2019), tpa1Δ cells show no increased sensitivity to MMS. Furthermore, unlike other genes that are involved in repairing MMS-induced lesions, we find that TPA1 does not exhibit a synthetic sick phenotype with Shu complex mutants upon MMS exposure (Figure S4).…”

“…3meC occurs primarily in ssDNA and can stall the replicative polymerases. Unlike bacteria and higher eukaryotes, yeast does not encode for an enzyme capable of directly repairing 3meC from ssDNA (Admiraal, Eyler et al, 2019, Sedgwick et al, 2007. Therefore, yeast rely on bypass mechanisms to complete DNA replication.…”

“…Furthermore, unlike other genes that are involved in repairing MMS-induced lesions, we find that TPA1 does not exhibit a synthetic sick phenotype with Shu complex mutants upon MMS exposure (Figure S4). Recently, the Mag1 glycosylase was shown to excise 3meC in dsDNA; therefore, initiating their repair through BER (Admiraal et al, 2019). However, Mag1 cannot excise 3meCs from ssDNA (Admiraal et al, 2019).…”

Transient kinetic analysis of oxidative dealkylation by the direct reversal DNA repair enzyme AlkB

“…Some organisms that lack an AlkB homolog have DNA glycosylases with expanded substrate repertoires that are able to initiate base excision repair of known AlkB substrates (17)(18)(19)(20), and we expected that such glycosylases would be useful for assaying AlkB and its homologs. The alkyladenine DNA glycosylase from Bacillus subtilis (bAAG; (21)) excises both the monoalkyl lesion 1mA and the exocyclic bridged lesion εA from DNA ( Figure S1); we therefore developed a DNA glycosylasecoupled assay for AlkB using bAAG ( Figure 2A).…”

“…A pET19-derived expression construct for E. coli AlkB (15) was transformed into BL21(DE3) E. coli, and protein was expressed and purified as previously described (20). Purified protein was concentrated to 650 µM as determined by the absorbance at 280 nm using the predicted extinction coefficient of 32430 M -1 cm -1 .…”