A Role for Saccharomyces cerevisiae Tpa1 Protein in Direct Alkylation Repair

Shivange, Gururaj; Kodipelli, Naveena; Mohan, Monisha; Anindya, Roy

doi:10.1074/jbc.m114.590216

Cited by 23 publications

(15 citation statements)

References 70 publications

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“…2). This led us to question the proposed direct participation of Tpa1 in repair of alkylated DNA (7) and to hypothesize that Mag1 may play a larger role in this process than has been appreciated. We showed that Mag1 excises alkylated bases present in duplex DNA that are known to be substrates of the DRR enzyme AlkB, including 3mC and 1mA.…”

Section: Discussionmentioning

confidence: 99%

“…S. cerevisiae also expresses Tpa1, which, like E. coli AlkB and many other proteins, is a member of the Fe(II)- and 2OG-dependent dioxygenase superfamily (6). Tpa1 has recently been reported to be a functional homolog of AlkB, acting as an oxidative dealkylase to repair alkylated DNA (7). However, Tpa1 is more closely related to Fe(II)/2OG-dependent dioxygenases that catalyze post-translational hydroxylation of proteins (8–10), and it has been reported to catalyze prolyl hydroxylation of ribosomal protein S23 and to regulate translation (11, 12).…”

Section: Introductionmentioning

confidence: 99%

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

Admiraal

Eyler

Baldwin

et al. 2019

Journal of Biological Chemistry

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Admiraal

Eyler

Baldwin

et al. 2019

Journal of Biological Chemistry

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“…DNA repair was assayed by measuring formaldehyde release as result of removal of methyl-adducts. Formaldehyde was detected by adding acetoacetanilide and ammonia directly to the reaction mix to form fluorescent compound with peak emission of 465 nm ( 27 ) (Supplementary Figure S1A). Concentration of released formaldehyde was determined from the formaldehyde standard curve (Supplementary Figure S1B and C).…”

Section: Resultsmentioning

confidence: 99%

RecA stimulates AlkB-mediated direct repair of DNA adducts

et al. 2016

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The Escherichia coli AlkB protein is a 2-oxoglutarate/Fe(II)-dependent demethylase that repairs alkylated single stranded and double stranded DNA. Immunoaffinity chromatography coupled with mass spectrometry identified RecA, a key factor in homologous recombination, as an AlkB-associated protein. The interaction between AlkB and RecA was validated by yeast two-hybrid assay; size-exclusion chromatography and standard pull down experiment and was shown to be direct and mediated by the N-terminal domain of RecA. RecA binding results AlkB–RecA heterodimer formation and RecA–AlkB repairs alkylated DNA with higher efficiency than AlkB alone.

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“…[25] Random mutations can also be used to give information on the effect of changing various residues in the active site. [26] An excellent example of directed evolution is the mutation of a transaminase for the asymmetric reduction step in the synthesis of Sitagliptin 16 (Scheme 10). Sitagliptin 16 (marketed as Januvia) is an antidiabetic drug developed and marketed by Merck & Co.…”

Section: Biotechnology: Metagenomics Directed Evolution and Rationalmentioning

confidence: 99%

The Impact of Recent Developments in Technologies which Enable the Increased Use of Biocatalysts

Foley

Maguire

2019

Eur J Org Chem

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While biocatalytic transformations are very powerful in enantioselective synthesis, frequently occurring under mild conditions, and proceed with extraordinary selectivity, there are practical challenges associated with the use of biocatalysis, such as limited substrate scope, stability, and reusability. Recent technological developments, for example immobilization, continuous flow, and molecular biology, all contribute towards enhancing the use of enzymes in synthesis.

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A Role for Saccharomyces cerevisiae Tpa1 Protein in Direct Alkylation Repair

Cited by 23 publications

References 70 publications

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

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

RecA stimulates AlkB-mediated direct repair of DNA adducts

The Impact of Recent Developments in Technologies which Enable the Increased Use of Biocatalysts

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