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Defining the impact of sumoylation on substrate binding and catalysis by thymine DNA glycosylase.

ABSTRACT: Thymine DNA glycosylase (TDG) excises thymine from mutagenic G·T mispairs generated by deamination of 5-methylcytosine (mC) and it removes two mC derivatives, 5-formylcytosine (fC) and 5-carboxylcytosine (caC), in a multistep pathway for DNA demethylation. TDG is modified by small ubiquitin-like modifier (SUMO) proteins, but the impact of sumoylation on TDG activity is poorly defined and the functions of TDG sumoylation remain unclear. We determined the effect of TDG sumoylation, by SUMO-1 or SUMO-2, on substrate binding and catalytic parameters. Single turnover experiments reveal that sumoylation dramatically impairs TDG base-excision activity, such that G·T activity is reduced by ?45-fold and fC and caC are excised slowly, with a reaction half-life of ?9 min (37°C). Fluorescence anisotropy studies reveal that unmodified TDG binds tightly to G·fC and G·caC substrates, with dissociation constants in the low nanomolar range. While sumoylation of TDG weakens substrate binding, the residual affinity is substantial and is comparable to that of biochemically-characterized readers of fC and caC. Our findings raise the possibility that sumoylation enables TDG to function, at least transiently, as reader of fC and caC. Notably, sumoylation could potentially facilitate TDG recruitment of other proteins, including transcription factors or epigenetic regulators, to these sites in DNA.

SUBMITTER: Coey CT

PROVIDER: S-EPMC6007377 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Defining the impact of sumoylation on substrate binding and catalysis by thymine DNA glycosylase.

Coey Christopher T CT Drohat Alexander C AC

Nucleic acids research 20180601 10

Thymine DNA glycosylase (TDG) excises thymine from mutagenic G·T mispairs generated by deamination of 5-methylcytosine (mC) and it removes two mC derivatives, 5-formylcytosine (fC) and 5-carboxylcytosine (caC), in a multistep pathway for DNA demethylation. TDG is modified by small ubiquitin-like modifier (SUMO) proteins, but the impact of sumoylation on TDG activity is poorly defined and the functions of TDG sumoylation remain unclear. We determined the effect of TDG sumoylation, by SUMO-1 or SU ...[more]

PMID: 29660017

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Project description:Repair of G·T mismatches arising from deamination of 5-methylcytosine (m(5)C) involves excision of thymine and restoration of a G·C pair via base excision repair (BER). Thymine DNA glycosylase (TDG) is one of two mammalian enzymes that can specifically remove thymine from G·T mispairs. While TDG can excise other bases, it maintains stringent specificity for a CpG context, suggesting deaminated m(5)C is an important biological substrate. Recent studies reveal TDG is essential for embryogenesis; it helps to maintain an active chromatin complex and initiates BER to counter aberrant de novo CpG methylation, which may involve excision of actively deaminated m(5)C. The relatively weak G·T activity of TDG has been implicated in the hypermutability of CpG sites, which largely involves C?T transitions arising from m(5)C deamination. Thus, it is important to understand how TDG recognizes and process substrates, particularly G·T mispairs. Here, we extend our detailed studies of TDG by examining the dependence of substrate binding and catalysis on pH, ionic strength, and temperature. Catalytic activity is relatively constant for pH 5.5-9, but falls sharply for pH>9 due to severely weakened substrate binding, and, potentially, ionization of the target base. Substrate binding and catalysis diminish sharply with increasing ionic strength, particularly for G·T substrates, due partly to effects on nucleotide flipping. TDG aggregates rapidly and irreversibly at 37°C, but can be stabilized by specific and nonspecific DNA. The temperature dependence of catalysis reveals large and unexpected differences for G·U and G·T substrates, where G·T activity exhibits much steeper temperature dependence. The results suggest that reversible nucleotide flipping is much more rapid for G·T substrates, consistent with our previous findings that steric effects limit the active-site lifetime of thymine, which may account for the relatively weak G·T activity. Our findings provide important insight into catalysis by TDG, particularly for mutagenic G·T mispairs.

Project description:Uracil DNA glycosylase (UNG) is a powerful DNA repair enzyme that has been shown to stabilize a glycosyl cation reaction intermediate and a related tight binding inhibitor using electrostatic interactions with the +1 and -1, but not the +2, phosphodiester group of the single-stranded DNA substrate Ap (2+)Ap (1+)Up (1-)ApA. These experimental results differed considerably from computational findings using duplex DNA, where the +2 phosphate was found to stabilize the transition state by approximately 5 kcal/mol, suggesting that UNG uses different catalytic strategies with single-stranded and double-stranded DNA substrates. In addition, the computational studies indicated that the conserved and positively charged His148 (which hydrogen bonds to the +2 phosphate) destabilized the glycosyl cation intermediate by 6-8 kcal/mol through anticatalytic electrostatic interactions. To evaluate these interesting proposals, we measured the kinetic effects of neutral methylphosphonate (MeP) stereoisomers at the +1 and +2 positions of a 12-mer dsDNA substrate and also the catalytic contribution and ionization state of His148. For MeP substitutions at the +1 position, single-turnover kinetic studies showed that the activation barrier was increased by 9.8 and 3.1 kcal/mol, corresponding to a stereoselectivity of nearly 40000-fold for the respective MeP isomers. Identical to the findings with ssDNA, MeP substitutions at the +2 position resulted in only small changes in the activation barrier (+/-0.3 kcal/mol), with little stereoselectivity ( approximately 4-fold). However, the H148A mutation destabilizes both the ground state and transition states by 2.4 and 4.3 kcal/mol, respectively. Thus, His148 is catalytic because it stabilizes the transition state to a greater extent (1.9 kcal/mol) than the ground state. Heteronuclear NMR studies established that His148 was neutral in the free enzyme at neutral pH, and in conformational exchange in a specific DNA complex containing uracil. We conclude that the +1 and +2 phosphate esters play identical catalytic roles in the reactions of single-stranded and double-stranded DNA substrates, and that His148 serves a catalytic role by positioning the substrate and catalytic water, or by an environmental effect.

Dataset Information

Defining the impact of sumoylation on substrate binding and catalysis by thymine DNA glycosylase.

Publications

Defining the impact of sumoylation on substrate binding and catalysis by thymine DNA glycosylase.

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