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DNA translocation by human uracil DNA glycosylase: role of DNA phosphate charge.

ABSTRACT: Human DNA repair glycosylases must encounter and inspect each DNA base in the genome to discover damaged bases that may be present at a density of <1 in 10 million normal base pairs. This remarkable example of specific molecular recognition requires a reduced dimensionality search process (facilitated diffusion) that involves both hopping and sliding along the DNA chain. Despite the widely accepted importance of facilitated diffusion in protein-DNA interactions, the molecular features of DNA that influence hopping and sliding are poorly understood. Here we explore the role of the charged DNA phosphate backbone in sliding and hopping by human uracil DNA glycosylase (hUNG), which is an exemplar that efficiently locates rare uracil bases in both double-stranded DNA and single-stranded DNA. Substitution of neutral methylphosphonate groups for anionic DNA phosphate groups weakened nonspecific DNA binding affinity by 0.4-0.5 kcal/mol per substitution. In contrast, sliding of hUNG between uracil sites embedded in duplex and single-stranded DNA substrates persisted unabated when multiple methylphosphonate linkages were inserted between the sites. Thus, a continuous phosphodiester backbone negative charge is not essential for sliding over nonspecific DNA binding sites. We consider several alternative mechanisms for these results. A model consistent with previous structural and nuclear magnetic resonance dynamic results invokes the presence of open and closed conformational states of hUNG. The open state is short-lived and has weak or nonexistent interactions with the DNA backbone that are conducive for sliding, and the populated closed state has stronger interactions with the phosphate backbone. These data suggest that the fleeting sliding form of hUNG is a distinct weakly interacting state that facilitates rapid movement along the DNA chain and resembles the transition state for DNA dissociation.

SUBMITTER: Schonhoft JD

PROVIDER: S-EPMC3628972 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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DNA translocation by human uracil DNA glycosylase: role of DNA phosphate charge.

Schonhoft Joseph D JD Kosowicz John G JG Stivers James T JT

Biochemistry 20130402 15

Human DNA repair glycosylases must encounter and inspect each DNA base in the genome to discover damaged bases that may be present at a density of <1 in 10 million normal base pairs. This remarkable example of specific molecular recognition requires a reduced dimensionality search process (facilitated diffusion) that involves both hopping and sliding along the DNA chain. Despite the widely accepted importance of facilitated diffusion in protein-DNA interactions, the molecular features of DNA tha ...[more]

PMID: 23506309

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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

DNA translocation by human uracil DNA glycosylase: role of DNA phosphate charge.

Publications

DNA translocation by human uracil DNA glycosylase: role of DNA phosphate charge.

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