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Human alkyladenine DNA glycosylase employs a processive search for DNA damage.

ABSTRACT: DNA repair proteins conduct a genome-wide search to detect and repair sites of DNA damage wherever they occur. Human alkyladenine DNA glycosylase (AAG) is responsible for recognizing a variety of base lesions, including alkylated and deaminated purines, and initiating their repair via the base excision repair pathway. We have investigated the mechanism by which AAG locates sites of damage using an oligonucleotide substrate containing two sites of DNA damage. This substrate was designed so that AAG randomly binds to either of the two lesions. AAG-catalyzed base excision creates a repair intermediate, and the subsequent partitioning between dissociation and diffusion to the second site can be quantified from the rates of formation of the different products. Our results demonstrate that AAG has the ability to slide for short distances along DNA at physiological salt concentrations. The processivity of AAG decreases with increasing ionic strength to become fully distributive at high ionic strengths, suggesting that electrostatic interactions between the negatively charged DNA and the positively charged DNA binding surface are important for nonspecific DNA binding. Although the amino terminus of the protein is dispensable for glycosylase activity at a single site, we find that deletion of the 80 amino-terminal amino acids significantly decreases the processivity of AAG. These observations support the idea that diffusion on undamaged DNA contributes to the search for sites of DNA damage.

SUBMITTER: Hedglin M

PROVIDER: S-EPMC2702167 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Human alkyladenine DNA glycosylase employs a processive search for DNA damage.

Hedglin Mark M O'Brien Patrick J PJ

Biochemistry 20081008 44

DNA repair proteins conduct a genome-wide search to detect and repair sites of DNA damage wherever they occur. Human alkyladenine DNA glycosylase (AAG) is responsible for recognizing a variety of base lesions, including alkylated and deaminated purines, and initiating their repair via the base excision repair pathway. We have investigated the mechanism by which AAG locates sites of damage using an oligonucleotide substrate containing two sites of DNA damage. This substrate was designed so that A ...[more]

PMID: 18839966

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Project description:Human uracil DNA glycosylase (hUNG) follows an extended reaction coordinate for locating rare uracil bases in genomic DNA. This process begins with diffusion-controlled engagement of undamaged DNA, followed by a damage search step in which the enzyme remains loosely associated with the DNA chain (translocation), and finally, a recognition step that allows the enzyme to efficiently bind and excise uracil when it is encountered. At each step along this coordinate, the enzyme must form DNA interactions that are highly specialized for either rapid damage searching or catalysis. Here we make extensive measurements of hUNG activity as a function of salt concentration to dissect the thermodynamic, kinetic, and electrostatic properties of key enzyme states along this reaction coordinate. We find that the interaction of hUNG with undamaged DNA is electrostatically driven at a physiological concentration of potassium ions (ΔGelect = -3.5 ± 0.5 kcal mol(-1)), with only a small nonelectrostatic contribution (ΔGnon = -2.0 ± 0.2 kcal mol(-1)). In contrast, the interaction with damaged DNA is dominated by the nonelectrostatic free energy term (ΔGnon = -7.2 ± 0.1 kcal mol(-1)), yet retains the nonspecific electrostatic contribution (ΔGelect = -2.3 ± 0.2 kcal mol(-1)). Stopped-flow kinetic experiments established that the salt sensitivity of damaged DNA binding originates from a reduction of kon, while koff is weakly dependent on salt. Similar findings were obtained from the salt dependences of the steady-state kinetic parameters, where the diffusion-controlled kcat/Km showed a salt dependence similar to kon, while kcat (limited by product release) was weakly dependent on salt. Finally, the salt dependence of translocation between two uracil sites separated by 20 bp in the same DNA chain was indistinguishable from that of kon. This result suggests that the transition-state for translocation over this spacing resembles that for DNA association from bulk solution and that hUNG escapes the DNA ion cloud during translocation. These findings provide key insights into how the ionic environment in cells influences the DNA damage search pathway.

Dataset Information

Human alkyladenine DNA glycosylase employs a processive search for DNA damage.

Publications

Human alkyladenine DNA glycosylase employs a processive search for DNA damage.

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