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Defining the functional footprint for recognition and repair of deaminated DNA.

Baldwin MR, O'Brien PJ - Nucleic Acids Res. (2012)

Bottom Line: AAG turnover is stimulated in the presence of APE1, indicating rapid exchange of AAG and APE1 at the abasic site produced by the AAG reaction.The coordinated reaction does not require an extended footprint, suggesting that each enzyme engages the site independently.Functional footprinting provides unique information relative to traditional footprinting approaches and is generally applicable to any DNA modifying enzyme or system of enzymes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.

ABSTRACT
Spontaneous deamination of DNA is mutagenic, if it is not repaired by the base excision repair (BER) pathway. Crystallographic data suggest that each BER enzyme has a compact DNA binding site. However, these structures lack information about poorly ordered termini, and the energetic contributions of specific protein-DNA contacts cannot be inferred. Furthermore, these structures do not reveal how DNA repair intermediates are passed between enzyme active sites. We used a functional footprinting approach to define the binding sites of the first two enzymes of the human BER pathway for the repair of deaminated purines, alkyladenine DNA glycosylase (AAG) and AP endonuclease (APE1). Although the functional footprint for full-length AAG is explained by crystal structures of truncated AAG, the footprint for full-length APE1 indicates a much larger binding site than is observed in crystal structures. AAG turnover is stimulated in the presence of APE1, indicating rapid exchange of AAG and APE1 at the abasic site produced by the AAG reaction. The coordinated reaction does not require an extended footprint, suggesting that each enzyme engages the site independently. Functional footprinting provides unique information relative to traditional footprinting approaches and is generally applicable to any DNA modifying enzyme or system of enzymes.

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Glycosylase activity of AAG towards hairpin DNA. (A) Maximal single-turnover rate constants (kmax) for excision of Hx are plotted as a function of lesion position. Differences could be because of changes in nucleotide flipping or N-glycosidic bond cleavage. (B) The relative specificity constants (kcat/KM) were determined by competition and are sensitive to any of the steps up to and including N-glycosidic bond cleavage. All rate constants were measured in at least three independent experiments, and the error bars indicate the mean ± [standard deviation (SD)]. The open bars for +1 and −1 substrates indicate the upper limit for the kcat/KM value because no competition was observed.
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gks952-F3: Glycosylase activity of AAG towards hairpin DNA. (A) Maximal single-turnover rate constants (kmax) for excision of Hx are plotted as a function of lesion position. Differences could be because of changes in nucleotide flipping or N-glycosidic bond cleavage. (B) The relative specificity constants (kcat/KM) were determined by competition and are sensitive to any of the steps up to and including N-glycosidic bond cleavage. All rate constants were measured in at least three independent experiments, and the error bars indicate the mean ± [standard deviation (SD)]. The open bars for +1 and −1 substrates indicate the upper limit for the kcat/KM value because no competition was observed.

Mentions: We first measured the single-turnover rate constants at saturating concentration of AAG for each of the DNA substrates. In all cases, the single-turnover reaction was saturated and followed a single exponential (representative data are shown in Figure 2A). The values of kmax for each of the substrates are plotted versus the lesion position in Figure 3A (Supplementary Table S1). Remarkably, lesions located only 2 bp away from the hairpin were relatively good substrates by this measure, and lesions that were directly adjacent to the hairpin were only ∼100-fold worse than the optimal substrates.Figure 3.


Defining the functional footprint for recognition and repair of deaminated DNA.

Baldwin MR, O'Brien PJ - Nucleic Acids Res. (2012)

Glycosylase activity of AAG towards hairpin DNA. (A) Maximal single-turnover rate constants (kmax) for excision of Hx are plotted as a function of lesion position. Differences could be because of changes in nucleotide flipping or N-glycosidic bond cleavage. (B) The relative specificity constants (kcat/KM) were determined by competition and are sensitive to any of the steps up to and including N-glycosidic bond cleavage. All rate constants were measured in at least three independent experiments, and the error bars indicate the mean ± [standard deviation (SD)]. The open bars for +1 and −1 substrates indicate the upper limit for the kcat/KM value because no competition was observed.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526306&req=5

gks952-F3: Glycosylase activity of AAG towards hairpin DNA. (A) Maximal single-turnover rate constants (kmax) for excision of Hx are plotted as a function of lesion position. Differences could be because of changes in nucleotide flipping or N-glycosidic bond cleavage. (B) The relative specificity constants (kcat/KM) were determined by competition and are sensitive to any of the steps up to and including N-glycosidic bond cleavage. All rate constants were measured in at least three independent experiments, and the error bars indicate the mean ± [standard deviation (SD)]. The open bars for +1 and −1 substrates indicate the upper limit for the kcat/KM value because no competition was observed.
Mentions: We first measured the single-turnover rate constants at saturating concentration of AAG for each of the DNA substrates. In all cases, the single-turnover reaction was saturated and followed a single exponential (representative data are shown in Figure 2A). The values of kmax for each of the substrates are plotted versus the lesion position in Figure 3A (Supplementary Table S1). Remarkably, lesions located only 2 bp away from the hairpin were relatively good substrates by this measure, and lesions that were directly adjacent to the hairpin were only ∼100-fold worse than the optimal substrates.Figure 3.

Bottom Line: AAG turnover is stimulated in the presence of APE1, indicating rapid exchange of AAG and APE1 at the abasic site produced by the AAG reaction.The coordinated reaction does not require an extended footprint, suggesting that each enzyme engages the site independently.Functional footprinting provides unique information relative to traditional footprinting approaches and is generally applicable to any DNA modifying enzyme or system of enzymes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.

ABSTRACT
Spontaneous deamination of DNA is mutagenic, if it is not repaired by the base excision repair (BER) pathway. Crystallographic data suggest that each BER enzyme has a compact DNA binding site. However, these structures lack information about poorly ordered termini, and the energetic contributions of specific protein-DNA contacts cannot be inferred. Furthermore, these structures do not reveal how DNA repair intermediates are passed between enzyme active sites. We used a functional footprinting approach to define the binding sites of the first two enzymes of the human BER pathway for the repair of deaminated purines, alkyladenine DNA glycosylase (AAG) and AP endonuclease (APE1). Although the functional footprint for full-length AAG is explained by crystal structures of truncated AAG, the footprint for full-length APE1 indicates a much larger binding site than is observed in crystal structures. AAG turnover is stimulated in the presence of APE1, indicating rapid exchange of AAG and APE1 at the abasic site produced by the AAG reaction. The coordinated reaction does not require an extended footprint, suggesting that each enzyme engages the site independently. Functional footprinting provides unique information relative to traditional footprinting approaches and is generally applicable to any DNA modifying enzyme or system of enzymes.

Show MeSH