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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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Stimulation of AAG by APE1 on asymmetric hairpins. (A) Multiple-turnover glycosylase activity was measured in the presence (light bars) and absence of APE1 (dark bars). The mean (SD) is shown for at least three independent experiments for each substrate. Arrows indicate those substrates for which the stimulated kcat value reaches the rate constant for excision of Hx. (B) Model for the displacement of AAG by APE1, whereby AAG leaves the abasic site to allow APE1 to bind. Tight binding by APE1 prevents rebinding of AAG and leaves it free to dissociate more quickly from undamaged DNA.
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gks952-F5: Stimulation of AAG by APE1 on asymmetric hairpins. (A) Multiple-turnover glycosylase activity was measured in the presence (light bars) and absence of APE1 (dark bars). The mean (SD) is shown for at least three independent experiments for each substrate. Arrows indicate those substrates for which the stimulated kcat value reaches the rate constant for excision of Hx. (B) Model for the displacement of AAG by APE1, whereby AAG leaves the abasic site to allow APE1 to bind. Tight binding by APE1 prevents rebinding of AAG and leaves it free to dissociate more quickly from undamaged DNA.

Mentions: We previously found that APE1 efficiently displaces AAG and stimulates its multiple-turnover glycosylase activity on substrates with upstream and downstream duplex regions that were as short as 6 bp by increasing the rate of AAG dissociation (14). This observation ruled out the possibility of a functional APE1–AAG complex that uses the full DNA binding surfaces of both proteins, but cannot rule out the possibility that the DNA binding sites are only partially used or partially overlapping. In the current work, we tested the stimulation of AAG by APE1 for substrates that have much shorter upstream and downstream duplex regions. Under the conditions used, the steady-state rate of AAG is limited by the rate of dissociation from the abasic DNA product. Representative data for the stimulation by APE1 are provided in Figure 2C. The dissociation rate constants in the presence and absence of APE1 are summarized in Figure 5 and Supplementary Table S1. The substrates with lesions immediately adjacent to the hairpins were omitted from this plot because the AAG reaction was no longer limited by product release. In many cases, the presence of APE1 accelerated the rate of product release to the point where AAG was limited by the rate of N-glycosidic bond cleavage (i.e. kcat = kmax). In these cases, the observed rate constant is a lower limit for the rate of AAG dissociation (indicated by arrows in Figure 5).Figure 5.


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

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

Stimulation of AAG by APE1 on asymmetric hairpins. (A) Multiple-turnover glycosylase activity was measured in the presence (light bars) and absence of APE1 (dark bars). The mean (SD) is shown for at least three independent experiments for each substrate. Arrows indicate those substrates for which the stimulated kcat value reaches the rate constant for excision of Hx. (B) Model for the displacement of AAG by APE1, whereby AAG leaves the abasic site to allow APE1 to bind. Tight binding by APE1 prevents rebinding of AAG and leaves it free to dissociate more quickly from undamaged DNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks952-F5: Stimulation of AAG by APE1 on asymmetric hairpins. (A) Multiple-turnover glycosylase activity was measured in the presence (light bars) and absence of APE1 (dark bars). The mean (SD) is shown for at least three independent experiments for each substrate. Arrows indicate those substrates for which the stimulated kcat value reaches the rate constant for excision of Hx. (B) Model for the displacement of AAG by APE1, whereby AAG leaves the abasic site to allow APE1 to bind. Tight binding by APE1 prevents rebinding of AAG and leaves it free to dissociate more quickly from undamaged DNA.
Mentions: We previously found that APE1 efficiently displaces AAG and stimulates its multiple-turnover glycosylase activity on substrates with upstream and downstream duplex regions that were as short as 6 bp by increasing the rate of AAG dissociation (14). This observation ruled out the possibility of a functional APE1–AAG complex that uses the full DNA binding surfaces of both proteins, but cannot rule out the possibility that the DNA binding sites are only partially used or partially overlapping. In the current work, we tested the stimulation of AAG by APE1 for substrates that have much shorter upstream and downstream duplex regions. Under the conditions used, the steady-state rate of AAG is limited by the rate of dissociation from the abasic DNA product. Representative data for the stimulation by APE1 are provided in Figure 2C. The dissociation rate constants in the presence and absence of APE1 are summarized in Figure 5 and Supplementary Table S1. The substrates with lesions immediately adjacent to the hairpins were omitted from this plot because the AAG reaction was no longer limited by product release. In many cases, the presence of APE1 accelerated the rate of product release to the point where AAG was limited by the rate of N-glycosidic bond cleavage (i.e. kcat = kmax). In these cases, the observed rate constant is a lower limit for the rate of AAG dissociation (indicated by arrows in Figure 5).Figure 5.

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
Related in: MedlinePlus