Defining the functional footprint for recognition and repair of deaminated DNA.
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.
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Michael R
MR
Baldwin
Patrick J
PJ
O'Brien
D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D045647:DNA Glycosylases / Q000378:metabolism*; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism*; D003641:Deamination; D006801:Humans; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D023302:Protein Footprinting / Q000379:methods
D009841:Oligonucleotides; D004247:DNA; C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase