Human AP endonuclease inefficiently removes abasic sites within G4 structures compared to duplex DNA.
Excision repair processes are essential to maintain genome stability. A decrease in efficiency and fidelity of these pathways at regions of the genome that can assume non-canonical DNA structures has been proposed as a possible mechanism to explain the increased mutagenesis and consequent diseased state frequently associated with these sites. Here we describe the development of a FRET-based approach to monitor the presence of G quadruplex (G4) DNA, a non-canonical DNA structure formed in runs of guanines, in damage-containing single-stranded and double-stranded DNA. Using this approach, we directly show for the first time that the presence within the G4 structure of an abasic site, the most common lesion spontaneously generated during cellular metabolism, decreases the efficiency of human AP endonuclease activity and that this effect is mostly the result of a decreased enzymatic activity and not of decreased binding of the enzyme to the damaged site. This approach can be generally applied to dissecting the biochemistry of DNA repair at non-canonical DNA structures.
Departments of Anatomy and Cell Biology.
Christopher
C
Broxson
Biochemistry and Molecular Biology.
Jaclyn N
JN
Hayner
Departments of Anatomy and Cell Biology.
Joshua
J
Beckett
Biochemistry and Molecular Biology.
Linda B
LB
Bloom
Departments of Anatomy and Cell Biology, Medicine, University of Florida College of Medicine, 1600 SW Archer Road, Gainesville, FL 32610, USA silviat@ufl.edu.
Silvia
S
Tornaletti
D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004249:DNA Damage; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D054856:G-Quadruplexes; D016259:Genes, myc; D011092:Polyethylene Glycols / Q000737:chemistry
D011092:Polyethylene Glycols; D004247:DNA; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase