Effects of backbone contacts 3' to the abasic site on the cleavage and the product binding by human apurinic/apyrimidinic endonuclease (APE1).
The mammalian apurinic/apyrimidinic (AP) endonuclease (APE1) is a multifunctional protein that plays essential roles in DNA repair and gene regulation. We decomposed the APEs into 12 blocks of highly conserved sequence and structure (molegos). This analysis suggested that residues in molegos common to all APEs, but not to the less specific nuclease, DNase I, would dictate enhanced binding to damaged DNA. To test this hypothesis, alanine was substituted for N226 and N229, which form hydrogen bonds to the DNA backbone 3' of the AP sites in crystal structures of the APE1/DNA complex. While the cleavage rate at AP sites of both N226A and N229A mutants increased, their ability to bind to damaged DNA decreased. The ability of a double mutant (N226A/N229A) to bind damaged DNA was further decreased, while the V(max) was almost identical to that of the wild-type APE1. A double mutant at N226 and R177, a residue that binds to the same phosphate as N229, had a significantly decreased activity and substrate binding. As the affinity for product DNA was decreased in all the mutants, the enhanced reaction rate of the single mutants could be due to alleviation of product inhibition of the enzyme. We conclude that hydrogen bonds to phosphate groups 3' to the cleavage site is essential for APE1's binding to the product DNA, which may be necessary for efficient functioning of the base excision repair pathway. The results indicate that the molego analysis can aid in the redesign of proteins with altered binding affinity and activity.
Izumi
Tadahide
T
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1079, USA. taizumi@utmb.edu
Schein
Catherine H
CH
Oezguen
Numan
N
Feng
Yanling
Y
Braun
Werner
W
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001216:Asparagine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007700:Kinetics; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D013379:Substrate Specificity / Q000235:genetics
D004268:DNA-Binding Proteins; D001216:Asparagine; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D000409:Alanine