Effects of Backbone Contacts 3‘ to the Abasic Site on the Cleavage and the Product Binding by Human Apurinic/Apyrimidinic Endonuclease (APE1)

Abstract

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.

The role of Asn-212 in the catalytic mechanism of human endonuclease APE1: Stopped-flow kinetic study of incision activity on a natural AP site and a tetrahydrofuran analogue

“…Stabilization of the kinked abasic substrate is mediated by interactions of one of the APE1 ␣-helixes with the minor groove, and by the insertion grooves to occupy the place of the flipped-out AP site. An extensive site-directed mutagenesis study of APE1 showed that the side chains of Asn-226, Asn-229 and Arg-177, interacting with two downstream AP-DNA phosphates, provide specific binding to abasic DNA [7]. Finally, the abasic sugar moiety is stabilized within the hydrophobic active site pocket, which includes the side chains of Phe-266, Trp-280 and Leu-282.…”

Molecular Recognition of DNA Damage Sites by Apurinic/Apyrimidinic Endonucleases

“…Comparing substrate/product binding of molegos common to DNase-1 showed that those distinctive for APES are not directly involved in cleavage, but establish protein-DNA interactions 3' to the abasic site. Site directed mutagenesis showed that one area we found probably contributes to APE'S ability to retain cleaved DNA after removal of the damaged bases [4]. These additional bonds enhance both specific binding to damaged DNA and the processivity of APE1.…”

“…Based on DNA binding assay with product DNA, we concluded that N226 and N229 are important for their product DNA binding. We conclude that the H-bond network at the DNA backbone just downstream of the abasic site is crucial for the product DNA binding, but also affect the substrate DNA binding [4]. **5'-CyS-labeled 26-mer DNA with a single tetrahydrofuran was used.…”

“…Inspection of this motif in the cocrystal structure of APEl led to two asparagine residues, N226 and N229, at close proximity of the downstream of abasic site [2]. The H-bond network of the substrate DNA and the two Asn residues resembles that of R177, which is important for APE 1 's product DNA binding [4,141. Substituting alanine at these positions enhanced AP-endonuclease activity, as was previously observed for the R177A mutant, which binds to the same site on damaged DNA.…”

“…Kinetics of these mutations showed that the enhanced reaction rate was accompanied by an increase in Km. Combining the mutations lead to progressive decreases in APE activity and ability to bind substrate DNA [4]. We examined catalytic (AP endonuclease) activity and DNA binding affinity of the missense mutant proteins, N226A and N229A, in which either of the Asn residue was changed to Ala- (Table I).…”