Abstract
Recent crystallographic studies reveal loops in human AP endonuclease 1 (APE1) that interact with the major and minor grooves of DNA containing apurinic/apyrimidinic (AP) sites. These loops are postulated to stabilize the DNA helix and the flipped out AP residue. The loop alpha8 interacts with the major groove on the 3' side of the AP site. To determine the essentiality of the amino acids that constitute the alpha8 loop, we created a mutant library containing random nucleotides at codons 222-229 that, in wild-type APE1, specify the sequence NPKGNKKN. Upon expression of the library (2 x 10(6) different clones) in Escherichia coli and multiple rounds of selection with the alkylating agent methyl-methane sulfonate (MMS), we obtained approximately 2 x 10(5) active mutants that complemented the MMS sensitivity of AP endonuclease-deficient E. coli. DNA sequencing showed that active mutants tolerated amino acid substitutions at all eight randomized positions. Basic and uncharged polar amino acids together comprised the majority of substitutions, reflecting the positively charged, polar character of the wild-type loop. Asn-222, Asn-226, and Asn-229 exhibited the least mutability, consistent with x-ray data showing that each asparagine contacts a DNA phosphate. Substitutions at residues 226-229, located nearer to the AP site, that reduced basicity or hydrogen bonding potential, increased Km 2- to 6-fold and decreased AP site binding; substitutions at residues 222-225 exhibited lesser effects. This initial mutational analysis of the alpha8 loop supports and extends the conclusion of crystallographic studies that the loop is important for binding of AP.DNA and AP site incision.
Highlights
Active mutants were obtained by transforming the library into AP endonuclease-deficient E. coli BW542 that lack ExoIII and endonuclease IV, and selecting for resistance to methylmethane sulfonate (MMS)
We have created a human AP endonuclease 1 (APE1) mutant library harboring amino acid substitutions in the eight-residue ␣8 loop that interacts with the DNA major groove on the 3Ј-side of the AP site
The amino acid composition of the loop is highly conserved from flies to mammals [18], our results indicate that every residue is replaceable and that active mutants can be selected that confer MMS resistance to AP endonuclease-deficient E. coli
Summary
To determine the essentiality of the amino acids that constitute the ␣8 loop, we created a mutant library containing random nucleotides at codons 222–229 that, in wild-type APE1, specify the sequence NPKGNKKN. DNA sequencing showed that active mutants tolerated amino acid substitutions at all eight randomized positions.
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