CRYSTAL STRUCTURE OF THE HUMAN DNA REPAIR ENZYME AP ENDONUCLEASE 1
Michael A. Gorman, Solange Morera, and Paul S. Freemont
Molecular Structure and Function Laboratory, Imperial
Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX,
UK.
gormanm@icrf.icnet.uk
Keywords: DNA repair, endonuclease, HAP1,
Ref-1, abasic sites, Base excision repair, mutagenesis,
Apurinic and apyrimidinic (AP) sites are produced by the hydrolysis of the N-glycosylic bond that link DNA bases to their deoxyribose sugar moieties. Spontaneous release of bases constitutes one of the most frequently occurring type of DNA damage under normal physiological conditions, with 103 - 104 bases being lost (and regenerated) per cell per day (1), thus representing a major challenge to the integrity of DNA. AP sites are also generated by various chemical or radiation-induced modifications of DNA bases, or by the action of DNA glycosylases, and are mutagenic in bacterial and mammalian systems (2). The repair of AP sites requires a number of enzymes which repair via a ëcut and patchí pathway (involving the regeneration of one nucleotide) or via a ëpatch and cutí pathway in which a number of nucleotides are re-synthesised (3). An AP endonuclease recognises the AP site and cleaves the DNA backbone of the defective strand. A DNA exonuclease then excises the AP deoxyribose-phosphate moiety leaving a gap of one nucleotide. The missing base is replaced by DNA polymerase and DNA ligase joins the cleaved strands together.
Human AP endonuclease 1 (HAP1) is the major AP endonuclease found in human cells and plays a pivotal role in both repair pathways (4). HAP1 is a class II AP endonuclease consisting of 318 amino acids with a molecular weight of 35.5 kDa. The N-terminal 62 amino acids, together with cysteine 65, have been associated with a unique role in mediating the reductive activation of oxidised transcriptional activators, such as Fos and Jun (5). The remaining primary structure is associated with the AP endonuclease catalytic activity and DNA recognition, which, when compared to the DNA repair domains of other AP endonucleases, shares a sequence identity ranging from 57% (Arp isolated from A. thaliana) to 27% (EXO III from E. coli) (6).
Presented is the determination of the X-ray crystal structure
of a 31 amino acid truncated but fully active form of HAP1 to
2.3A resolution (7). The HAP1 structure represents the first
structure of a eukaryotic AP endonuclease. Using the structural
identity and amino acid sequence homology between HAP1, EXO III
(the E. coli homologue of HAP1), and DNase I, a new mode
of AP site recognition is proposed, which involves a DNA
extra-helical deoxyribose moiety (see figure).