Chinese hamster AP endonuclease operates by a two‐metal ion assisted catalytic mechanism

Borjigin, Mandula; Arenaz, P.; Stec, Boguslaw

doi:10.1016/j.febslet.2011.12.025

Cited by 1 publication

(1 citation statement)

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“…Calcium typically acts as an inhibitor of enzymes in phosphodiester hydrolysis, because it has similar coordination chemistry to Mg 2+ , but its larger ionic radius and reduced electronegativity mean that it is unable to support catalysis (17). Titrations of different metal ions can lead to synergistic effects, which have been interpreted as the requirement for two metal ions in catalysis (16,18,19). We performed reactions of NApe with varying ratios of Mg 2+ and Ca 2+ , while maintaining a constant chloride concentration to minimize ionic strength effects (Fig.…”

Section: Resultsmentioning

confidence: 99%

Structural basis for the recognition and cleavage of abasic DNA in Neisseria meningitidis

Lü

Šilhán

MacDonald

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Base excision repair (BER) is a highly conserved DNA repair pathway throughout all kingdoms from bacteria to humans. Whereas several enzymes are required to complete the multistep repair process of damaged bases, apurinic-apyrimidic (AP) endonucleases play an essential role in enabling the repair process by recognizing intermediary abasic sites cleaving the phosphodiester backbone 5′ to the abasic site. Despite extensive study, there is no structure of a bacterial AP endonuclease bound to substrate DNA. Furthermore, the structural mechanism for AP-site cleavage is incomplete. Here we report a detailed structural and biochemical study of the AP endonuclease from Neisseria meningitidis that has allowed us to capture structural intermediates providing more complete snapshots of the catalytic mechanism. Our data reveal subtle differences in AP-site recognition and kinetics between the human and bacterial enzymes that may reflect different evolutionary pressures.X-ray crystallography | genome stability | prokaryote A basic (apurinic-apyrimidic, AP) sites within DNA pose a serious threat to all living organisms; if unrepaired, they lead to stalled replication, increased mutations, and an overall loss of genomic integrity. For example human cells defective in AP-site processing are nonviable (1). AP sites occur through either the spontaneous loss of nucleotide bases via chemical breakage of the N-glycosylic bond or as intermediates in the repair of DNA lesions such as oxidatively damaged bases, spontaneously deaminated cytosines, or alkylation of bases (2). It is estimated that 10 4 abasic sites are produced per day per cell in eukaryotes and if left unrepaired will result in significant mutagenic loading on the cell (3, 4). All living organisms have therefore evolved specific enzymes that recognize and repair damaged or missing DNA bases known as the base excision repair (BER) pathway (2).In BER, DNA glycosylases recognize and remove damaged bases to produce abasic sites. The resultant abasic sites are then recognized by AP endonucleases that bind and catalyze a magnesium-dependent 5′ cleavage of the phosphodiester backbone, producing a 3′-OH. Other bifunctional DNA glycosylases cleave the resulting abasic site via an AP lyase mechanism, which leaves a 3′ unsaturated aldehyde (5). In either case, DNA backbone cleavage marks the AP site for subsequent repair by DNA polymerases and ligases. Two main families of AP endonucleases have been identified: the major human AP endonuclease (APEX1) and Escherichia coli homolog ExoIII belong to the ExoIII family of AP endonucleases and are highly conserved (∼51% sequence homology; ∼29% sequence identity). The other is the EndoIV family, named after the E. coli enzyme, and they cleave abasic sites in a Zn 2+ -dependent reaction. Both E. coli and Saccharomyces cerevisiae possess both ExoIII and EndoIV type enzymes, whereas humans have two ExoIII family enzymes.Molecular and biochemical details of abasic DNA recognition and backbone cleavage have mainly focused on the human APEX1 enz...

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Section: Resultsmentioning

confidence: 99%