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

Abstract

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...

Capturing snapshots of APE1 processing DNA damage

“…N212 also plays a key role in coordinating the nucleophilic water, consistent with the dramatic loss of activity with the N212A mutant 47 . The position of this water is similar to that observed with other nucleases such as TDP2 48 , endonuclease IV (Nfo) 18 , and the Neisseria meningitides APE1 49 . As with APE1, these nucleases utilize side-chain contacts to facilitate cleavage of the DNA backbone by hydrogen bonding to a water molecule.…”

Structural insights into the ligand-binding hot spots of APEX1: an in silico analysis

“…1a). There is a profound understanding of how APEX1 locates scanning DNA (Carey and Strauss, 1999), substrate binding (Li and Wilson, 2014) and mechanism of phosphodiester cleavage (Lu et al, 2012). But areas such as inhibitor binding and important hot spot residues in ligand interaction were not studied extensively for APEX1.…”

fH-dependent complement evasion by disease-causing meningococcal strains with absent fHbp genes or frameshift mutations

“…N. meningitidis has evolved a number of mechanisms to evade complement-mediated bacteriolysis, including elaboration of polysaccharide capsules, lipooligosaccharide structure and sialylation [24, 37], and recruitment of the complement down-regulating molecule, factor H (fH) [12, 38]. Binding of fH to the bacterial surface leads to inactivation of C3b (to iC3b), and degradation of the C3bBb (the C3 convertase of the alternative pathway) through an interaction between fH and factor I [39, 40]. Both mechanisms result in decreased formation of the membrane attack complex, and decreased bacteriolysis.…”