Uncoupling of the apyrimidinic/apurinic endonucleolytic and 3′→5′ exonucleolytic activities of the Nfo protein of Mycoplasma pneumoniae through mutation of specific amino acid residues

Abstract

The DNA recombination and repair machineries of Mycoplasma pneumoniae and Mycoplasma genitalium were predicted to consist of a set of~11 proteins. The function of one of these proteins was inferred from its homology with proteins belonging to the Endo IV enzyme family. The members of this family function in the repair of apyrimidinic/apurinic (AP) sites in DNA. As such activity may be crucial in the mycoplasmal life cycle, we set out to study the Endo IV-like proteins encoded by M. pneumoniae and M. genitalium. Both proteins, termed Nfo Mpn and Nfo Mge , respectively, were assessed for their ability to interact with damaged and undamaged DNA. In the absence of divalent cations, both proteins exhibited specific cleavage of AP sites. Surprisingly, the proteins also recognized and cleaved cholesteryl-bound deoxyribose moieties in DNA, showing that these Nfo proteins may also function in repair of large DNA adducts. In the presence of Mg 2+ , Nfo Mpn and Nfo Mge also showed 39A59 exonucleolytic activity. By introduction of 13 single point mutations at highly conserved positions within Nfo Mpn , two major types of mutants could be distinguished: (i) mutants that showed no, or limited, AP cleavage activity in the presence of EDTA, but displayed significant levels of AP cleavage activity in the presence of Mg 2+ ; these mutants displayed no, or very low, exonucleolytic activity; and (ii) mutants that only demonstrated marginal levels of AP site cleavage activity in the presence of Mg 2+ and did not show exonucleolytic activity. Together, these results indicated that the AP endonucleolytic activity of the Nfo Mpn protein can be uncoupled from its 39A59 exonucleolytic activity.

Identification of a Novel Cobamide Remodeling Enzyme in the Beneficial Human Gut Bacterium Akkermansia muciniphila

“…6B) (86,90,93,94,96). Significant losses in activity have been observed in site-directed mutants of metal binding residues in AP endonuclease 2 superfamily members (87,(98)(99)(100)(101)(102)(103)(104)(105)(106). Similarly, E. coli strains expressing cbiR alleles with single mutations in many of these conserved residues had little to no AdoCbl hydrolysis activity (Fig.…”

Plasmodium falciparum Apn1 homolog is a mitochondrial base excision repair protein with restricted enzymatic functions

“…S1), yet it lacks NIR activity. Similarly, all eight amino acids of Mycoplasma pneumoniae Nfo implicated in the loss of 3′–5′ exonuclease activity while retaining AP endonuclease function in the presence of metal ions , are conserved in Pf Apn1. Thus, the uncoupling of AP endonuclease/phosphatase and exonuclease/NIR activities in Pf Apn1 indicates involvement of residues that have not so far been investigated for a role in exonuclease and NIR activities.…”

The mechanisms underlying antigenic variation and maintenance of genomic integrity in Mycoplasma pneumoniae and Mycoplasma genitalium

“…Nfo Mpn and Nfo Mge were also found to possess 3′ → 5′ exonuclease activity in the presence of Mg 2+ . In addition, both proteins were shown to recognize and remove larger DNA lesions, such as cholesteryl-modified bases in the DNA (Estevao et al 2014).…”