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.
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 NfoMpn and NfoMge, 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+), NfoMpn and NfoMge also showed 3'→5' exonucleolytic activity. By introduction of 13 single point mutations at highly conserved positions within NfoMpn, 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 NfoMpn protein can be uncoupled from its 3'→5' exonucleolytic activity.
Laboratory of Pediatrics, Pediatric Infectious Diseases and Immunity, Erasmus MC-Sophia Children's Hospital, Rotterdam, The Netherlands.
Silvia
S
Estevão
Laboratory of Pediatrics, Pediatric Infectious Diseases and Immunity, Erasmus MC-Sophia Children's Hospital, Rotterdam, The Netherlands.
Pieternella E
PE
van der Spek
Laboratory of Pediatrics, Pediatric Infectious Diseases and Immunity, Erasmus MC-Sophia Children's Hospital, Rotterdam, The Netherlands.
Annemarie M C
AMC
van Rossum
Erasmus University College, Department Life Sciences, Erasmus University Rotterdam, The Netherlands.|Laboratory of Pediatrics, Pediatric Infectious Diseases and Immunity, Erasmus MC-Sophia Children's Hospital, Rotterdam, The Netherlands.
Cornelis
C
Vink
D019943:Amino Acid Substitution; D000596:Amino Acids / Q000235:genetics*; D045643:DNA Repair Enzymes / Q000235:genetics* / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000235:genetics* / Q000378:metabolism*; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D009177:Mycoplasma pneumoniae / Q000201:enzymology* / Q000235:genetics
D000596:Amino Acids; D050505:Mutant Proteins; D003851:Deoxyribonucleases; D045643:DNA Repair Enzymes