1887

Abstract

The DNA recombination and repair machineries of and 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 and . Both proteins, termed Nfo and Nfo, 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, Nfo and Nfo also showed 3′→5′ exonucleolytic activity. By introduction of 13 single point mutations at highly conserved positions within Nfo, 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; 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 and did not show exonucleolytic activity. Together, these results indicated that the AP endonucleolytic activity of the Nfo protein can be uncoupled from its 3′→5′ exonucleolytic activity.

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2014-06-01
2019-10-19
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