Abstract
3′-5′ exonucleases are frequently found to be associated to polymerases or helicases domains in the same enzyme or could function as autonomous entities. Here we uncovered that Candida albicans Pif1 (CaPif1) displays a 3′-5′ exonuclease activity besides its main helicase activity. These two latter activities appear to reside on the same polypeptide and the new exonuclease activity could be mapped to the helicase core domain. We clearly show that CaPif1 displays exclusively exonuclease activity and unambiguously establish the directionality of the exonuclease activity as the 3′-to-5′ polarity. The enzyme appears to follow the two-metal-ion driven hydrolyzing activity exhibited by most of the nucleases, as shown by its dependence of magnesium and also by the identification of aspartic residues. Interestingly, an excellent correlation could be found between the presence of the conserved residues and the exonuclease activity when testing activities on Pif1 enzymes from eight fungal organisms. In contrast to others proteins endowed with the double helicase/exonuclease functionality, CaPif1 differs in the fact that the two activities are embedded in the same helicase domain and not located on separated domains. Our findings may suggest a biochemical basis for mechanistic studies of Pif1 family helicases.
Highlights
3′-5′ exonucleases are frequently found to be associated to polymerases or helicases domains in the same enzyme or could function as autonomous entities
CaPif[1] helicase-catalyzed DNA unwinding using electrophoretic mobility shift assay (EMSA), we found that in addition to the expected unwound ssDNA product, a significant degradation of fluorescein labeled oligonucleotides appeared in the bottom of gels, suggesting that CaPif[1] protein could possess some nuclease activity
Since CaPif[1] was expressed and purified from E. coli and knowing that E. coli contains several 3′to 5′exonucleases including exonuclease I, exonuclease VII, and RecJ, capable of degrading single-stranded DNA19, we performed additional experiments to address whether the observed nuclease activity is intrinsic to CaPif[1], or just stems from a contaminating nuclease resulting from unspecific copurification
Summary
3′-5′ exonucleases are frequently found to be associated to polymerases or helicases domains in the same enzyme or could function as autonomous entities. Different from the above mentioned helicase-associated exonucleases in which the helicase and exonuclease domains possess structurally autonomous domains or motifs, and each helicase and exonuclease domain being active in isolation, the residues implicated in exonuclease active sites of CaPif[1] appear to be embedded in Pif[1] helicase domains, indicating that the helicase and exonuclease activities are not structurally separable. It appears that this property is well conserved, at least, among the fungal species
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