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An array of basic residues is essential for the nucleolytic activity of the PHP domain of bacterial/ archaeal PolX DNA polymerases

AuthorsRodríguez, Guillermo ; Martín, María Teresa; de Vega, Miguel
Issue Date9-Jul-2019
PublisherNature Publishing Group
CitationScientific Reports
AbstractBacterial/archaeal family X DNA polymerases (PolXs) have a C-terminal PHP domain with an active site formed by nine histidines and aspartates that catalyzes 3′-5′ exonuclease, AP-endonuclease, 3′-phosphodiesterase and 3′-phosphatase activities. Multiple sequence alignments have allowed us to identify additional highly conserved residues along the PHP domain of bacterial/archaeal PolXs that form an electropositive path to the catalytic site and whose potential role in the nucleolytic activities had not been established. Here, site directed mutagenesis at the corresponding Bacillus subtilis PolX (PolXBs) residues, Arg469, Arg474, Asn498, Arg503 and Lys545, as well as to the highly conserved residue Phe440 gave rise to enzymes severely affected in all the nucleolytic activities of the enzyme while conserving a wild-type gap-filling activity, indicating a function of those residues in DNA binding at the PHP domain. Altogether, the results obtained with the mutant proteins, the spatial arrangement of those DNA binding residues, the intermolecular transference of the 3′-terminus between the PHP and polymerization active sites, and the available 3D structures of bacterial PolXs led us to propose the requirement to a great degree of a functional/structural flexibility to coordinate the synthetic and degradative activities in these enzymes
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