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

Guillermo Rodríguez,Miguel De Vega,María Teresa Martín

doi:10.1038/s41598-019-46349-8

Abstract

Bacterial/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.

Highlights

Genome stability maintenance is critical to all forms of life
Whereas in the case of the eukaryotic Polλ, Polμ, terminal deoxyribonucleotide transferase (TdT) and yeast Pol[4], the Polβ-like core is linked to an additional N-terminal BRCT domain, critical to interactions with nonhomologous end joining (NHEJ) and V(D)J recombination factors[5,8,9,11,12,16], in bacterial/archaeal PolXs is fused to a C-terminal Polymerase Histidinol Phosphatase (PHP) domain[17,18]
In the tertiary structure of the PHP domain the above residues are arranged to form a solvent exposed catalytic active site that is located nearby the molecular surface of the domain (Fig. 1c)

Summary

Introduction

Genome stability maintenance is critical to all forms of life. the enormous variety of DNA damages has imposed the evolution of specific DNA repair pathways where a plethora of specific enzymatic activities repair those lesions that otherwise could cause a blockage of essential biological processes as genome replication and transcription[1]. Whereas in the case of the eukaryotic Polλ, Polμ, terminal deoxyribonucleotide transferase (TdT) and yeast Pol[4], the Polβ-like core is linked to an additional N-terminal BRCT domain, critical to interactions with NHEJ and V(D)J recombination factors[5,8,9,11,12,16], in bacterial/archaeal PolXs is fused to a C-terminal Polymerase Histidinol Phosphatase (PHP) domain[17,18] In the latter case, extensive biochemical analysis carried out in the PolX from the bacteria Bacillus subtilis (PolXBs) and Thermus thermophilus (ttPolX) have shown that the C-terminal PHP domain contains a nine-residue active site that binds divalent cations to catalyze nucleolytic activities[19,20,21,22,23]. Considering both, the results shown here and the available structures of ttPolX ternary complexes[24] and of the apo PolX from Deinococcus radiodurans (PolXDr) we discuss the functional flexibility required to coordinate the synthetic and degradative activities of these enzymes

Methods

Results

Conclusion