The PHP domain of PolX from Staphylococcus aureus aids high fidelity DNA synthesis through the removal of misincorporated deoxyribo-, ribo- and oxidized nucleotides

Shilpi Nagpal,Deepak T Nair

doi:10.1038/s41598-021-83498-1

Abstract

The X family is one of the eight families of DNA polymerases (dPols) and members of this family are known to participate in the later stages of Base Excision Repair. Many prokaryotic members of this family possess a Polymerase and Histidinol Phosphatase (PHP) domain at their C-termini. The PHP domain has been shown to possess 3′–5′ exonuclease activity and may represent the proofreading function in these dPols. PolX from Staphylococcus aureus also possesses the PHP domain at the C-terminus, and we show that this domain has an intrinsic Mn2+ dependent 3′–5′ exonuclease capable of removing misincorporated dNMPs from the primer. The misincorporation of oxidized nucleotides such as 8oxodGTP and rNTPs are known to be pro-mutagenic and can lead to genomic instability. Here, we show that the PHP domain aids DNA replication by the removal of misincorporated oxidized nucleotides and rNMPs. Overall, our study shows that the proofreading activity of the PHP domain plays a critical role in maintaining genomic integrity and stability. The exonuclease activity of this enzyme can, therefore, be the target of therapeutic intervention to combat infection by methicillin-resistant-Staphylococcus-aureus.

Highlights

DNA Polymerases have been classified into eight families, the A, B, C, D, X, Y, reverse transcriptase and PrimPol on the basis of sequence/structural similarity of the domains which catalyze the nucleotide polymerization reaction[1,2,3,4]
Our studies show that the PHP domain of PolX from S. aureus aids high fidelity DNA synthesis through the removal of misincorporated deoxyribo, ribo, and oxidized nucleotides
Multiple sequence alignment of the protein encoded by gene SACOL1153 with other conserved members of the PolX family confirmed the presence of structurally conserved PolX like core and a PHP domain (Fig. 1). saPolX shares about 32% sequence identity with ttPolX with 98% sequence coverage

Summary

Introduction

DNA Polymerases (dPols) have been classified into eight families, the A, B, C, D, X, Y, reverse transcriptase and PrimPol on the basis of sequence/structural similarity of the domains which catalyze the nucleotide polymerization reaction[1,2,3,4]. Among these families, the X- family DNA polymerases (PolX) are present in many organisms with representatives reported in archaea, viruses, bacteria, and eukaryotes[4,5,6,7,8,9,10,11]. In the case of bacterial X family polymerase, both ttPolX and bsPolX show low frequency of incorporation of 8oxodGTP irrespective of the templating nucleotide. ttPolX and bsPolX incorporate 8oxodGTP opposite dA with 15- and 218-times lower efficiency, respectively than dTTP opposite d A63,64

Methods

Results

Conclusion