Biochemical characterization of UvrD helicase and its 1 interplay with DNA mismatch repair proteins in 2 Neisseria gonorrhoeae

Abstract

UvrD helicase belongs to type I superfamily of helicases. It participates in Nucleotide Excision Repair (NER) and DNA Mismatch Repair (MMR). It also participates in the rolling circle replication. It has been shown that UvrD can dismantle several proteins from DNA such as RecA and Tus protein. By unwinding the recombination intermediates and dismantling RecA from DNA, it acts as an anti- recombinase. UvrD has also been shown to physically interact with RNA polymerase and helps in the transcription coupled repair. In addition to these roles, UvrD helicase also contributes to the virulence of many pathogenic bacteria. In Mycobacterium tuberculosis, the inactivation of uvrD1 gene reduced its persistence in a mouse model of tuberculosis infection. Inactivation of uvrD gene in Neisseria meningitis increases the rate of phase variation. The uvrD null mutants of Haemophilus influenza exhibit a high degree of UV sensitivity and reduced level of host cell reactivation and decreased phage recombination. Biochemical studies of Helicobacter pylori UvrD (HpUvrD) shows that it can unwind DNA duplex using the energy derived from GTP hydrolysis and deletion of the C- terminal 63 residues disrupted the oligomerisation of HpUvrD. (30). Similar studies with Haemophilus influenzae UvrD reveal that the C- terminal 48 residues are important for its oligomerisation (30). In spite of these important roles in the virulence of pathogenic bacteria, a few UvrD helicase from pathogenic bacteria have been bio-chemically characterized. In this study, UvrD helicase from N. gonorrohoeae (NgoUvrD) has been bio-chemically characterized. NgoUvrD was cloned into pET14b vector between NdeI and BamHI. The protein was heterologously expressed as N- terminal His6 tag. NgoUvrD was then purified with Ni2+-NTA affinity chromatography followed by Heparin Sepharose chromatography. Size exclusion chromatography indicates that NgoUvrD behaves as dimer in solution. NgoUvrD has been found to unwind varied range of substrates like DNA with 5’ overhang, DNA with 3’ overhang, Holliday junction and blunt end duplex DNA. However, for blunt end duplex DNA the unwinding efficiency of NgoUvrD decreases with the length of DNA. Moreover, streptavidin displacement assay demonstrates that NgoUvrD translocates in 3’ to 5’ direction. Despite this how it can unwind DNA substrates with 5’ or 3’ overhangs with equal efficiency is yet to be studied.