An Adenosine Triphosphate- Dependent 5'-3' DNA Helicase From sk1-Like Lactococcus lactis F13 Phage

Magdalena Chmielewska-Jeznach; Kamil Steczkiewicz; Kamil Kobyłecki; Jacek K Bardowski; Agnieszka K Szczepankowska

doi:10.3389/fmicb.2022.840219

An Adenosine Triphosphate- Dependent 5'-3' DNA Helicase From sk1-Like Lactococcus lactis F13 Phage

Front Microbiol. 2022 Mar 15:13:840219. doi: 10.3389/fmicb.2022.840219. eCollection 2022.

Authors

Magdalena Chmielewska-Jeznach¹, Kamil Steczkiewicz¹, Kamil Kobyłecki¹, Jacek K Bardowski¹, Agnieszka K Szczepankowska¹

Affiliation

¹ Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland.

Abstract

Here, we describe functional characterization of an early gene (gp46) product of a virulent Lactococcus lactis sk1-like phage, vB_Llc_bIBBF13 (abbr. F13). The GP46 _F13 protein carries a catalytically active RecA-like domain belonging to the P-loop NTPase superfamily. It also retains features characteristic for ATPases forming oligomers. In order to elucidate its detailed molecular function, we cloned and overexpressed the gp46 gene in Escherichia coli. Purified GP46 _F13 protein binds to DNA and exhibits DNA unwinding activity on branched substrates in the presence of adenosine triphosphate (ATP). Size exclusion chromatography with multi-angle light scattering (SEC-MALS) experiments demonstrate that GP46 _F13 forms oligomers, and further pull-down assays show that GP46 _F13 interacts with host proteins involved in replication (i.e., DnaK, DnaJ, topoisomerase I, and single-strand binding protein). Taking together the localization of the gene and the obtained results, GP46 _F13 is the first protein encoded in the early-expressed gene region with helicase activity that has been identified among lytic L. lactis phages up to date.

Keywords: DNA replication; DNA unwinding; Lactococcus lactis; P-loop NTPase; sk1-like bacteriophage.