Investigation of Stoichiometry of T4 Bacteriophage Helicase Loader Protein (gp59)

Tae-Hee Lee,Sri Ranjini Arumugam,Stephen J. Benkovic

doi:10.1074/jbc.m109.029926

Tae-Hee Lee, Sri Ranjini Arumugam

Open Access

https://doi.org/10.1074/jbc.m109.029926

Copy DOI

Abstract

The T4 bacteriophage helicase loader (gp59) is one of the main eight proteins that play an active role in the replisome. gp59 is a small protein (26 kDa) that exists as a monomer in solution and in the crystal. It binds preferentially to forked DNA and interacts directly with the T4 helicase (gp41), single-stranded DNA-binding protein (gp32), and polymerase (gp43). However, the stoichiometry and structure of the functional form are not very well understood. There is experimental evidence for a hexameric structure for the helicase (gp41) and the primase (gp61), inferring that the gp59 structure might also be hexameric. Various experimental approaches, including gel shift, fluorescence anisotropy, light scattering, and fluorescence correlation spectroscopy, have not provided a clearer understanding of the stoichiometry. In this study, we employed single-molecule photobleaching (smPB) experiments to elucidate the stoichiometry of gp59 on a forked DNA and to investigate its interaction with other proteins forming the primosome complex. smPB studies were performed with Alexa 555-labeled gp59 proteins and a forked DNA substrate. Co-localization experiments were performed using Cy5-labeled forked DNA and Alexa 555-labeled gp59 in the presence and absence of gp32 and gp41 proteins. A systematic study of smPB experiments and subsequent data analysis using a simple model indicated that gp59 on the forked DNA forms a hexamer. In addition, the presence of gp32 and gp41 proteins increases the stability of the gp59 complex, emphasizing their functional role in T4 DNA replication machinery.

Highlights

The helicase loader functions to load the helicase onto a DNA strand that is coated with gp32
Based on its similarity to the members of the high mobility group (HMG) family of proteins, a model structure has been proposed by Mueser et al (8) for gp59 bound to forked DNA in which the N-terminal domain of gp59 binds to the duplex DNA, whereas the lagging strand passes through a narrow groove that lies between the N- and C-terminal domains and the leading strand binds to the bottom surface of the C-terminal domain
We have experimentally investigated the stoichiometry and interaction of gp59 on its primary forked DNA substrate in the presence of gp32 and gp41 proteins using the single-molecule photobleaching4 method

Summary

Introduction

The helicase loader (gp59) functions to load the helicase onto a DNA strand that is coated with gp. The gp protein is known to exist as a monomer in solution and in an x-ray crystal structure (7, 8) It binds to several types of DNA substrates, preferring a replication fork (i.e. forked DNA) (9). The dissociation constant (Kd) observed for gp binding to a gp32-DNA complex is ϳ2 nM (10). Functional assays using single-turnover and steady-state kinetics showed that DNA unwinding by the helicase is enhanced by 200-fold in the presence of gp, where maximum unwinding is observed when the proteins exist in equal stoichiometry (16). The electron microscopic studies on the structure of the gp41-gp complex revealed two hexamers in a 1:1 stoichiometry (19). We have experimentally investigated the stoichiometry and interaction of gp on its primary forked DNA substrate in the presence of gp and gp proteins using the single-molecule photobleaching (smPB) method.

Methods

Results

Conclusion