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

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), singlestranded 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.T4 bacteriophage is one of the major model systems for acquiring information on DNA replication and repair processes. The T4 replisome is composed of eight major proteins that are subgrouped into the holoenzyme (gp43 polymerase, gp45 clamp, and gp44/62 clamp loader) and the primosome (gp41 helicase, gp59 helicase loader, gp32 single-stranded DNA-binding protein, and gp41 primase). Replication takes place through the coordinated function of all these proteins (1).The helicase loader (gp59) functions to load the helicase onto a DNA strand that is coated with gp32. In addition, gp59 inhibits the initiation of polymerization by gp43 (2, 3). Furthermore, it acts in recombination and recombination-dependent DNA replication (4, 5) and functions as a gatekeeper in origin-dependent replication in vivo (6). The gp59 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). 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.An interaction between gp59 and g...

Assembly and Dynamics of Gp59-Gp32-Single-stranded DNA (ssDNA), a DNA Helicase Loading Complex Required for Recombination-dependent Replication in Bacteriophage T4

“…D, reactions were repeated using the same conditions and the same range of concentrations as in C except that the Gp59 was removed from the reaction. Gp59 to etheno-modified ssDNA and 2-aminopurine-labeled fork DNAs as well as hexamer formation on fork DNA (13,21,33). The presence of the Alexa Fluor 546 label significantly stabilizes Gp59-DNA interactions because fluorescence quenching of labeled fork, ssDNA, or dsDNA was not reduced by the addition of equimolar unlabeled DNA (data not shown).…”

Mutational Analysis of the T4 Gp59 Helicase Loader Reveals Its Sites for Interaction with Helicase, Single-stranded Binding Protein, and DNA

“…Small angle x-ray scattering analysis of a gp59/SSB heterodimer agrees with the gp59/ SSB contact (36). It should be noted that single molecule photobleaching experiments have suggested that the active form of gp59, like that of helicase, is a hexamer (35). Thus, it may be that each monomer of gp59 makes contact with individual monomers of helicase and/or SSB.…”

A novel method to accurately locate and count large numbers of steps by photobleaching

“…, 2011), transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-regulatory proteins (Hastie et al. , 2013), T4 bacteriophage helicase loader protein (Arumugam et al. , 2009), bacterial oxidative phosphorylation complexes (Llorente-Garcia et al.…”