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), 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.

FIGURE 1.

Structures of the forked DNA substrates used in this study. 1, forked DNA (10/40/40-mer) with biotin at the 5′-end of the leading strand for the smPB study using unlabeled substrate (unlabeled forked DNA); 2, forked DNA (34/62/50-mer) with Cy5 dye at the 5′-end of the primer and biotin at the 3′-end of the lagging strand (Cy5-labeled forked DNA) for the smPB study with labeled substrate and gp59-Alexa 555.

FIGURE 2.

Analysis of photobleaching steps of protein-forked DNA complexes. a–c, the smPB fluorescence images were collected at a 100-ms exposure time with 3 × 3-pixel binning with an EMCCD camera. The fluorescence images were converted into fluorescence traces using mathematical codes written in IDL. Representative plots of fluorescence intensity versus time (ms) show photobleaching steps 1, 2, and 6. a.u., arbitrary units.

FIGURE 3.

Histogram plot of smPB events. The histogram represents the smPB experiment done with Cy5-labeled forked DNA (FkD-Cy5) with gp59-Alexa 555 and other proteins. Histograms show the normalized events versus number of photobleaching steps. Similar histograms were built for smPB experiments with unlabeled forked DNA with the same proteins (data not shown). The experiments were done in triplicate, and the errors assigned. The population of the hexamer seen is indicated by the circle.

FIGURE 4.

Plots of smPB experiments. The following plots show the experimental and fitted data for various smPB experiments. The data were fitted to a monomer model using Equation 4 as described under “Materials and Methods.” The normalized number of events and their corresponding photobleaching steps were provided as the input, whereas [A], K, and σ were used as fitting parameters. A–D, unlabeled forked DNA + gp59 and other proteins (i.e. gp32 and gp41); E–H, gp59 and other proteins alone; I–L, Cy5-labeled forked DNA and gp59 and other proteins.

FIGURE 5.

Schematic representation of oligomers of gp59 on a replication fork. a, hexamer of gp59; b, random oligomer of gp59; c, gp59 at the forked DNA and co-localized on gp32; d, ternary complex between a hexamer of gp59 and gp32 and gp41 in the presence of forked DNA.