Investigation of stoichiometry of T4 bacteriophage helicase loader protein (gp59).
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.
Arumugam
Sri Ranjini
SR
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Lee
Tae-Hee
TH
Benkovic
Stephen J
SJ
D017122:Bacteriophage T4 / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism; D019915:DNA Primase / Q000737:chemistry / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D038761:Photobleaching; D011485:Protein Binding / Q000502:physiology; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; C091225:gene 59 protein, Enterobacteria phage T4; C060035:gp32 protein, Enterobacteria phage T4; D019915:DNA Primase; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases
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