title: How a holoenzyme for DNA replication is formed.
abstract: Loading of the phage T4 sliding clamp gp45 by the gp44/62 clamp loader onto DNA to form the holoenzyme and their disassembly pathways were investigated using FRET-based single-molecule and ensemble kinetic studies. gp44/62-mediated assembly of gp45 onto the DNA involves a rate-limiting conformational rearrangement of the gp45-gp44/62-DNA complex. Single-molecule measurements revealed the intermediates in gp45 loading and their interconversion, suggesting that the assembly is not concerted but is broken down into many small kinetic steps. Two populations of the gp45-gp44/62-DNA complex are formed on the end-blocked DNA that are poised to form the holoenzyme with the polymerase. In the absence of a polymerase, the two clamp populations dissociated from the DNA along with gp44/62 with distinct rates. In the presence of polymerase, holoenzyme assembly involved the recruitment of the polymerase to the gp45-gp44/62-DNA complex mediated by the chaperoning activity of gp44/62. This transient multiprotein complex then decomposed through an ATP hydrolysis-dependent exit of gp44/62 leaving the holoenzyme on DNA. The rate of dissociation of the holoenzyme from the DNA is sensitive to whether the DNA ends are blocked, underscoring its mobility on the DNA.
authors.affiliation: Department of Chemistry, Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Senthil K
authors.initials: SK
authors.lastname: Perumal
authors.forename: Wenhui
authors.initials: W
authors.lastname: Ren
authors.forename: Tae-Hee
authors.initials: TH
authors.lastname: Lee
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Benkovic
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology*; D004261:DNA Replication / Q000235:genetics / Q000502:physiology*; D004279:DNA, Viral / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D020035:Holoenzymes / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances / Q000378:metabolism*; D057054:Molecular Imaging; D011487:Protein Conformation; D014759:Viral Envelope Proteins / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D004279:DNA, Viral; D020035:Holoenzymes; D046911:Macromolecular Substances; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C074855:gene 44 protein, Enterobacteria phage T4; C088659:gp62 protein, bacteriophage T4