Fluorescence Monitoring of T4 Polymerase Holoenzyme Accessory Protein Interactions During Loading of the Sliding Clamp onto the Template–Primer Junction

• Published in: Journal of molecular biology Vol. 264; no. 3; pp. 426 - 439
• Main Authors: Latham, Gary J., Pietroni, Paola, Dong, Feng, Young, Mark C., von Hippel, Peter H.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 06.12.1996
• Subjects: Adenosine Triphosphatases - metabolism; Amino Acid Sequence; anisotropy; ATPase; Bacteriophage T4 - enzymology; Bacteriophage T4 - genetics; Bacteriophage T4 - physiology; Base Sequence; Deoxyribonucleotides - metabolism; DNA Primers; DNA Replication - physiology; DNA-Directed DNA Polymerase; fluorescence; Fluorescence Polarization; Fluorescent Dyes; Maleimides; Models, Genetic; Molecular Sequence Data; Naphthalenesulfonates; replication; Templates, Genetic; Trans-Activators - genetics; Trans-Activators - metabolism; Viral Proteins - genetics; Viral Proteins - metabolism; Virus Replication; T4; replication; fluorescence; ATPase; anisotropy
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1996.0651

Assembly of the T4 polymerase holoenzyme requires coordinated interactions among the core polymerase and the clamp loader (gp44/62) and sliding clamp (gp45) accessory proteins. Here we describe the creation of a mutant of gp45 that can be uniquely modified by fluorescent probes within each protein monomer at a site-specific cysteine residue. The fluorescently labeled gp45 was shown to have the same biological activity as the wild-type protein. These strike “labeled” gp45 adducts were then used in steady-state and fluorescence polarization studies to monitor the interaction of gp45 with the gp44/62 clamp-loading complex and template–primer DNA in the presence and absence of ATP, and of the non-hydrolyzable analog, adenosine 5′-O-(3-thiotriphosphate). We find that a complex of ATP-activated gp44/62 with appropriately labeled gp45 shows significant fluorescent enhancement (and an increase in fluores- cent anisotropy), that can be partially reversed by interaction with template–primer DNA. Fluorescence-monitored binding curves between gp45 and ATP-activated gp44/62 reveal that the two protein complexes bind with a 1:1 stoichiometry. Analysis shows that these methods can be used to follow the ATP-driven loading of gp45 onto the template–primer by the gp44/62 clamp-loading complex, and in combination with the kinetic data presented in the companion article, provide insight into the rate-limiting steps during clamp assembly on template–primer DNA. A reaction pathway for this processivity clamp-loading process is proposed.