Fluorescence monitoring of T4 polymerase holoenzyme accessory protein interactions during loading of the sliding clamp onto the template-primer junction.
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 fluorescent 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.
Latham
G J
GJ
Institute of Molecular Biology, Department of Chemistry, University of Oregon, Eugene 97403-1229, USA.
Pietroni
P
P
Dong
F
F
Young
M C
MC
von Hippel
P H
PH
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D017931:DNA Primers; D004261:DNA Replication / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase; D003854:Deoxyribonucleotides / Q000378:metabolism; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D008301:Maleimides; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates; D013698:Templates, Genetic; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D017931:DNA Primers; D003854:Deoxyribonucleotides; D005456:Fluorescent Dyes; D008301:Maleimides; D009282:Naphthalenesulfonates; D015534:Trans-Activators; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; C074555:gene 45 protein, Enterobacteria phage T4; C006421:1,5-I-AEDANS; C003944:N-(3-pyrene)maleimide; D004259:DNA-Directed DNA Polymerase; D000251:Adenosine Triphosphatases