Fluorescence monitoring of T4 polymerase holoenzyme accessory protein interactions during loading of the sliding clamp onto the template-primer junction

被引:30
作者
Latham, GJ
Pietroni, P
Dong, F
Young, MC
vonHippel, PH
机构
[1] UNIV OREGON,INST MOL BIOL,EUGENE,OR 97403
[2] UNIV OREGON,DEPT CHEM,EUGENE,OR 97403
关键词
T4; replication; ATPase; fluorescence; anisotropy;
D O I
10.1006/jmbi.1996.0651
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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. (C) 1996 Academic Press Limited
引用
收藏
页码:426 / 439
页数:14
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