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Home Specificity and interactions of the DNA polymerase accessory proteins of bacteriophage T4 and RB69

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Specificity and interactions of the DNA polymerase accessory proteins of bacteriophage T4 and RB69

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Disclaimer	Access requires a license to the Dissertations and Theses (ProQuest) database.
Link to File	http://libproxy.tulane.edu:2048/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:9906410
Title	Specificity and interactions of the DNA polymerase accessory proteins of bacteriophage T4 and RB69
Author	Yeh, Lu-Shu
School	Tulane University
Academic Field	Biology, Molecular Biology, Genetics Biology, Microbiology
Abstract	The DNA replicase of bacteriophage T4 is a loosely assembled multiprotein complex that consists of at least nine polypeptides. The work described in this dissertation focused on elucidating the roles of sliding-clamp loading proteins, which form a subcomplex of the replicase holoenzyme assembly We have cloned and sequenced the genes encoding the clamp loader (genes 44 and 62) and the sliding clamp (gene 45) of bacteriophage RB69, a distant relative of T4. The biological and enzymological properties of these proteins from both phages were compared. Although clamp loader complexes can be exchanged between the T4 and RB69 biological systems, subunits of the clamp loaders are not interchangeable. RB69 gp44 and T4 gp62 can only assemble into an inactive complex while complexes of T4 gp44 and RB69 gp62 are partially active in vivo. This incompatibility was being utilized as a marker to localize the important residue(s) in gp44 and gp62 that are involved in generating a biologically active gp44/62 complex. We have localized these residues to Q171(T4)/K171(RB69) in gp44 and A36(T4)/P36(RB69) in gp62 Studies of ATPase activities of T4 and RB69 clamp loaders revealed differences in biological activities between them, and shed new light on the roles of clamp loader subunits in the clamp loading event. RB69 clamp loader has intrinsically higher ATPase activity than the corresponding T4 protein. Although gp62 has been proposed to mediate the interaction between clamp loader and sliding clamp (gp45), our results indicate that it is gp44 that mediates the clamp loader-sliding clamp interaction. We also showed a direct relationship between the putative ATP-binding domain (GKT box) and the biological activity of clamp loader. The 'GKT box' of gp44 is intolerant to amino-acid substitution, whether conservative or non-conservative. In vitro ATPase assays demonstrated that a conservative substitution (K56R) in 'GKT box' resulted in an $\sim$80% decrease of ATPase activity compared to the wild-type protein Our study raises new questions about the role of gp62 in clamp loading. Plasmid constructs for overproduction and purification of His-tagged gp62 are now available to pursue a detailed in vitro analysis of the gp44-gp62-gp45 interaction
Language	eng
Advisor(s)	Karam, Jim D
Degree Date	1998
Degree	Ph.D
Publisher	Tulane University
Publication Date	1998
Source	Source: 278 p., Dissertation Abstracts International, Volume: 59-09, Section: B, page: 4612
Identifier	See 'reference url' on the navigation bar.
Rights	Copyright is in accordance with U.S. Copyright law
Contact Information	digitallibrary@tulane.edu
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Home Specificity and interactions of the DNA polymerase accessory proteins of bacteriophage T4 and RB69

Specificity and interactions of the DNA polymerase accessory proteins of bacteriophage T4 and RB69

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