Characterization of herpes simplex virus Type I helicase-primase: Subunit assembly and function

Persistent Link:
http://hdl.handle.net/10150/284310
Title:
Characterization of herpes simplex virus Type I helicase-primase: Subunit assembly and function
Author:
Constantin, Nicoleta
Issue Date:
2000
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Protein-protein interactions participate in the assembly, regulation, and processivity of all molecular "machines." In this dissertation, a series of genetic and biochemical approaches have been used to analyze the protein-protein interactions which participate in the formation of the Herpes Simplex Virus Type 1 replisome. The emphasis of these studies lies on the herpes helicase-primase complex, its assembly, regulation, and processivity. The herpes helicase-primase is a heterotrimeric complex encoded by the viral UL5, UL52, and UL8 genes. The yeast two-hybrid system was used to generate a protein linkage map of the herpes replisome and to characterize the interactions among the three subunits of the helicase-primase (UL5, UL52, and UL8). Deletion analysis and co-immunoprecipitation, were used to show that a 548 amino acid carboxy-terminal fragment of UL52 interacts with UL8, while a 350 amino acid N-terminal fragment is required for interaction with UL5 and may also be involved in the regulation of the strength of interaction with UL8. Comparative sequence analysis suggested that the functional interactions among the subunits of the helicase-primase encoded by alpha-herpesviruses may differ from those of the helicase-primases encoded by the beta- and gamma-herpesviruses. Expression and purification of the UL5 subunit of the helicase-primase in the absence of UL52 resulted in an inactivating but reversible catalytic deficiency in UL5. UL52 corrected this deficiency when added subsequently. Based on these results, a proposal is suggested here that UL52 regulates the activity of UL5 by inducing conversion of UL5 from an inactive to an active conformation, and/or by contributing amino acid residues to the catalytic site. Finally, evidence from a combination of biochemical approaches suggested that the HSV-1 helicaseprimase is a monomeric ATPase with a non-globular shape and that it belongs to the group of helicases which use an inchworming mechanism for unwinding DNA.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Biology, Molecular.; Biology, Microbiology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Biochemistry amd Molecular biophysics
Degree Grantor:
University of Arizona
Advisor:
Dodson, Mark S.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleCharacterization of herpes simplex virus Type I helicase-primase: Subunit assembly and functionen_US
dc.creatorConstantin, Nicoletaen_US
dc.contributor.authorConstantin, Nicoletaen_US
dc.date.issued2000en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractProtein-protein interactions participate in the assembly, regulation, and processivity of all molecular "machines." In this dissertation, a series of genetic and biochemical approaches have been used to analyze the protein-protein interactions which participate in the formation of the Herpes Simplex Virus Type 1 replisome. The emphasis of these studies lies on the herpes helicase-primase complex, its assembly, regulation, and processivity. The herpes helicase-primase is a heterotrimeric complex encoded by the viral UL5, UL52, and UL8 genes. The yeast two-hybrid system was used to generate a protein linkage map of the herpes replisome and to characterize the interactions among the three subunits of the helicase-primase (UL5, UL52, and UL8). Deletion analysis and co-immunoprecipitation, were used to show that a 548 amino acid carboxy-terminal fragment of UL52 interacts with UL8, while a 350 amino acid N-terminal fragment is required for interaction with UL5 and may also be involved in the regulation of the strength of interaction with UL8. Comparative sequence analysis suggested that the functional interactions among the subunits of the helicase-primase encoded by alpha-herpesviruses may differ from those of the helicase-primases encoded by the beta- and gamma-herpesviruses. Expression and purification of the UL5 subunit of the helicase-primase in the absence of UL52 resulted in an inactivating but reversible catalytic deficiency in UL5. UL52 corrected this deficiency when added subsequently. Based on these results, a proposal is suggested here that UL52 regulates the activity of UL5 by inducing conversion of UL5 from an inactive to an active conformation, and/or by contributing amino acid residues to the catalytic site. Finally, evidence from a combination of biochemical approaches suggested that the HSV-1 helicaseprimase is a monomeric ATPase with a non-globular shape and that it belongs to the group of helicases which use an inchworming mechanism for unwinding DNA.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBiology, Molecular.en_US
dc.subjectBiology, Microbiology.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineBiochemistry amd Molecular biophysicsen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorDodson, Mark S.en_US
dc.identifier.proquest9992144en_US
dc.identifier.bibrecord.b41175359en_US
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