Molecular and Biochemical Analyses of a Three-Subunit Euryarchaeal Clamp Loader Complex
Chen, Yi-Hsing
This item is only available for download by members of the University of Illinois community. Students, faculty, and staff at the U of I may log in with your NetID and password to view the item. If you are trying to access an Illinois-restricted dissertation or thesis, you can request a copy through your library's Inter-Library Loan office or purchase a copy directly from ProQuest.
Permalink
https://hdl.handle.net/2142/83613
Description
Title
Molecular and Biochemical Analyses of a Three-Subunit Euryarchaeal Clamp Loader Complex
Author(s)
Chen, Yi-Hsing
Issue Date
2008
Doctoral Committee Chair(s)
Cann, Isaac K.O.
Department of Study
Animal Sciences
Discipline
Animal Sciences
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
DNA polymerase requires two processing factors, sliding clamp and clamp loader, to direct rapid and accurate DNA replication. In eukaryotes, the ring-shaped sliding clamp, proliferating cell nuclear antigen (PCNA), encircles double-stranded DNA within its central hole and tethers the DNA polymerases onto DNA. A spiral-shaped pentameric protein, replication factor C (RFC), functions as the clamp loader, which installs the sliding clamp onto primer-template junction of DNA in an ATP-dependent manner. The critical clamp loader acts as molecular switch from distributive to processive DNA synthesis. Here, we describe a novel form of clamp loader from the euryarchaeon Methanosarcina acetivorans (Mac). The M. acetivorans clamp loader comprises two similar small subunits (MacRFCS1 and MacRFCS2) and one large subunit (MacRFCL). Since the simplest RFC is made up of two different subunits and the most complex form is made up of five different subunits, the MacRFC may represent a critical link in the evolution of complex clamp loaders from simple forms in the archaeal/eukaryotic sister lineage. MacRFCS1 was found to form oligomers in solution but not MacRFCS2. The clamp loader complex stimulated PCNA-dependent DNA elongation by a cognate DNA polymerase. The site-directed mutagenesis in the Walker A and SRC motifs indicated that both motifs in MacRFCS1 are essential for clamp loading. A densitometric method suggested that the spatial distribution of MacRFC complex is similar to that of the E. coli clamp loader which has the same 3:1:1 ratio and is made of three gamma subunits (motor), a delta' subunit (stator) and a delta subunit (wrench). Furthermore, our studies on this archaeal three-subunits clamp loader have provided important biochemical, molecular, and evolutionary insights into the function of RFC, an indispensable molecular switch in archaeal/eukaryotic DNA replication.
Use this login method if you
don't
have an
@illinois.edu
email address.
(Oops, I do have one)
IDEALS migrated to a new platform on June 23, 2022. If you created
your account prior to this date, you will have to reset your password
using the forgot-password link below.
