Molecular and Biochemical Analyses of a Three-Subunit Euryarchaeal Clamp Loader Complex

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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Animal Sciences

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2008

Author dc:creator

• Chen, Yi-Hsing

Contributors dc:contributor

• Cann, Isaac K.O.

• Chemistry, Biochemistry

Language dc:language

eng

Identifier

(MiAaPQ)AAI3337731

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/83613