This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Google Scholar
Right arrow Articles by Chen, Y.-H.
Right arrow Articles by Cann, I. K. O.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, Y.-H.
Right arrow Articles by Cann, I. K. O.

 Previous Article  |  Next Article 

Journal of Bacteriology, November 2009, p. 6539-6549, Vol. 191, No. 21
0021-9193/09/$08.00+0     doi:10.1128/JB.00414-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Molecular Analyses of a Three-Subunit Euryarchaeal Clamp Loader Complex from Methanosarcina acetivorans{triangledown} ,{dagger}

Yi-Hsing Chen,1 Yuyen Lin,1 Aya Yoshinaga,2 Benazir Chhotani,3 Jenna L. Lorenzini,3 Alexander A. Crofts,3 Shou Mei,3 Roderick I. Mackie,1,4 Yoshizumi Ishino,2 and Isaac K. O. Cann1,3,4*

Department of Animal Sciences,1 Department of Microbiology,3 Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801,4 Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, Fukuoka-shi, Japan 812-85812

Received 26 March 2009/ Accepted 24 August 2009

Chromosomal DNA replication is dependent on processive DNA synthesis. Across the three domains of life and in certain viruses, a toroidal sliding clamp confers processivity to replicative DNA polymerases by encircling the DNA and engaging the polymerase in protein/protein interactions. Sliding clamps are ring-shaped; therefore, they have cognate clamp loaders that open and load them onto DNA. Here we use biochemical and mutational analyses to study the structure/function of the Methanosarcina acetivorans clamp loader or replication factor C (RFC) homolog. M. acetivorans RFC (RFCMa), which represents an intermediate between the common archaeal RFC and the eukaryotic RFC, comprises two different small subunits (RFCS1 and RFCS2) and a large subunit (RFCL). Size exclusion chromatography suggested that RFCS1 exists in oligomeric states depending on protein concentration, while RFCS2 exists as a monomer. Protein complexes of RFCS1/RFCS2 formed in solution; however, they failed to stimulate DNA synthesis by a cognate DNA polymerase in the presence of its clamp. Determination of the subunit composition and previous mutational analysis allowed the prediction of the spatial distribution of subunits in this new member of the clamp loader family. Three RFCS1 subunits are flanked by an RFCS2 and an RFCL. The spatial distribution is, therefore, reminiscent of the minimal Escherichia coli clamp loader that exists in space as three {gamma}-subunits (motor) flanked by the {delta}' (stator) and the {delta} (wrench) subunits. Mutational analysis, however, suggested that the similarity between the two clamp loaders does not translate into the complete conservation of the functions of individual subunits within the RFCMa complex.

* Corresponding author. Mailing address: Department of Animal Sciences, 1207 West Gregory Drive, University of Illinois at Urbana-Champaign, Urbana, IL 61801. Phone: (217) 333-2090. Fax: (217) 333-8286. E-mail: icann{at}illinois.edu

{triangledown} Published ahead of print on 28 August 2009.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, November 2009, p. 6539-6549, Vol. 191, No. 21
0021-9193/09/$08.00+0     doi:10.1128/JB.00414-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»