Molecular analyses of a three-subunit euryarchaeal clamp loader complex from Methanosarcina acetivorans

Yi Hsing Chen, Yuyen Lin, Aya Yoshinaga, Benazir Chhotani, Jenna L. Lorenzini, Alexander A. Crofts, Shou Mei, Roderick I. Mackie, Yoshizumi Ishino, Isaac K.O. Cann

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Abstract

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 ringshaped; 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 γ-subunits (motor) flanked by the δ′ (stator) and the δ (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.

Original languageEnglish
Pages (from-to)6539-6549
Number of pages11
JournalJournal of bacteriology
Volume191
Issue number21
DOIs
Publication statusPublished - Nov 2009

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

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    Chen, Y. H., Lin, Y., Yoshinaga, A., Chhotani, B., Lorenzini, J. L., Crofts, A. A., Mei, S., Mackie, R. I., Ishino, Y., & Cann, I. K. O. (2009). Molecular analyses of a three-subunit euryarchaeal clamp loader complex from Methanosarcina acetivorans. Journal of bacteriology, 191(21), 6539-6549. https://doi.org/10.1128/JB.00414-09