Molecular cloning and expression of the Saccharomyces cerevisiae RFC3 gene, an essential component of replication factor C

Xiangyang Li, Peter M.J. Burgers

Research output: Contribution to journalArticle

55 Scopus citations

Abstract

Yeast replication factor C (RF-C) is a multipolypeptide complex required for processive DNA replication by DNA polymerases δ and ε. The gene encoding the 40-kDa subunit of the Saccharomyces cerevisiae RF-C (RFC3) has been cloned. The RFC3 gene is required for yeast cell growth and has been mapped to the left arm of chromosome XIV. The deduced amino acid sequence of the RFC3 gene shows a high homology to the 36-, 37-, and 40-kDa subunits of human RF-C (also called activator 1), with the highest homology to the 36- kDa subunit. Among the conserved regions are the A motif of ATP binding proteins; the 'DEAD box,' common to DNA helicases and other ATPases; and the 'RFC box,' an ≃15-amino acid domain virtually identical in the yeast and human RF-C subunits. Limited homology to the functional homologs of the Escherichia coli replication apparatus was also observed. The steady-state mRNA levels of RFC3 do not change significantly during the mitotic cell cycle of yeast. The intact form of the RFC3 gene product (Rfc3p) has been overproduced in E. coli and purified to homogeneity. Purified Rfc3p has an ATPase activity that is markedly stimulated by single-stranded DNA but not by double-stranded DNA or RNA.

Original languageEnglish
Pages (from-to)868-872
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number3
DOIs
StatePublished - Feb 1 1994

Keywords

  • DNA polymerase δ
  • DNA polymerase ε
  • DNA replication
  • proliferating cell nuclear antigen

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