Abstract
We introduced nine site-directed mutations into seven conserved fission yeast proliferative cell nuclear antigen (PCNA) residues, Leu2, Asp63, Arg64, Gly69, Gln201, Glu259, and Glu260, either as single or as double mutants. Both the recombinant wild type and mutant PCNAs were able to form homotrimers in solution and to sustain growth of a null pcna strain (Deltapcna). Wild type Schizosaccharomyces pombe PCNA and PCNA proteins with mutations in Asp63, Gln201, Glu259, or Glu260 to Ala were able to stimulate DNA synthetic activity and to enhance the processivity of calf thymus DNA polymerase delta holoenzyme similar to calf thymus PCNA. Mutations of Leu2 to Val or Arg64 to Ala, either singly or as a double mutant, yielded PCNA mutant proteins that had reduced capacity in enhancing the processivity of DNA polymerase delta but showed no deficiency in stimulation of the ATPase activity of replication factor C. S. pombe Deltapcna strains sustained by these two mutant-pcna alleles had moderate defects in growth and displayed elongated phenotypes. These cells, however, were not sensitive to UV irradiation. Together, these in vitro and in vivo studies suggest that the side chains of Leu2 and Arg64 in one face of the PCNA trimer ring structure are two of the several sites involved in tethering DNA polymerase delta for processive DNA synthesis during DNA replication.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adenosine Triphosphatases / metabolism
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Alleles
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Amino Acid Sequence
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Animals
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Cattle
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Chromatography, Gel
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Conserved Sequence
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DNA Polymerase III
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DNA-Binding Proteins / metabolism
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DNA-Directed DNA Polymerase / metabolism*
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Genes, Fungal
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Genotype
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Homeodomain Proteins*
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Kinetics
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Macromolecular Substances
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Minor Histocompatibility Antigens
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Models, Structural
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Mutagenesis, Site-Directed
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Point Mutation
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Proliferating Cell Nuclear Antigen / chemistry*
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Proliferating Cell Nuclear Antigen / isolation & purification
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Proliferating Cell Nuclear Antigen / metabolism*
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Protein Structure, Secondary*
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Proto-Oncogene Proteins c-bcl-2*
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Recombinant Proteins / chemistry
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Replication Protein C
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Repressor Proteins*
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Saccharomyces cerevisiae Proteins*
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Schizosaccharomyces / genetics
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Schizosaccharomyces / metabolism*
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Schizosaccharomyces / radiation effects
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Thymus Gland / metabolism
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Ultraviolet Rays
Substances
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BCL2-related protein A1
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DNA-Binding Proteins
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Homeodomain Proteins
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MATA1 protein, S cerevisiae
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Macromolecular Substances
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Minor Histocompatibility Antigens
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Proliferating Cell Nuclear Antigen
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Proto-Oncogene Proteins c-bcl-2
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Recombinant Proteins
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Repressor Proteins
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Saccharomyces cerevisiae Proteins
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DNA Polymerase III
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DNA-Directed DNA Polymerase
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Adenosine Triphosphatases
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Replication Protein C