Abstract
Replication factor C (RF-C), a complex of five polypeptides, is essential for cell-free SV40 origin-dependent DNA replication and viability in yeast. The cDNA encoding the large subunit of human RF-C (RF-Cp145) was cloned in a Southwestern screen. Using deletion mutants of RF-Cp145 we have mapped the DNA binding domain of RF-Cp145 to amino acid residues 369-480. This domain is conserved among both prokaryotic DNA ligases and eukaryotic poly(ADP-ribose) polymerases and is absent in other subunits of RF-C. The PCNA binding domain maps to amino acid residues 481-728 and is conserved in all five subunits of RF-C. The PCNA binding domain of RF-Cp145 inhibits several functions of RF-C, such as: (i) in vitro DNA replication of SV40 origin-containing DNA; (ii) RF-C-dependent loading of PCNA onto DNA; and (iii) RF-C-dependent DNA elongation. The PCNA binding domain of RF-Cp145 localizes to the nucleus and inhibits DNA synthesis in transfected mammalian cells. In contrast, the DNA binding domain of RF-Cp145 does not inhibit DNA synthesis in vitro or in vivo. We therefore conclude that amino acid residues 481-728 of human RF-Cp145 are critical and act as a dominant negative mutant of RF-C function in DNA replication in vivo.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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DNA Replication / physiology*
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DNA, Complementary / genetics
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DNA-Binding Proteins / chemistry
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DNA-Binding Proteins / metabolism*
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Depression, Chemical
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Homeodomain Proteins*
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Humans
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Leukemia-Lymphoma, Adult T-Cell / pathology
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Macromolecular Substances
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Minor Histocompatibility Antigens
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Molecular Sequence Data
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Peptide Fragments / metabolism
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Proliferating Cell Nuclear Antigen / metabolism*
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Protein Binding
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Protein Conformation
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Protein Structure, Tertiary
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Proto-Oncogene Proteins c-bcl-2*
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Recombinant Fusion Proteins / metabolism
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Replication Protein C
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Repressor Proteins*
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins*
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Sequence Alignment
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Sequence Deletion
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Sequence Homology, Amino Acid
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Transfection
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Tumor Cells, Cultured
Substances
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BCL2-related protein A1
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DNA, Complementary
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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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Peptide Fragments
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Proliferating Cell Nuclear Antigen
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Proto-Oncogene Proteins c-bcl-2
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Recombinant Fusion Proteins
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Repressor Proteins
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Saccharomyces cerevisiae Proteins
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Replication Protein C