A conserved domain of the large subunit of replication factor C binds PCNA and acts like a dominant negative inhibitor of DNA replication in mammalian cells.
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.
Fotedar
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R
Institut de Biologie Structurale J.-P. Ebel, Grenoble, France.
Mossi
R
R
Fitzgerald
P
P
Rousselle
T
T
Maga
G
G
Brickner
H
H
Messier
H
H
Kasibhatla
S
S
Hübscher
U
U
Fotedar
A
A
D000595:Amino Acid Sequence; D004261:DNA Replication / Q000502:physiology*; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D003864:Depression, Chemical; D018398:Homeodomain Proteins; D006801:Humans; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000473:pathology; D046911:Macromolecular Substances; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051818:Replication Protein C; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014407:Tumor Cells, Cultured
C096816:BCL2-related protein A1; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D046911:Macromolecular Substances; D015778:Minor Histocompatibility Antigens; D010446:Peptide Fragments; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D051818:Replication Protein C