title: Cloning of the large subunit of activator 1 (replication factor C) reveals homology with bacterial DNA ligases.
abstract: We have cloned a gene encoding a DNA-binding protein by Southwestern screening of a murine cDNA library with a double-stranded oligonucleotide containing the sequence from the bidirectional promoter of the alpha 1 and alpha 2 collagen IV genes. The middle portion of this 1131-amino acid protein has a region homologous to bacterial DNA ligases, and the more carboxyl portion contains several domains homologous to p40, p38, p37, and p36.5 subunits of activator 1 (A1, also called replication factor C), a human replication protein complex. Western blotting revealed that antiserum generated against part of the recombinant protein reacted specifically with the 145-kDa component of the purified human A1 complex, indicating that it is the murine counterpart of the A1 p145. Characterization of the DNA-binding activity of the recombinant fusion protein by gel mobility-shift assay revealed that it had a preference for a run of pyrimidines on one strand. Deletion analysis using recombinant proteins revealed that the DNA ligase-like domain was required for DNA-binding activity. The finding that the region required for the binding of murine A1 p145 to DNA has similarity to a domain found in DNA ligases suggests that this region may be utilized by both proteins in recognizing DNA.
authors.affiliation: Laboratory of Developmental Biology, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892.
authors.forename: P D
authors.initials: PD
authors.lastname: Burbelo
authors.forename: A
authors.initials: A
authors.lastname: Utani
authors.forename: Z Q
authors.initials: ZQ
authors.lastname: Pan
authors.forename: Y
authors.initials: Y
authors.lastname: Yamada
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003094:Collagen / Q000235:genetics; D011088:DNA Ligases / Q000737:chemistry / Q000235:genetics*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D004622:Embryo, Mammalian; D006367:HeLa Cells; D018398:Homeodomain Proteins; D006801:Humans; D007106:Immune Sera; D015151:Immunoblotting; D046911:Macromolecular Substances; D051379:Mice; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D019253:Proto-Oncogene Proteins c-bcl-2; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid
chemical_list: C096816:BCL2-related protein A1; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D007106:Immune Sera; C096063:MATA1 protein, S cerevisiae; D046911:Macromolecular Substances; D015778:Minor Histocompatibility Antigens; D009838:Oligodeoxyribonucleotides; D019253:Proto-Oncogene Proteins c-bcl-2; D012097:Repressor Proteins; C497566:Rfc2 protein, mouse; C497567:Rfc3 protein, mouse; C497568:Rfc4 protein, mouse; C497569:Rfc5 protein, mouse; D029701:Saccharomyces cerevisiae Proteins; D003094:Collagen; D051818:Replication Protein C; D011088:DNA Ligases