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 DNAbinding protein by Southwestern screening of a murine cDNA library with a double-stranded oligonucleotide containing the sequence from the bidirectional promoter of the al and a2 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 (Al, 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 Al complex, indicating that it is the murine counterpart of the Al 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 Al 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.DNA replication is a highly controlled process regulated by a large number of proteins and enzymes which form multiple protein-DNA and protein-protein complexes (1). The DNA polymerases involved in replication have the ability to rapidly duplicate very long stretches of DNA without dissociation (1). Reconstitution experiments using purified human and yeast proteins have revealed that the ability of DNA polymerases 8 and e to replicate large stretches of DNA is not inherent in the DNA polymerases but rather is due to a number of associated accessory proteins dedicated to maintaining the processive action of these DNA polymerases (2-4).The use of multisubunit proteins to clamp the DNA polymerase onto primed DNA is not restricted to eukaryotes but is a universal process, also occurring in bacteriophage T4 and Escherichia coli (reviewed in refs. 5-7). In each system a multisubunit polymerase accessory protein complex [eukaryotic activator 1 (Al, also called replication factor C, RF-C), T4 gene products 44/62 (g44/62), E. coli ycomplex] functions to recognize the primed template and load a second polymerase accessory protein [eukaryotic proliferating-cell nuclear antigen (PCNA), T4 g45, E. coli /3] on the DNA in the presence of ATP. In E. coli, the y complex (y66'x subunits) places a dimer of the clamp protein (,8 subunit) on the DNA, which then recruits the DNA polymerase III core (8-10). The (3 dimer has been shown to form a ring-shaped clamp around the DNA (11). In T4, ATP is required for the association of the g44/62 (4:1) complex and the g45 protein on primed DNA, and ATP hydrolysis is required to add the g43 DNA poly-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise...

A Mammalian Bromodomain Protein, Brd4, Interacts with Replication Factor C and Inhibits Progression to S Phase

“…Among several proteins tested, we found that RFC-140, the large subunit of RFC (4,5,44), interacts with Brd4. Extracts from NIH 3T3 cells were first precipitated with anti-Brd4 antibody, and the precipitated materials were tested for RFC-140 by immunoblot analysis.…”

The Dna Replication Fork in Eukaryotic Cells

“…Functional homologs exist in bacteria, some bacteriophages, and Archea (95). The cDNAs encoding the individual subunits of human and the yeast S. cerevisiae RFC have been cloned, and all five yeast genes are essential for cell viability (96)(97)(98)(99)(100)(101)(102)(103)(104). Sequence comparisons show a high degree of similarity among all five subunits, and based on these similarities and the conserved sequences found among species, short stretches of amino acid sequences called RFC boxes I-VIII have been defined (104).…”

Clamping down on clamps and clamp loaders

“…To support these affirmations, the cloning and expression RF-C, PCNA, RP-A and ATP selectively stimulates DNA syn-of the large subunit of RF-C revealed that this subunit preferenthesis by polymerases δ and ε [26]. The possibility to isolate tially binds to dsDNA compared with ssDNA [18,31,32]. stable polymerases δ and ε holoenzymes, assembled on DNA by Using a modified PCNA form that could be phosphorylated RF-C, PCNA and ATP, suggests a direct interaction of the auxil-in vitro and a dsDNA template with a defined gap of 45 nucleoiary proteins with the polymerases [26].…”