CLONING OF THE LARGE SUBUNIT OF ACTIVATOR-1 (REPLICATION FACTOR-C) REVEALS HOMOLOGY WITH BACTERIAL-DNA LIGASES

We have cloned a gene encoding a DNA-binding protein by Southwestern s creening of a murine cDNA library with a double-stranded oligonucleoti de containing the sequence from the bidirectional promoter of the alph a1 and alpha2 collagen IV genes. The middle portion of this 1131-amino acid protein has a region homologous to bacterial DNA ligases, and th e more carboxyl portion contains several domains homologous to p40, p3 8, p37, and p36.5 subunits of activator 1 (A1, also called replication factor C), a human replication protein complex. Western blotting reve aled 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 p14 5. Characterization of the DNA-binding activity of the recombinant fus ion protein by gel mobility-shift assay revealed that it had a prefere nce for a run of pyrimidines on one strand. Deletion analysis using re combinant proteins revealed that the DNA ligase-like domain was requir ed 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 prote ins in recognizing DNA.