A ROLLING CIRCLE REPLICATION INITIATOR PROTEIN WITH A NUCLEOTIDYL-TRANSFERASE ACTIVITY ENCODED BY THE PLASMID-PGT5 FROM THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS-ABYSSI

The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi presents similarities to plasmids from the pC194 family that replicat e by the rolling circle mechanism. These plasmids encode a replication initiator protein, which activates the replication origin by nicking one of the two DNA strands. The gene encoding the putative Rep protein of pGT5 (Rep75) has been cloned and overexpressed in Escherichia coli , and the recombinant protein has been purified to homogeneity. Rep75 exhibits a highly thermophilic nicking-closing activity in vitro on si ngle-stranded oligonucleotides containing the putative double-stranded replication origin sequence of pGT5. Gel shift analyses on single-str anded oligonucleotides indicate that Rep75 recognizes the single-stran ded DNA region upstream of the nicking site via non-covalent interacti on and remains covalently linked to the 5'-phosphate of the downstream fragment after nicking. Besides these expected activities, Rep75 cont ains a dATP (and ATP) terminal transferase activity at the 3'-OH extre mity of the nicking site, which had not been reported previously for p roteins of this type. Rep75, which is the first replication initiator protein characterized in an archaeon, offers an attractive new model f or the study of rolling circle replication.