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In this paper we report the cloning and sequencing of two small plasmids, pTAUp and pTADw, from the Antarctic Gram-negative Psychrobacter sp strain TA144. The observation that pTAUp contains a putative Rep-coding gene (Psyrep) suggested that its duplication occurs via a rolling-circle replication mechanism. This hypothesis was confirmed by the identification of the pTAUp single-stranded DNA form. The putative pTAUp plus origin of replication was found at the 3′ end of the Psyrep by using an in vivo complementation assay. Structural similarities at the level of (i) gene organization, (ii) protein sequence, and (iii) nick site sequences strongly suggest that the psychrophilic enzyme belongs to a new subfamily of replication enzymes.