Identification and in vitro deoxynucleotidylation of the terminal protein of the linear plasmid pAL1 of Arthrobacter nitroguajacolicus Rü61a.

The plasmid pAL1 of Arthrobacter nitroguajacolicus Rü61a is a linear replicon, characterized by inverted terminal repeats and terminal proteins (TPs) covalently bound to its 5'-ends. Previous sequence analysis and predictions of possible secondary structures formed by telomeric 3'-overhangs indicated significant differences of the 'left' and 'right' telomere of pAL1, raising the question of whether each terminus is recognized by a specific protein. The genes pAL1.102 and pAL1.103, located close to a terminus, code for possible DNA-binding proteins; however, only the pORF102 protein encoded by pAL1.102 shows a weak similarity to known TPs of Streptomyces linear replicons. pORF102, purified from recombinant A. nitroguajacolicus Rü61a as a fusion with maltose-binding protein (MBP), was specifically associated with terminal pAL1 DNA, whereas MBP-pORF103 was devoid of DNA, suggesting that pORF102 represents the protein attached to both ends of the linear plasmid. In electrophoretic mobility shift assays, the MBP-pORF102 protein was not capable of specifically recognizing telomeric DNA sequences. Consistent with its proposed role as a protein primer in DNA synthesis, pORF102 was deoxynucleotidylated in vitro with dCMP, complementary to the 3'-ends (... GCAGG) of pAL1.