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.
Institute of Molecular Microbiology and Biotechnology, University of Münster, Münster, Germany.
Stephan
S
Kolkenbrock
Susanne
S
Fetzner
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D003853:Deoxyribonucleosides / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D003853:Deoxyribonucleosides; D011993:Recombinant Fusion Proteins