Mutational analysis of the specific priming signal essential for DNA replication of the broad host-range plasmid RSF1010.
To analyze the RSF1010-specific priming mechanism, a library of randomly mutagenized ssiA sequences was constructed by chemical synthesis using mixed nucleotide phosphoramidites. Synthetic ssiA sequences with the single base-substitutions were assayed for the SSI activity in E. coli JM109 expressing RepB' primase. It was demonstrated that the activity of ssiA was damaged markedly by single base-substitutions within the possible stem-loop structure and its 3'-flanking region. It is conceivable that these domains are critical in recognition and primer synthesis by RepB' primase.
Honda
Y
Y
Department of Agricultural Chemistry, Kyoto University, Japan.
Akioka
T
T
Takebe
S
S
Tanaka
K
K
Miao
D
D
Higashi
A
A
Nakamura
T
T
Taguchi
Y
Y
Sakai
H
H
Komano
T
T
D001482:Base Composition; D001483:Base Sequence; D004261:DNA Replication / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D015183:Restriction Mapping
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides