The Escherichia coli (E. coli) DNA replication is initiated at the origin of replication, oriC. The newly replicated DNA are hemimethylated, where the parental DNA strand is methylated and the new DNA strand is unmethylated. The new DNA remains hemimethylated for up to 1/3 of the cell cycle by a process known as sequestration. The SeqA protein has been identified as an important factor in sequestration, by binding to the hemimethylated origins and preventing reinitiation of replication. The SeqA protein is therefore known as a negative modulator of initiation of replication in E.coli.
It has been assumed that SeqA does not operate alone, and perhaps there are proteins that interact with it, thus promoting SeqA’s functions. Biochemical experiments have identified three membrane proteins that were present in wild type E. coli cells, but not in ∆seqA mutant cells. These proteins might be potential interacting partners for the SeqA protein.
Here we have used a genetic screen as an independent method to attempt to identify SeqA interacting partners. The genetic screen involved making an E.coli genomic library and transforming it into a temperature-sensitive strain with double mutation, seqA4∆recA. The seqA4∆recA strain was viable at 42°C, but not at 30°C. DNA fragments from the genomic library were cloned into a multicopy plasmid and transformants were then selected at 30°C. The cells that would form colonies at 30°C would therefore contain a multicopy suppressor gene coding for a SeqA interacting protein. We found that the seqA4∆recA strain accumulated suppressors when it was transformed with an empty plasmid (background frequency). This made the screen extremely challenging. The suppression-mediated growth was observed after 2 days of incubation.
Furthermore, some preliminary results were found. During investigation of the background frequency of suppression, an interesting phenotype was observed where the vector (pUN121) was reduced in size. It is suggested that this suppressor mutation may affect replication fork repair or degradation.