Abstract
SeqA is a protein that negatively regulates replication initiation in Escherichia coli by binding to hemimethylated GATC sites near the origin of replication. Several functions of SeqA have been well characterized, but some of the biochemical mechanisms for these functions have not been examined in detail. In order to study some of these mechanisms, we created several mutants using the Linker Scan method, as well as some site directed mutants and XL1-Red mutagenized mutants. These mutants were screened for sensitivity to DNA damaging agents. The DNA binding and dimerization abilities were then studied using flow cytometry, fluorescence microscopy, and pull down interaction experiments. This study yielded several mutants of interest, primarily using the Linker Scan method, which disturb the structure in separate locations in the protein: one elongates the N-terminal protein binding domain, another eliminates the C-terminal DNA binding domain entirely, and two appear to disrupt the structure in a small part of the C-terminal domain. These mutants will be useful for further studies of SeqA that measure the relative importance of the different domains to better understand the overall mechanisms and functions of the protein.