Genetic studies of replication restart in Escherichia coli

Faithful DNA replication is essential for all organisms to maintain genetic integrity. During the DNA replication, replications forks are frequently stalled or collapsed due to the encounter of DNA lesions or blocking proteins. These events can occur anywhere on the chromosome which is away from the origin of replication. For survival, cells require a number of proteins to repair the damages and restart the replication near or at the damaged site. In Escherichia coli, a group of proteins called primosomal proteins consisting of PriA, PriB, PriC, Rep, DnaT, DnaC, DnaB and DnaG are required for directing DnaB replicative helicase back onto DNA substrates. The main difference between chromosomal replication at oriC and replication restart is the former process is initiated by DnaA recognizing a specific sequence of oriC while the latter process can be initiated by PriA recognizing a specific DNA structure. The mechanism of replication restart is highly ordered and well regulated, and to date, this mechanism has yet to be fully understood. This lab uses a genetic tool to understand replication restart in vivo and discovered that replication restart in E. coli can be explained by a multiple replication restart pathway model (Sandler, 2000). This dissertation presents work that advances our knowledge of replication restart by studying Rep and PriB using a genetic approach. Although it has been shown that Rep and PriB are important for replication restart, the contribution of these two proteins in vivo is still not clearly understood. In the case of Rep, this study provided the characterization of three rep mutants: a rep null mutant, a rep defective in a regulation of Rep monomer helicase activity and a rep ATP hydrolysis deficient mutant. Although these rep mutants showed a similar phenotype, there were some differences such as the phenotypes when combined with other mutations and spontaneous suppressors. In this study, a rep null mutant exhibited an unexpected phenotype including high basal levels of SOS expression and cell filamentation when combined with priB. The priB rep double mutant developed a spontaneous suppressor mapped in dnaC region. A rep mutant defective in an autoinhibition of Rep monomer helicase activity showed a more detrimental phenotype than a rep null mutant when combined with priB suggesting that this mutant Rep inappropriately removes PriC and completes with PriA. The other rep mutant, an ATPase deficient mutant, showed a similar phenotype to that of a rep null mutant as a single mutant and a double mutant with priB, however, spontaneous suppressors developed in the regions close to priC and dnaC, but not in the either of these genes. Lastly, PriB mutants that biochemical evidence has shown some effects on PriA, ssDNA and DnaT binding were tested for effects on replication restart in vivo. The studies of these PriB mutants lead to a model of the regulation of replication restart via PriA-PriB pathway which can be explained by a hand-off mechanism for primosomal assembly

BIOACTIVE ACTIVITY OF A RECOMBINANT LONGAN (Dimocarpus longan LOUR.) SEED PEPTIDE

Background: Consumption of antioxidants has been evident to prevent diseases caused by free radicals damage. Antioxidants can be found in the form of peptide in various natural sources. From our previous study, to overcome obstacles of direct longan seed hydrolysate extraction, the recombinant Longan1 peptide which contains 4 repeats of ISYVVPVYIAEITPKTFRGGF linked by D was produced from Escherichia coli. The in vitro bioactive properties of this recombinant peptide were characterized.  Methods: The recombinant and chemically synthesized Longan1 peptides were tested for bioactive activity including, DPPH, ABTS, and nitric oxide radical scavenging assays, the ability to protect plasmid DNA from hydroxyl radicals, anti-proliferative activity to several cancer cell lines, and antiinflammatory effects in cell culture level. Results: The recombinant peptide revealed antioxidative activities, including DPPH, ABTS, and nitric oxide radical scavenging activity, which are similar to the chemically-synthesized one. However, the recombinant peptide exhibited higher in vitro ability to protect DNA from hydroxyl radicals. The IC50 value of the recombinant Longan1 peptide could only be calculated through the assay of antiproliferation of stomach KATO-III cancer cell line, while IC50 value from the chemically synthesized peptide could not be calculated in any tested cell lines. Finally, the anti-inflammatory effect determined by the inhibition of nitric oxide production from macrophages RAW 264.7 activated by LPS revealed that the recombinant Longan1 peptide could inhibit nitric oxide production from macrophage cells, whereas the chemically-synthesized one could not. Conclusion: With all these properties, the recombinant Longan1 peptide seems to have bioactivity that can possibly be a candidate for further medical application or supplementary products. &nbsp

A novel dnaC mutation that suppresses priB rep mutant phenotypes in Escherichia coli K-12

973-98

Localization of RecA in Escherichia coli K-12 using RecA-GFP

1074-108