Abstract/Details

Genetic studies of replication restart in <i>Escherichia coli</i>


2008 2008

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Abstract (summary)

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.

Indexing (details)


Subject
Microbiology
Classification
0410: Microbiology
Identifier / keyword
Biological sciences; DNA replication; Replication restart
Title
Genetic studies of replication restart in <i>Escherichia coli</i>
Author
Boonsombat, Ruethairat
Number of pages
136
Publication year
2008
Degree date
2008
School code
0118
Source
DAI-B 69/12, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
ISBN
9780549915263
Advisor
Sandler, Steven
University/institution
University of Massachusetts Amherst
Department
Microbiology
University location
United States -- Massachusetts
Degree
Ph.D.
Source type
Dissertations & Theses
Language
English
Document type
Dissertation/Thesis
Dissertation/thesis number
3336940
ProQuest document ID
304565668
Copyright
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
http://search.proquest.com/docview/304565668
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