The bacteria Deinococcus radiodurans (D. rad) is able to survive multiple double stranded breaks to its DNA with no detriment to its health as its genome can be fully repaired in a matter of hours. During bacterial DNA replication, points of DNA damage can cause replication to cease, requiring an origin independent replisome loading pathway to resume the replication. This pathway is called replication restart , a highly conserved pathway in bacteria. In the E. coli replication restart pathway primosome proteins function by binding to the DNA fork and facilitating the loading of the DnaB helicase onto the replication fork. The E. coli primosome protein A (PriA) acts as a helicase to unwind a portion of the double stranded DNA at forks without leading strand gaps, while primosome protein C helps facilitate DnaB loading at replication forks with a leading strand gap. Previous experiments have shown that the D. rad PriA does not seem to have any activity as a helicase. Additionally, the D. rad PriA is much larger than other bacterial PriAs indicating that it may have a different functionality. D. rad does not have a PriC protein and because replication restart pathways are crucial to the health of bacterial cells it is possible that D. rad PriA might have additional functionality to compensate for the lack of PriC. The purpose of this project was to test the ability of the D. rad replication restart proteins to load D. rad DnaB onto DNA forks with a leading strand gap, an activity that PriC would accomplish in E. coli. The required proteins were cultured in specialized E. coli bacteria and then extracted and purified. Helicase assays were then used to test the ability of PriA to load DnaB onto DNA forks with a leading strand gap with the results indicating that no DnaB-mediated unwinding occurred.
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Chemistry | Physical Sciences and Mathematics
Ryan, Michael, "Investigating Bacterial Replication Restart: Can D.rad PriA Load D.rad DnaB onto DNA Forks with a Leading Strand Gap?" (2014). Honors Theses. 31.