The Mre11/Rad50 complex has been implicated in the early steps of DNA double-strand break (DSB) repair through homologous recombination in several organisms. However, the enzymatic properties of this complex are incompatible with the generation of 3’ single-stranded DNA for recombinase loading and strand exchange. In thermophilic Archaea, the mre11 and rad50 genes cluster in an operon with genes encoding a bidirectional DNA helicase, HerA, and a 5’ to 3’ exonuclease, NurA, suggesting these four enzymes function in a common pathway. I show that purified Mre11 and Rad50 from Pyrococcus furiosus act cooperatively with HerA and NurA to resect the 5’ strand at a DNA end under physiological conditions in vitro where HerA and NurA alone do not show detectable activity. Furthermore, I demonstrate that HerA and NurA physically interact, and this interaction stimulates both helicase and nuclease activities. The products of HerA/NurA long-range resection are oligonucleotide products and HerA/NurA activity demonstrates both sequence specificity and a preference to cut at a specific distance from the DNA end. I demonstrate a novel activity of Mre11/Rad50 to make an endonucleolytic cut on the 5’ strand, which is consistent with a role for the Mre11 nuclease in the removal of 5’ protein conjugates. I also show that Mre11/Rad50 stimulates HerA/NurA-mediated resection through two different mechanisms. The first involves an initial Mre11 nucleolytic processing event of the DNA to generate a 3’ ssDNA overhang, which is then resected by HerA/NurA in the absence of Mre11/Rad50. The second mechanism likely involves local unwinding of the DNA end in a process dependent on Rad50 ATPase activity. I propose that this unwinding step facilitates binding of HerA/NurA to the DNA end and efficient resection of the break. Furthermore, the binding affinity of NurA for 3’ overhang and unwound DNA end substrates partially explains the efficiency of the two resection mechanisms. Lastly, 3’ single-stranded DNA generated by these enzymes can be used by the Archaeal RecA homolog RadA to catalyze strand exchange. This work elucidates how the conserved Mre11/Rad50 complex promotes DNA end resection in Archaea, and may serve as a model for DSB processing in eukaryotes. / text
| Identifer | oai:union.ndltd.org:UTEXAS/oai:repositories.lib.utexas.edu:2152/ETD-UT-2010-05-1001 |
| Date | 26 January 2011 |
| Creators | Hopkins, Ben Barrett |
| Source Sets | University of Texas |
| Language | English |
| Detected Language | English |
| Type | thesis |
| Format | application/pdf |
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