Browsing by Subject "Rad50"

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Effect of nucleotide binding on Rad50 conformational state, multimeric state, and DNA binding ability
(2009-05) Estrin, Eric; Tanya T. Paull
The ability of an organism to swiftly repair double-stranded breaks (DSBs) in DNA is a crucial process that, if absent, can result in genomic instability. The Mre11/Rad50 protein complex is highly conserved and plays a key role in sensing, processing, and repairing DNA DSBs. Rad50 is known to be necessary for the DNA end-bridging catalyzed by the MRN complex, and association of the broken DNA ends is essential to prevent loss of chromosome fragments in vivo. The Rad50 protein contains a large coiled-coil domain and ATP-binding motifs, with an overall structure similar to the Structural Maintenance of Chromosomes (SMC) family of proteins. Rad50 undergoes ATP-dependent homodimerization, which creates a potential DNA binding cleft. Recently, Rad50 has been shown to have adenylate kinase activity in addition to the previously known ATPase activity. It is still unknown what role ATP-induced dimerization and conformational changes play in pfRad50’s various activities. We hypothesize that dimerization is needed for DNA binding, and that Rad50 requires large conformational changes for proper function in its part in pfMR exonuclease activity. Here, we use site-directed mutagenesis to create Rad50 mutants that have cysteines placed in structurally relevant portions of the protein. With these cysteines, we used disulfide crosslinking, fluorescence, and Fluorescence Resonance Energy Transfer (FRET) to detect if changes in conformation or multimeric state of Rad50 are necessary for adenylate kinase activity, ATPase activity, and DNA binding.
Investigation of the mechanistic basis for the role of Rad50 in double-strand break repair
(2006) Bhaskara, Venugopal; Paull, Tanya T.
Members of the Rad52 epistasis group, which includes a heterotrimeric complex, formed by Mre11, Rad50 and Nbs1 (Xrs2 in yeast) helps in the protection of cell’s genetic content from DNA doublestrand breaks. The Rad50 component of the human Mre11/Rad50/Nbs1 (Xrs2 in yeast) complex (MRN(X)) belongs to the ABC superfamily of ATPases and is conserved among all organisms and contains Walker A (N-Terminus) and Walker B (C-Terminus) ATPase domains connected by a long coiled-coil region. We show for the first time that Rad50 shows adenylate kinase activity (ATP + AMP ↔ 2ADP) and that this activity is important for tethering of DNA ends. We further show that Rad50 can catalyze “reverse” adenylate kinase activity (2ADP → ATP + AMP) and this activity is stimulated in the presence of linear DNA ends. We also show that the signature motif of Rad50 is essential for all ATP-dependent activities in vivo and in vitro.
The P. furiosus Mre11/Rad50 complex facilitates 5’ strand resection by the HerA helicase and NurA nuclease at a DNA double-strand break
(2010-05) Hopkins, Ben Barrett; Paull, Tanya T.; Dudley, Jaquelin P.; Graham, David E.; Jayaram, Makkuni; Yin, Whitney Y.
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.