UVM Theses and Dissertations
Format:
Print
Author:
Chen, Jianhong
Dept./Program:
Biochemistry
Year:
2011
Degree:
PhD
Abstract:
Homologous recombination-mediated (HR) DJA double-strand breaks (DSBs) repairing pathway plays plays crucial roles in meiotic sister-chromatid segregation, immune system development, recovery of stalled DNA replication fork upon DNA damage, and telomere length maintenance in the absence of telomerase. Defect in any HR components have been implicated in chromosome aneuploidy, a wide variety of cancers predispostion, and immune disease.
RAD51-catalyzed DNA strand exchange refction occurs as the core reaction in HR-mediated DSBs repair. Rad51-ssDNA filament assembly allosterically activates RAD51's ATPase activity, and is essential for Rad51-catalyzed DNA strand exchange. Previous studies of yeast RAD51 indicated that residue His352 occupies an important position at the filament interface, where it could relay allosteric signals between subunits/active sites. To test this hypothesis, we mutated His352, performed biochemical studies of the mutants, and solved the crystal structure of interactions to salt. At very low salt concentrations, H352A displaces RPA from ssDNA, hydrolyzes ATP, and performs DNA strand exchange on full-length M13 substrates.
These activities disappear at salt concentrations [greater than or equal to] 80 mM NaC1. In contrast, the H352Y mutation stabilizes Rad5I-ssDNA binding, but the binding is uncoupled from catalytic activity at normal salt and pH. H352Y binds to ATP, but shows little ssDNA-dependent ATPase and no DNA strand exchange activity on full-length substrates. H352Y cannot displace RPA from ssDNA, indicating that ATP hydrolysis is important for this activity. Combined, the data suggest that H352Y is stuck in a high-affinity ssDNA-binding state with limited catalytic turnover. This idea is supported by the crystal structure H352Y, which reveals a right-handed helical filament in a high-pitch (130 Å) conformation with P₆(1) symmetry. The helical pitch is identical to that reported Rad51-I345T, a gain-of-function mutant with enhanced ssDNA-binding activity. The H352Y structure differs from I345T in that the former lost the alternative interaction interface.
Meanwhile,3 tumor-derived human RAD51 variants, Dl49N, G15lD, and R150Q, were analyzed with their biochemical behaviors in this study. All the three mutants exhibited comparable DNA strand exchange activity to the wild-type hRad5l under optimum situation. The ATP-dependent steady state ATP hydrolysis rate assay, however, indicated that both Kcat/Km and Km of G151D and R150Q were affected. Consistently, G151D was defective and R150Q showed severely affected DNA strand exchange activity under ATP stringent condition. ssDNA binding profile further revealed some fundamental differences between the 3 mutants and wild-type hRAD51 proteins: the G151D required nucleotide presence to bind to ssDNA while the others didn't; the four hRAD51 proteins differed with each other in their ability in forming the high molecular weight (HMW) and low molecular weight (LMW) co-aggregates of protein/ssDNA complex. Preliminary structural analysis excluded the possibility of mis-folding of the hRAD51 mutants. Interestingly, the three mutations were mapped to the same loop on the surface of monomeric hRAD51 which is responsible for hRAD51/p53 interaction. Therefore, we suggested that the three mutations exert their tumorigenic effect through affecting interaction of hRAD5l with the other proteins or interfering in organization the high order structure of hRAD51 proteins.
RAD51-catalyzed DNA strand exchange refction occurs as the core reaction in HR-mediated DSBs repair. Rad51-ssDNA filament assembly allosterically activates RAD51's ATPase activity, and is essential for Rad51-catalyzed DNA strand exchange. Previous studies of yeast RAD51 indicated that residue His352 occupies an important position at the filament interface, where it could relay allosteric signals between subunits/active sites. To test this hypothesis, we mutated His352, performed biochemical studies of the mutants, and solved the crystal structure of interactions to salt. At very low salt concentrations, H352A displaces RPA from ssDNA, hydrolyzes ATP, and performs DNA strand exchange on full-length M13 substrates.
These activities disappear at salt concentrations [greater than or equal to] 80 mM NaC1. In contrast, the H352Y mutation stabilizes Rad5I-ssDNA binding, but the binding is uncoupled from catalytic activity at normal salt and pH. H352Y binds to ATP, but shows little ssDNA-dependent ATPase and no DNA strand exchange activity on full-length substrates. H352Y cannot displace RPA from ssDNA, indicating that ATP hydrolysis is important for this activity. Combined, the data suggest that H352Y is stuck in a high-affinity ssDNA-binding state with limited catalytic turnover. This idea is supported by the crystal structure H352Y, which reveals a right-handed helical filament in a high-pitch (130 Å) conformation with P₆(1) symmetry. The helical pitch is identical to that reported Rad51-I345T, a gain-of-function mutant with enhanced ssDNA-binding activity. The H352Y structure differs from I345T in that the former lost the alternative interaction interface.
Meanwhile,3 tumor-derived human RAD51 variants, Dl49N, G15lD, and R150Q, were analyzed with their biochemical behaviors in this study. All the three mutants exhibited comparable DNA strand exchange activity to the wild-type hRad5l under optimum situation. The ATP-dependent steady state ATP hydrolysis rate assay, however, indicated that both Kcat/Km and Km of G151D and R150Q were affected. Consistently, G151D was defective and R150Q showed severely affected DNA strand exchange activity under ATP stringent condition. ssDNA binding profile further revealed some fundamental differences between the 3 mutants and wild-type hRAD51 proteins: the G151D required nucleotide presence to bind to ssDNA while the others didn't; the four hRAD51 proteins differed with each other in their ability in forming the high molecular weight (HMW) and low molecular weight (LMW) co-aggregates of protein/ssDNA complex. Preliminary structural analysis excluded the possibility of mis-folding of the hRAD51 mutants. Interestingly, the three mutations were mapped to the same loop on the surface of monomeric hRAD51 which is responsible for hRAD51/p53 interaction. Therefore, we suggested that the three mutations exert their tumorigenic effect through affecting interaction of hRAD5l with the other proteins or interfering in organization the high order structure of hRAD51 proteins.