The process of homologous recombination promotes error-free repair of double-strand breaks and is essential for meiosis. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Herein, we review recent genetic, biochemical, and structural analyses of the genes and proteins involved in recombination.
Type IB topoisomerases and tyrosine recombinases are structurally homologous strand transferases that act through DNA-(3'-phosphotyrosyl)-enzyme intermediates. A constellation of conserved amino acids (Arg-130, Lys-167, Arg-223, and His-265 in vaccinia topoisomerase) catalyzes transesterification of tyrosine to the scissile phosphodiester. We used 5'-bridging phosphorothiolate-modified DNAs to implicate Lys-167 as a general acid catalyst. The lower pKa of the 5'-S leaving group versus 5'-O restored activity to the K167A mutant, whereas there was no positive thio effect for mutants R223A and H265A. The lysine is located atop a flexible hairpin loop, and it shifts into the minor groove upon DNA binding. Coupling of conformational changes in a general acid loop to covalent catalysis of phosphoryl transfer is one of several mechanistic features shared by the topoisomerase/recombinase and protein phosphatase superfamilies.
The Mre11-Rad50-Xrs2 complex is involved in DNA double-strand break repair, telomere maintenance, and the intra-S phase checkpoint. The Mre11 subunit has nuclease activity in vitro, but the role of the nuclease in DNA repair and telomere maintenance remains controversial. We generated six mre11 alleles with substitutions of conserved residues within the Mre11-phosphoesterase motifs and compared the phenotypes conferred, as well as exonuclease activity and complex formation, by the mutant proteins. Substitutions of Asp16 conferred the most severe DNA repair and telomere length defects. Interactions between Mre11-D16A or Mre11-D16N and Rad50 or Xrs2 were severely compromised, whereas the mre11 alleles with greater DNA repair proficiency also exhibited stable complex formation. At all of the targeted residues, alanine substitution resulted in a more severe defect in DNA repair compared to the more conservative asparagine substitutions, but all of the mutant proteins exhibited ,2% of the exonuclease activity observed for wild-type Mre11. Our results show that the structural integrity of the Mre11-Rad50-Xrs2 complex is more important than the catalytic activity of the Mre11 nuclease for the overall functions of the complex in vegetative cells.
Type IB topoisomerases cleave and rejoin DNA through a DNA-(3-phosphotyrosyl)-enzyme intermediate. A constellation of conserved amino acids (Arg-130, Lys-167, Arg-223, and His-265 in vaccinia topoisomerase) catalyzes the attack of the tyrosine nucleophile (Tyr-274) at the scissile phosphodiester. Previous studies implicated Arg-223 and His-265 in transition state stabilization and Lys-167 in proton donation to the 5-O of the leaving DNA strand. Here we find that Arg-130 also plays a major role in leaving group expulsion. The rate of DNA cleavage by vaccinia topoisomerase mutant R130K, which was slower than wild-type topoisomerase by a factor of 10 ؊4.3 , was stimulated 2600-fold by a 5-bridging phosphorothiolate at the cleavage site. The catalytic defect of the R130A mutant was also rescued by the 5-S modification (190-fold stimulation), albeit to a lesser degree than R130K. We surmise that Arg-130 plays dual roles in transition state stabilization and general acid catalysis. Whereas the R130A mutation abolishes both functions, R130K permits the transition state stabilization function (via contact of lysine with the scissile phosphate) but not the proton transfer function. Our results show that the process of general acid catalysis is complex and suggest that Lys-167 and Arg-130 comprise a proton relay from the topoisomerase to the 5-O of the leaving DNA strand.Type IB DNA topoisomerases relax DNA supercoils via a reaction pathway entailing noncovalent binding of the enzyme to duplex DNA, cleavage of one DNA strand with formation of a covalent DNA-(3Ј-phosphotyrosyl)-protein intermediate, strand passage, and strand religation (1, 2). Tyrosine recombinases use a similar transesterification mechanism to form and resolve Holliday junctions. The catalytic domains of topo 1 IB and tyrosine recombinases adopt a common fold composed of eight ␣ helices and a three-stranded antiparallel  sheet (3-9). The constituents of the active site occupy similar positions in the topo IB and recombinase tertiary structures.Four conserved amino acid side chains (e.g. Arg-130, Lys-167, Arg-223, and His-265 in the vaccinia topoisomerase) catalyze the attack of the active site tyrosine nucleophile (Tyr-274) on the scissile phosphodiester (10 -12). Mutational, stereochemical, and structural data for vaccinia and nuclear topoisomerase IB and tyrosine recombinases suggest that the two arginines and the histidine contact the nonbridging oxygens of the scissile phosphodiester and that these interactions serve to stabilize a proposed pentacoordinate phosphorane transition state (3, 5, 9, 10 -13).Recently we used 5Ј-bridging phosphorothiolate-modified DNAs to implicate Lys-167 of vaccinia topoisomerase as a general acid catalyst of the DNA cleavage reaction (14). The hypothesis was that if the expulsion of the 5Ј-oxygen of the leaving DNA strand was indeed catalyzed by a general acid on the topoisomerase, then the requirement for the general acid ought to be alleviated by introducing a 5Ј-bridging phosphorothiolate at the scissile phosphodiest...
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