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.

AbstractHemophilia is treated by IV replacement therapy with Factor VIII (FVIII) or Factor IX (FIX), either on demand to resolve bleeding, or as prophylaxis. Improved treatment may be provided by drugs designed for subcutaneous and less frequent administration with a reduced risk of inhibitor formation. Tissue factor pathway inhibitor (TFPI) down-regulates the initiation of coagulation by inhibition of Factor VIIa (FVIIa)/tissue factor/Factor Xa (FVIIa/TF/FXa). Blockage of TFPI inhibition may facilitate thrombin generation in a hemophilic setting. A high-affinity (KD = 25pM) mAb, mAb 2021, against TFPI was investigated. Binding of mAb 2021 to TFPI effectively prevented inhibition of FVIIa/TF/FXa and improved clot formation in hemophilia blood and plasma. The binding epitope on the Kunitz-type protease inhibitor domain 2 of TFPI was mapped by crystallography, and showed an extensive overlap with the FXa contact region highlighting a structural basis for its mechanism of action. In a rabbit hemophilia model, an intravenous or subcutaneous dose significantly reduced cuticle bleeding. mAb 2021 showed an effect comparable with that of rFVIIa. Cuticle bleeding in the model was reduced for at least 7 days by a single intravenous dose of mAb 2021. This study suggests that neutralization of TFPI by mAb 2021 may constitute a novel treatment option in hemophilia.

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.

All eukaryotic forms of DNA topoisomerase I contain an extensive and highly charged N-terminal domain. This domain contains several nuclear localization sequences and is essential for in vivo function of the enzyme. However, so far no direct function of the N-terminal domain in the in vitro topoisomerase I reaction has been reported. In this study we have compared the in vitro activities of a truncated form of human topoisomerase I lacking amino acids 1-206 (p67) with the full-length enzyme (p91). Using these enzyme forms, we have identified for the first time a direct role of residues within the N-terminal domain in modulating topoisomerase I catalysis, as revealed by significant differences between p67 and p91 in DNA binding, cleavage, Eukaryotic topoisomerase I (topo I)1 is a monomeric enzyme that plays a major role in important cellular processes by regulating the topology of DNA. The enzyme relaxes negative and positive supercoils arising as a consequence of DNA processes such as DNA transcription, replication, recombination, and chromosome condensation (1). Mechanistically, topo I acts by introducing transient singlestrand breaks into the DNA double helix. The catalytic cycle can be subdivided into several steps including: (i) non-covalent DNA binding, (ii) cleavage, (iii) strand rotation, (iv) religation, and (v) enzyme turnover. The cleavage and religation events constitute two reverse phosphoryl transfer (transesterification) reactions. During the cleavage reaction, an active-site tyrosine residue of the enzyme is used as a nucleophile to break a phosphodiester bond of the DNA backbone, generating a covalent enzyme-(3Ј-phosphotyrosyl)-DNA linkage and a free 5Ј-hydroxyl group (2-4). This 5Ј-hydroxyl group provides the nucleophile for the religation reaction that restores intact DNA.The solved crystal structure of an N-terminal-truncated version of the human topo I together with proteolytic analyses show that the enzyme is organized into four structural domains. These consist of an N-terminal domain (amino acids 1-206), a core domain (amino acids 207-635), a linker domain (amino acids 636 -712), and a C-terminal domain (amino acids 713-765) (2, 3). The C-terminal domain contains the active-site tyrosine (Tyr 723 ), which together with the catalytic residues Arg 488 , Lys 532 , Arg 590 , and His 632 of the core domain constitutes the active site of the enzyme (4 -8). Structural data show that the core and C-terminal domains form a clamp structure that wraps around the DNA and, together with the helix-turnhelix linker domain (1), contacts DNA in a region extending 4 base pairs upstream and 9 base pairs downstream of the cleavage site (3). Based on this structural information of the human topo I-DNA complex, a model for strand rotation (topoisomerization) has been proposed. According to this "controlled rotation" model, rotation of the cleaved strand around the intact strand is partially hindered by contacts between the rotating DNA and part of the core and linker domains (3). The involvement of the linker ...

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...

We report that diverse species of bacteria encode a type IB DNA topoisomerase that resembles vaccinia virus topoisomerase. Deinococcus radiodurans topoisomerase IB (DraTopIB), an exemplary member of this family, relaxes supercoiled DNA in the absence of a divalent cation or ATP. DraTopIB has a compact size (346 aa) and is a monomer in solution. Mutational analysis shows that the active site of DraTopIB is composed of the same constellation of catalytic side chains as the vaccinia enzyme. Sequence comparisons and limited proteolysis suggest that their folds are conserved. These findings imply an intimate evolutionary relationship between the poxvirus and bacterial type IB enzymes, and they engender a scheme for the evolution of topoisomerase IB and tyrosine recombinases from a common ancestral strand transferase in the bacterial domain. Remarkably, bacteria that possess topoisomerase IB appear to lack DNA topoisomerase III. The type IB family of DNA topoisomerases includes eukaryotic nuclear topoisomerase I and the topoisomerases of poxviruses (1, 2). The type IB enzymes relax DNA supercoils via a multistep 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. A constellation of conserved amino acid side chains (Arg-130, Lys-167, Lys-220, Arg-223, and His-265 in vaccinia virus topoisomerase) catalyzes the attack of the active site tyrosine nucleophile (Tyr-274) on the scissile phosphodiester. The RKKRH ''catalytic pentad'' is conserved in all members of the topo IB family. The Arg-130, Lys-167, Arg-223, and His-265 components of the pentad each lend a 10 2 -10 5 enhancement of the transesterification rate (3-8). The Int family of site-specific DNA recombinases (now called tyrosine recombinases) use a similar mechanism to catalyze formation and resolution of Holliday junctions. The catalytic pentad of the tyrosine recombinases (RKHRH) characteristically contains a histidine at the third position occupied by lysine in topo IB (9).topo IB and tyrosine recombinases consist of two domains that form a C-shaped protein clamp around duplex DNA. The carboxyl-terminal catalytic domains of topo IB and tyrosine recombinases adopt a common fold composed of eight ␣-helices and a three-strand antiparallel ␤-sheet (6, 10-15). The constituents of the catalytic pentad occupy similar positions in the tertiary structures of topo IB and tyrosine recombinases. Thus, it is proposed that topo IB and tyrosine recombinases evolved from a common ancestral DNA strand transferase (11), in which case we can consider them distinct branches of an enzyme superfamily defined by a DNA-3Ј-phosphotyrosyl intermediate.Remarkably, there is no structural similarity at all between the amino-terminal domains of type IB topoisomerases and those of the tyrosine recombinases. For example, the amino-terminal domain of vaccinia topoisomerase consists almost entirely of ␤-strands, whereas the amino t...

The antitumor compounds camptothecin and its derivatives topotecan and irinotecan stabilize topoisomerase I cleavage complexes by inhibiting the religation reaction of the enzyme. Previous studies, using radiolabeled camptothecin or affinity labeling reagents structurally related to camptothecin, suggest that the agent binds at the topoisomerase I-DNA interface of the cleavage complexes, interacting with both the covalently bound enzyme and with the +1 base. In this study, we have investigated the molecular mechanism of camptothecin action further by taking advantage of the ability of topoisomerase I to couple non-DNA nucleophiles to the cleaved strand of the covalent enzyme-DNA complexes. This reaction of topoisomerase I was originally observed at moderate basic pH where active cleavage complexes mediate hydrolysis or alcoholysis by accepting water or polyhydric alcohol compounds as substitutes for a 5'-OH DNA end in the ligation step. Here, we report that a H2O2-derived nucleophile, presumably, the peroxide anion, facilitates the release of topoisomerase I from the cleavage complexes at neutral pH, and we present evidence showing that this reaction is mechanistically analogous to DNA ligation. We find that camptothecin, topotecan, and SN-38 (the active metabolite of irinotecan) inhibit H2O2 ligation mediated by cleavage complexes not containing DNA downstream of the cleavage site, indicating that drug interaction with DNA 3' to the covalently bound enzyme is not strictly required for the inhibition, although the presence of double-stranded DNA in this region enhances the drug effect. The results suggest that camptothecins prevent ligation by blocking the active site of the covalently bound enzyme.