▪ Abstract DNA topoisomerases solve the topological problems associated with DNA replication, transcription, recombination, and chromatin remodeling by introducing temporary single- or double-strand breaks in the DNA. In addition, these enzymes fine-tune the steady-state level of DNA supercoiling both to facilitate protein interactions with the DNA and to prevent excessive supercoiling that is deleterious. In recent years, the crystal structures of a number of topoisomerase fragments, representing nearly all the known classes of enzymes, have been solved. These structures provide remarkable insights into the mechanisms of these enzymes and complement previous conclusions based on biochemical analyses. Surprisingly, despite little or no sequence homology, both type IA and type IIA topoisomerases from prokaryotes and the type IIA enzymes from eukaryotes share structural folds that appear to reflect functional motifs within critical regions of the enzymes. The type IB enzymes are structurally distinct from a...

DNA topoisomerases: structure, function, and mechanism.

DNA topoisomerases are the magicians of the DNA world — by allowing DNA strands or double helices to pass through each other, they can solve all of the topological problems of DNA in replication, transcription and other cellular transactions. Extensive biochemical and structural studies over the past three decades have provided molecular models of how the various subfamilies of DNA topoisomerase manipulate DNA. In this review, the cellular roles of these enzymes are examined from a molecular point of view.

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Cellular roles of DNA topoisomerases: a molecular perspective.

DNA topoisomerases and their poisoning by anticancer and antibacterial drugs.

Homologous recombination (HR) serves to eliminate deleterious lesions, such as double-stranded breaks and interstrand crosslinks, from chromosomes. HR is also critical for the preservation of repli- cation forks, for telomere maintenance, and chromosome segrega- tion in meiosis I. As such, HR is indispensable for the maintenance of genome integrity and the avoidance of cancers in humans. The HR reaction is mediated by a conserved class of enzymes termed recombinases. Two recombinases, Rad51 and Dmc1, catalyze the pairing and shuffling of homologous DNA sequences in eukaryotic cells via a filamentous intermediate on ssDNA called the presynaptic filament. The assembly of the presynaptic filament is a rate-limiting process that is enhanced by recombination mediators, such as the breast tumor suppressor BRCA2. HR accessory factors that facil- itate other stages of the Rad51- and Dmc1-catalyzed homologous DNA pairing and strand exchange reaction have also been identified. Recent progress on elucidating the mechanisms of action of Rad51 and Dmc1 and their cohorts of ancillary factors is reviewed here.

https://www.med.nyu.edu/klein/PDFs/annurev.biochem.77.061306.125255.pdf

Mechanism of eukaryotic homologous recombination.

DNA double-strand breaks (DSBs) are cytotoxic lesions that can result in mutagenic events or cell death if left unrepaired or repaired inappropriately. Cells use two major pathways for DSB repair: nonhomologous end joining (NHEJ) and homologous recombination (HR). The choice between these pathways depends on the phase of the cell cycle and the nature of the DSB ends. A critical determinant of repair pathway choice is the initiation of 5′-3′ resection of DNA ends, which commits cells to homology-dependent repair, and prevents repair by classical NHEJ. Here, we review the components of the end resection machinery, the role of end structure, and the cell-cycle phase on resection and the interplay of end processing with NHEJ.

Double-Strand Break End Resection and Repair Pathway Choice

▪ Abstract 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.

Recombination proteins in yeast.

Catalytic mechanism of DNA topoisomerase IB.

Hemostatic effect of a monoclonal antibody mAb 2021 blocking the interaction between FXa and TFPI in a rabbit hemophilia model.

THE RAD50, XRS2, and MRE11 genes were first identified in screens for ionizing radiation (IR)-sensitive or meiotic recombination-defective mutants in Saccharomyces cerevisiae (Symington 2002). Subsequent studies revealed similar defects in meiosis, repair of IR-induced DNA damage, telomere length, nonhomologous end joining (NHEJ), and the intra-S phase checkpoint in mre11, rad50, and xrs2 null mutants (Haber 1998; D'Amours and Jackson 2002). The corresponding proteins form a high-affinity complex with an ∼2:2:1 stoichiometry of Mre11, Rad50, and Xrs2 (Anderson et al. 2001; Chen et al. 2001). Both Mre11 and Rad50 are conserved and are homologous to the Escherichia coli SbcD and SbcC proteins, respectively (Sharples and Leach 1995). Functional analogs of the Xrs2 subunit are found only in eukaryotes, e.g., the human Nbs1 protein, and the sequences of these display very limited interspecies homology (Carney et al. 1998; Varon et al. 1998; Chahwan et al. 2003; Ueno et al. 2003). Mre11 contains five sequence motifs found in a superfamily of phosphoesterases that, apart from E. coli SbcD, also includes di-metal Ser/Thr protein phosphatases. Conserved residues within these motifs are required for Mre11 nuclease activity in vitro (Furuse et al. 1998; Usui et al. 1998; Moreau et al. 1999). Mre11 displays 3′–5′ ssDNA, 3′–5′ dsDNA exonuclease, and ssDNA endonuclease activities with the endonuclease activity acting preferentially at ssDNA/dsDNA transitions (Furuse et al. 1998; Paull and Gellert 1998; Usui et al. 1998; Moreau et al. 1999; Trujillo and Sung 2001). Biochemical analyses of the Mre11-Rad50 complex suggest that one function of the complex is to tether DNA ends together (Chen et al. 2001; de Jager et al. 2001; Hopfner et al. 2002), and it has been suggested that this tethering activity could hold sister chromatids in close proximity for efficient repair (Wiltzius et al. 2005). The rapid recruitment of the Mre11-Rad50-Xrs2 (MRX) complex to double-strand breaks (DSBs) in vivo is critical for signaling DNA damage to downstream effectors, such as Tel1 and Mec1 in yeast or ATM in mammalian cells (Nelms et al. 1998; Grenon et al. 2001; Usui et al. 2001; Nakada et al. 2003; Uziel et al. 2003; Costanzo et al. 2004; Lisby et al. 2004).

The rapid recruitment of the Mre11 complex to DSBs is also suggestive of an early role in the repair of DSBs. Studies with the mre11 null (mre11Δ) mutant have shown a defect in the 5′–3′ resection of DSBs suggesting a direct role of Mre11 in the nucleolytic resection process (Lee et al. 1998; Tsubouchi and Ogawa 1998). Although the exonuclease activity of Mre11 is of the opposite polarity to that observed for DSB resection (White and Haber 1990), the endonuclease activity of Mre11 could potentially function in concert with a helicase unwinding the DNA duplex and the Mre11 endonuclease resecting the 5′ ends.

Analysis of mre11 nuclease-defective (mre11-nd) mutants has led to conflicting views on the role of the Mre11 nuclease in end resection because of the different phenotypes conferred by these alleles. All of the mre11-nd mutants share the property of sporulation deficiency due to an inability to process Spo11-induced DSBs during meiosis, as well as synthetic mitotic lethality with rad27Δ (Furuse et al. 1998; Tsubouchi and Ogawa 1998; Usui et al. 1998; Moreau et al. 1999; Debrauwere et al. 2001). The Mre11-58 protein has two point mutations in phosphoesterase motif IV (H213Y, L225I), consistently lacks nuclease activity in vitro, and is furthermore defective in complex formation with Rad50 and Xrs2 (Usui et al. 1998). The mre11-58 allele confers a phenotype similar to that of an mre11Δ allele for sensitivity to ionizing radiation, processing of HO-induced DSBs, and sensitivity to HU and telomere length, but is proficient for meiotic DSB formation (Tsubouchi and Ogawa 1998; Usui et al. 1998; D'Amours and Jackson 2001). The mre11-D16A mutant (phosphoesterase motif I) is less sensitive to IR than the mre11Δ mutant and the telomeres are of intermediate length between the mre11Δ and MRE11 strains (Furuse et al. 1998). This mutant is proficient for end-joining repair of cohesive ends, and the purified Mre11-D16A protein interacts with Rad50 and Xrs2 in vitro (Lewis et al. 2004). However, the recruitment of Rad50 to telomeres is impaired in the mre11-D16A strain and there is a defect in complex formation between the Schizosaccharomyces pombe Rad32-D25A protein (equivalent to Mre11-D16A) and Rad50 in vivo (Tomita et al. 2003; Takata et al. 2005). In contrast, the mre11-D56N and mre11-H125N strains (phosphoesterase motifs II and III, respectively) show only a three- to fourfold decrease in survival to 500 Gy IR compared to MRE11 strains, exhibit no defect in processing HO-induced DSBs, and have telomeres of the same length as MRE11 strains (Moreau et al. 1999; Llorente and Symington 2004). These results have led to the suggestion that the Mre11-D56N and Mre11-H125N proteins retain sufficient residual nuclease activity to process DSBs in vivo (Lewis et al. 2004). However, in genetic assays for endonucleolytic processing of hairpin structures in yeast, the mre11-D56N and mre11-H125N strains were as defective as the mre11Δ strain, indicating that there is little, if any, residual endonuclease activity (Rattray et al. 2001; Lobachev et al. 2002; Farah et al. 2005). Biochemical studies of the human Mre11-3 protein, which has two amino acid substitutions within phosphoesterase motif III, have shown a complete loss of nuclease activity even though in the crystal structure of the Pyrococcus furiosus Mre11-3, the overall protein fold and active site conformation is the same as those in the wild-type protein (Arthur et al. 2004). In vivo, the yeast mre11-3 mutant shows similar sensitivity to IR as the mre11-H125N strain and normal resection of an HO-induced DSB (Bressan et al. 1998; Moreau et al. 1999; Lee et al. 2002).

Another possibility to account for the more severe phenotypes conferred by the mre11-D16A allele is a defect in complex assembly, as shown for the mre11-58 allele (Usui et al. 1998). Although the Mre11-D16A protein binds to Rad50 and Xrs2 in vitro, it is possible that a subtle defect in complex formation may not have been apparent at high protein concentrations (Lewis et al. 2004). To test these possibilities, we have compared several phenotypes, nuclease activity, and complex formation of six mre11 mutants with substitutions of invariant residues within phosphoesterase motifs I, II, and III. All six mutants were equally impaired for the exonuclease activity of Mre11. Consistent with previous studies (Furuse et al. 1998; Lewis et al. 2004), we find that substitutions of Asp16 within motif I confer the most severe DNA repair and telomere length defects. However, these defects appear to be related to a deficiency in complex formation as interactions between Rad50 or Xrs2 and Mre11-D16A or Mre11-D16N are severely compromised, whereas the mre11 alleles with less severe DNA repair and telomere maintenance defects exhibit stable complex formation.

https://www.genetics.org/content/genetics/early/2005/09/02/genetics.105.049478.full.pdf

Mutations in Mre11 phosphoesterase motif I that impair Saccharomyces cerevisiae Mre11-Rad50-Xrs2 complex stability in addition to nuclease activity.

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.

https://www.pnas.org/content/pnas/99/4/1853.full.pdf

Berit Olsen Krogh

Papers

Recombination proteins in yeast.

Catalytic mechanism of DNA topoisomerase IB.

Hemostatic effect of a monoclonal antibody mAb 2021 blocking the interaction between FXa and TFPI in a rabbit hemophilia model.

Mutations in Mre11 phosphoesterase motif I that impair Saccharomyces cerevisiae Mre11-Rad50-Xrs2 complex stability in addition to nuclease activity.

A poxvirus-like type IB topoisomerase family in bacteria