Processive Recombination by Wild-type Gin and an Enhancer-independent Mutant

Crisona, Nancy J.; Kanaar, Roland; González, Thalia; Zechiedrich, Lynn; Klippel, Anke; Cozzarelli, Nicholas R.

doi:10.1006/jmbi.1994.1671

Cited by 83 publications

(44 citation statements)

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“…DNA was detected by ethidium bromide staining or by Southern blotting. Knotted DNA for electron microscopy (EM) analysis was purified from agarose gels as described (45).…”

Section: Methodsmentioning

confidence: 99%

“…The variance of the topoisomer distribution ( 2 ) equals w 2 /4. EM Analysis of DNA Knot Chirality-Knotted DNA was prepared for EM to visualize DNA crossings as described previously (45). Briefly, purified nicked DNA knots were denatured with glyoxal, coated with RecA protein in the presence of ATP, fixed with 0.2% glutaraldehyde for 15 min, and purified by size exclusion over Sepharose CL-4B.…”

Section: Methodsmentioning

confidence: 99%

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The Saccharomyces cerevisiae Smc2/4 Condensin Compacts DNA into (+) Chiral Structures without Net Supercoiling

Stray

Crisona

Belotserkovskii

et al. 2005

Journal of Biological Chemistry

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Smc2/4 forms the core of the Saccharomyces cerevisiae condensin, which promotes metaphase chromosome compaction. To understand how condensin manipulates DNA, we used two in vitro assays to study the role of SMC (structural maintenance of chromosome) proteins and ATP in reconfiguring the path of DNA. The first assay evaluated the topology of knots formed in the presence of topoisomerase II. Unexpectedly, both wild-type Smc2/4 and an ATPase mutant promoted (؉) chiral knotting of nicked plasmids, revealing that ATP hydrolysis and the non-SMC condensins are not required to compact DNA chirally. The second assay measured Smc2/4-dependent changes in linking number (Lk). Smc2/4 did not induce (؉) supercoiling, but instead induced broadening of topoisomer distributions in a cooperative manner without altering Lk 0 . To explain chiral knotting in substrates devoid of chiral supercoiling, we propose that Smc2/4 directs chiral DNA compaction by constraining the duplex to retrace its own path. In this highly cooperative process, both (؉) and (؊) loops are sequestered (about one per kb), leaving net writhe and twist unchanged while broadening Lk. We have developed a quantitative theory to account for these results. Additionally, we have shown at higher molar stoichiometries that Smc2/4 prevents relaxation by topoisomerase I and nick closure by DNA ligase, indicating that Smc2/4 can saturate DNA. By electron microscopy of Smc2/4-DNA complexes, we observed primarily two protein-laden bound species: long flexible filaments and uniform rings or "doughnuts." Close packing of Smc2/4 on DNA explains the substrate protection we observed. Our results support the hypothesis that SMC proteins bind multiple DNA duplexes.SMC (structural maintenance of chromosome) proteins are the central components of several multiprotein complexes that help to organize chromosomes throughout the cell cycle (1-5). Understanding the properties of the SMC proteins may illuminate their essential function in cohesin, condensin, "compensin," repair complexes containing Smc5/6, and recombination complexes (6 -10). Disruption of SMC function in prokaryotes and eukaryotes alike leads to chromosome instability, defects in repair and recombination, and death under conditions of rapid growth (11-15). The budding yeast Smc2 and Smc4 proteins form a heterodimeric ATPase (16) that, in combination with the non-SMC proteins Brn1, Ycg1, and Ycs4, comprises the holo-condensin enzyme. Holo-condensin drives global chromosome condensation at mitosis (4, 17, 18) and specific condensation of the rDNA locus at anaphase (19). The Xenopus 8 S condensin FF and the Schizosaccharomyces pombe Cut3/14 complex are examples of other stable condensin SMC pairs whose properties have been studied in some detail (20 -23).The SMC proteins are large (ϳ150 kDa) and contain five structural motifs that include N-and C-terminal globular domains, a central hinge domain (H), and two long ␣-helices (␣ N and ␣ C ) arranged as follows: N-␣ N -H-␣ C -C. Coiled-coil pairing of ␣ N and ␣ C folds a single...

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“…DNA was detected by ethidium bromide staining or by Southern blotting. Knotted DNA for electron microscopy (EM) analysis was purified from agarose gels as described (45).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The Saccharomyces cerevisiae Smc2/4 Condensin Compacts DNA into (+) Chiral Structures without Net Supercoiling

Stray

Crisona

Belotserkovskii

et al. 2005

Journal of Biological Chemistry

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“…After reaction with Int, fully nicked DNA catenanes were generated by treatment with DNase I in the presence of ethidium bromide. Electrophoresis then extraction from low melting agarose allowed recovery of single catenane isomers (36).…”

Section: Methodsmentioning

confidence: 99%

Contrasting Enzymatic Activities of Topoisomerase IV and DNA Gyrase from Escherichia coli

Ullsperger

Cozzarelli

1996

Journal of Biological Chemistry

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133

122

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DNA gyrase and topoisomerase IV (Topo IV) have distinct roles as unlinking enzymes during DNA replication despite 40% sequence identity between them. DNA gyrase unlinks replicating DNA by introducing negative supercoils while Topo IV decatenates the two daughter molecules. For this study, we measured the rates of unlinking of various topoisomers of DNA by DNA gyrase and Topo IV. Each enzyme has marked preferences for certain strand-passage reactions. DNA gyrase is a relatively poor decatenase, catalyzing strand-passage events that result in supercoiling at rates several orders of magnitude faster than those causing decatenation. Topo IV, in contrast, decatenates linked circles 10 -40 times more quickly than it removes the intramolecular crossings from supercoiled DNA. Supercoiled catenanes are unlinked at an even more increased rate by Topo IV. Thus, the supercoils augment decatenation rather than compete with catenane crossings for their removal. Knot crossings and the crossings of multiply interlinked catenanes are also preferentially removed by Topo IV. This ability of Topo IV to selectively unlink catenated molecules mirrors its key role in decatenation of replicating chromosomes in vivo.

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“…Although knots and catenanes are considered to be unfavorable for most DNA metabolic processes (Ishii et al 1991;Portugal and Rodriguez-Campos 1996;Sundin and Varshavsky 1981), the formation of writhe, knots, and catenanes can bring distant sites close to one another to facilitate processes that require long-range genomic interactions (Liu et al 2009). For example, writhe can promote enhancer-promoter communication (Liu et al 2001) or regulate recombination processes (Crisona et al 1994;Merickel and Johnson 2004). Writhe is typically accommodated by changes in local DNA geometry that can facilitate the binding of proteins due to either a local distortion, such as bending, of DNA or the higher probability of juxtaposition of two DNA segments (Fogg et al 2012;Zechiedrich and Osheroff 2010) (Fig.…”

Section: Dna Twist (Torsion)-dependent Protein Activitymentioning

confidence: 99%

The dynamic interplay between DNA topoisomerases and DNA topology

Seol

Neuman

2016

Biophys Rev

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Topological properties of DNA influence its structure and biochemical interactions. Within the cell, DNA topology is constantly in flux. Transcription and other essential processes, including DNA replication and repair, not only alter the topology of the genome but also introduce additional complications associated with DNA knotting and catenation. These topological perturbations are counteracted by the action of topoisomerases, a specialized class of highly conserved and essential enzymes that actively regulate the topological state of the genome. This dynamic interplay among DNA topology, DNA processing enzymes, and DNA topoisomerases is a pervasive factor that influences DNA metabolism in vivo. Building on the extensive structural and biochemical characterization over the past four decades that has established the fundamental mechanistic basis of topoisomerase activity, scientists have begun to explore the unique roles played by DNA topology in modulating and influencing the activity of topoisomerases. In this review we survey established and emerging DNA topology-dependent protein-DNA interactions with a focus on in vitro measurements of the dynamic interplay between DNA topology and topoisomerase activity.

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Processive Recombination by Wild-type Gin and an Enhancer-independent Mutant

Cited by 83 publications

References 0 publications

The Saccharomyces cerevisiae Smc2/4 Condensin Compacts DNA into (+) Chiral Structures without Net Supercoiling

The Saccharomyces cerevisiae Smc2/4 Condensin Compacts DNA into (+) Chiral Structures without Net Supercoiling

Contrasting Enzymatic Activities of Topoisomerase IV and DNA Gyrase from Escherichia coli

The dynamic interplay between DNA topoisomerases and DNA topology

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