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Early Ideas & Insights


1985 - DNA tracking motors generate helical unwinding and overwinding waves
A DNA tracking complex that does not turn relative to the double helix will produce concomitant DNA unwinding (behind) and DNA overwinding (in front).

J. Roca (hand-drawn model), 13th International Congress of Biochemistry - Amsterdam (1985

J. Roca, Doctoral Thesis (hand-drawn model)- University of Barcelona (1988)

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Hand-drawn Roca 1985

DNA unwinding & overwinding by fixed tracking motors - Roca 1985
1988 - Regulation of Topo I by a factor that blocks swiveling of the cleaved DNA complex.
J. Roca (hand-drawn model), Doctoral Thesis - University of Barcelona (1988 )
Plausible mechanisms that regulate DNA topology.



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Hand-drawn Roca 1985
Inhibition of topo I:cleaved DNA complex swiveling

1988 - DNA gyrase inverts a (+) into a (-) DNA supercoil by passing the wraped duplex throughout the entire enzyme inteface
J. Roca (hand-drawn), Doctoral Thesis - University of Barcelona (1988)



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Hand-drawn Roca 1985

Transported DNA crosses the entire dimer interface of gyrase
1988 - DNA gyrase can extrude and fasten DNA loops upon sliding the captured T-segment relative to the G-segment.
J. Roca (hand-drawn), Doctoral Thesis - University of Barcelona (1988)

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Hand-drawn Roca 1985

Loops extrusion mechanism mediated by DNA gyrase
2001 - A DNA "knitting" activity shapes chromosome and DNA loop architecture
Specific knotting (knitting) of the base of DNA loops by topo II might sufice to maintain chromosome architecture.



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Hand-drawn Roca 1985

DNA loops emerging from a "knitted" DNA scafold
2002 - DNA supercoiling  by "plectonases":  Powerful  motors that twist chromatin loops
DNA supercoiling  by "plectonases":  Powerful  motors that twist chromatin loops... ...

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Plectonase Roca 2002

Plectonase Mechanism- Roca 2002
2005 - Gate padlocks regulate the catalytic and structural functions of type-2 topoisomerases
Molecular padlocks impair oppening of the entry "N-gate" and the exit "C-gate" of Type-2 topoisomerases

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Gate Lockers Roca 2000

......Gate lockers......


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Seminal   Findings


  The DNA transport mechanism of type-2 topoisomerases
The G-segment binds inside first & the T-segment is captured by a clamp after
The capture of a DNA double helix by an ATP-dependent protein clamp: A key step in DNA transport by type II DNA topoisomerases.
Joaquim Roca and James C. Wang.
Cell 833-840 (1992)


On the Simultaneous Binding of Eukaryotic Topoisomerase II to a Pair of Double-Stranded DNA.
Joaquim Roca, James Berger, and James Wang.
J. Biol. Chem. 268, 14250-14255 (1993).

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Cross Invert Roca 1997
Type-2 topoisomerases "invert" DNA-crossings


Cross Invert Roca 1997

A Cleaved G-segment:Topo complex is gated to allow passage of a T-segment

The N-Gate to get in the T-segment, the C-gate to get it out
DNA transport by a type II DNA topoisomerase: Evidence in favor of a two-gate mechanism.
Joaquim Roca and James C. Wang.
Cell 77, 609-616. (1994).


DNA transport by type II DNA topoisomerases: Direct evidence of a two gate mechanism.
Joaquim Roca, James Berger, Sephen Harrison, and James Wang.
Proc. Natl. Acad. Sci. USA 93, 4057 - 4062,  (1996).


The path of the DNA along the dimer interface of topoisomerase II. 
Joaquim Roca.
J. Biol. Chem. 279,  25783–25788 (2004).

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The two gate mechanism IC
Entry N-gate (pink), DNA-gate (yellow), Exit C-gate (blue)


T segmetn hold
The T-segment can be hold on the central cavity of the topo II closed clamp

The DNA transport preferences of topoisomerase II
The geometry of -double and triple- DNA crossings determine T-segment capture probability
The probabilities of supercoil removal and decatenation by yeast DNA topoisomerase II.Joaquim Roca and  James Wang.
Genes to Cells1, 17-27 (1996)

Varying levels of positive and negative supercoiling differently affect the efficiency with which topoisomerase II catenates and decatenates DNA. 
Joaquim Roca
J. Mol. Biol.305, 441-450, (2001)

Asymmetric removal of supercoils suggests how topoisomerase II simplifies DNA topology.
Sonia Trigueros, Javier Salceda, Ignacio Bermúdez, Xavier Fernández and Joaquim Roca.
J. Mol. Biol. 335, 723-731 (2004)

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G-T angles

G-T angles determine +/- supercoil relaxation preferences

3 segment interaction

3 segment interaction determines catenation/decatenation preferences
TOPO II proof-reads DNA topology before releasing the T-segment by the C-gate

Topoisomerase II minimizes DNA entanglements by proofreading DNA topology after DNA strand passage. 
Belen Martinez-Garcia, Xavier Fernandez, Ofelia Diaz-Ingelmo, Antonio Rodriguez-Campos, Chaysavanh Manichanh and Joaquim Roca.
Nucleic Acids Res 42: 1821-30. (2014)



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Proofreading DNA topology

Proofreading DNA topology

Topoisomerase II inhibitors & poisons
Bisdioxopiperazines (ICRF-193) inhibit topoisomerase II by locking the N-gate
Antitumor bisdioxopiperazines inhibit yeast DNA topoisomerase II by trapping the enzyme in the form of a closed protein clamp. 
Joaquim Roca, Ishida R,  Berger J,  Andoh T,  and  Wang, J.
Proc. Natl. Acad. Sci. USA  91, 1781-1785. (1994)



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ICRF-193 mech

......pie figurahh......

Topoisomerase activity at eukarytic chromatin
Topoisomerase I and II differently tune (-) and (+) DNA torsional stress at eukaryotic chromatin

Topoisomerase II, not topoisomerase I, is the proficient relaxase of nucleosomal DNA. 
Javier Salceda, Xavier Fernández, and Joaquim Roca.
EMBO Journal 25, 2575-2583 (2006)



Chromatin regulates DNA torsional energy via topoisomerase II-mediated relaxation of positive supercoils.
Xavier Fernandez, Ofelia Diaz-Ingelmo, Belen Martinez-Garcia, Joaquim Roca.
EMBO Journal  33: 1492-501(2014)


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Hand-drawn Roca 1985

Topo I and Topo II Mechanistic Constraints to Relax Nucleosomal Fibers

Differential Relaxation Kinetics of (+)S and (-)S chromatin by Topo I and Topo II

Relax (+)S chromatin
Differential Relaxation Kinetics of (+)S and (-)S chromatin by Topo I and Topo II

DNA topoisomerase -activities and effects- beyond relaxing DNA torsional stress
DNA topoisomerase II activity in nonreplicating, transcriptionally inactive, chicken late spermatids.
Joaquim Roca and Cristobal Mezquita.
EMBO Journal. 8, p 1855-1860 (1989)




Circular  minichromosomes become highly recombinogenic in topoisomerase-deficient yeast Cells. Sonia Trigueros and Joaquim Roca.
J. Biol. Chem. 276, 2243-2249  (2001)




Failure to relax negative supercoiling of DNA  is a primary cause of mitotic hyper-recombination in topoisomerase-deficient yeast cells.
Sonia Trigueros and Joaquim Roca
J. Biol. Chem. 277, 37207-37221  (2002)




A GyrB-GyrA fusion protein expressed in yeast cells is able to remove DNA supercoils but cannot substitute eukaryotic topoisomerase II.
Sonia Trigueros and Joaquim Roca.
Genes to Cells 7, 249-257 (2002)




Topoisomerase II is required for the production of long Pol II gene transcripts in yeast.
Ricky Joshi, Benjamin Piña and Joaquim Roca
Nucleic Acids Res. 40: 7907-7915 (2012)




Topoisomerase II regulates yeast genes with singular chromatin architectures. Nikolaou C, Bermúdez I, Manichanh C, García-Martinez J, Guigó R, Pérez-Ortín JE and Roca J.
Nucleic Acids Res. 41, 9243-9256 (2013)



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Spermatogenesis
Topo II-induced DNA-strand breaks peaks in late spermatids


Spermatogenesis
Relaxation of (-)s by Topo I or E.coli TopA prevent plasmid multimerization


Spermatogenesis
Topo II inhibition impairs synthesis of long RNA transcripts


Spermatogenesis
Architecture of gene promoters regulated by Topo II



Deciphering the 3D path of DNA by KNOT analysis
DNA packaging in viral capsids

Knotting probability of DNA molecules confined in restricted volumes: DNA knotting in Phage capsids. Javier Arsuaga, Mariel Vazquez, Sonia Trigueros, De Witt Sumners, and Joaquim Roca
Proc. Natl. Acad. Sci. USA 99, 5373-5377  (2002)



DNA knots reveal a chiral organization of DNA in phage capsids. Javier Arsuaga, Mariel Vazquez, Paul McGuirk, Sonia Trigueros, De Witt Sumners and Joaquim Roca.
Proc. Natl. Acad. Sci. USA 102, 9165-9169  (2005)



Fractal Dimension of DNA Knots.Erika Ercolini, Francesco Valle, Jozef Adamcik, Ralf Metzler, Paolo De Los Rios, Joaquim Roca and Giovanni Dietler. 
Physical Review Letters 98, 058102:1-4, (2007)



Production of highly knotted DNA by means of cosmid circularization inside phage capsids. Sonia Trigueros and Joaquim Roca.
BMC Biotechnology 7:94 (2007)


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Phage knots
Predominance of toroidal knots (51) over achiral (41) and twsit (52) knots




Phage knots
Simulation of DNA paths producing achiral (41) and toroidal (51) knots


Topo II produces DNA knots in eukaryotic chromatin
DNA knots occur in intracellular chromatin.
Valdes, A., Segura, J., Dyson, S., Martinez-Garcia, B., and Roca, J.
Nucleic Acids Res 46, 650-660 (2018)


Transcriptional supercoiling boosts topoisomerase II-mediated knotting of intracellular DNA.
Antonio Valdés, Lucia Coronel, Belén Martínez-García, Joana Segura, Sílvia Dyson, Ofelia Díaz-Ingelmo, Cristian Micheletti, and Joaquim Roca
Nucleic Acids Res. Published: 05 June 2019


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Chromatin Knots
Chromatin DNA Knots

Chromatin Knots
Positive supercoiling generated during transcription boost DNA knotting in vivo and can stall RNA Pol-II


SMCs: Mechanism and functions
Interplay of SMCs and Topoisomerase II in DNA entanglement
Condensin minimizes topoisomerase II-mediated entanglements of DNA in vivo
Sílvia Dyson, Joana Segura, Belén Martínez-García, Antonio Valdés, and Joaquim Roca
The EMBO Journal 40, 1, e105393 (2021) 

 

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Condensin & Topo II

......pie figurahh......
DNA Loop Extrusion Mechanism of SMCs


Condensin pinches a short negatively supercoiled DNA loop during each round of ATP usage
Belén Martínez-García†, Sílvia Dyson†, Joana Segura†, Alba Ayats, Erin E Cutts, Pilar Gutierrez-Escribano, Luís Aragón, Joaquim Roca
The EMBO Journal e111913 (2023)


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Pinch and Merge Mechanism

The "Pinch and Merge" mechanism of SMC complexes to extrude DNA loops


Introducing "TOPOLOMICS" : Genome-wide analysis of DNA topology



The "UNCONSTRAINED TOPOLOME": Genome-wide analysis of -unconstrained- DNA twist and writhe
Genome-wide analysis of chromosomal DNA topology by means of psoralen-DNA photobinding.
Ignacio Bermúdez, José García-Martínez, José Pérez-Ortín, Joaquim Roca.
Nucleic Acids Res 38, e182 (2010)

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Psoralen:DNA mapping

......pie figurahh......

Positional dependence of transcriptional inhibition by DNA torsional stress in yeast chromosomes.
Ricky Joshi , Benjamin Piña, and Joaquim Roca.
EMBO Journal 29, 740–748 (2010)

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(+)S DNA mapping
......pie figurahh......


The "CONSTRAINED TOPOLOME": Genome-wide analysis of -constrained- DNA twist and writhe

DNA Topology and Global Architecture of Point Centromeres.
Diaz-Ingelmo, O., Martinez-Garcia, B., Segura, J., Valdes, A., and Roca, J.
Cell Rep 13, 667-677 (2015)


Intracellular nucleosomes constrain a DNA linking number difference of -1.26 that reconciles the Lk paradox.
Joana Segura, Ricky S. Joshi, Ofelia Díaz-Ingelmo, Antonio Valdés, Silvia Dyson, Belén Martínez-García and Joaquim Roca.
Nature Communications Sep 28;9(1):3989 (2018)


Nucleosomal DNA has topological memory.
Segura, Díaz-Ingelmo, Martínez-García, Ayats-Fraile, Nikolaou and Roca
bioRxiv DOI:10.1101/2023.05.21.541612.
Nature Com -under review- (2024)


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Topology of Point Centromeres
DNA topology of point centromeres


DNA Topology of Nucleosomes
DNA topology of in vivo nucleosomes

Topology of Point Centromeres
Nucleosomal DNA has an intrinsic topology that is imprinted by their genomic origin




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Methods Developed & Novel Approaches



Separation of rooster spermatogenic nuclei by means of centrifugal elutriation.
Jacint Boix and Joaquim Roca.
Cytometry 14, 465-467 (1993)
Nuclei elutriation
Elutriation of cell nuclei from different stages of spermatogenesis

 


A hit-and-run system for targeted genetic manipulations in yeast.
Joaquim Roca, Marc Gartenberg, Yasiji Oshima and James Wang.
Nucleic Acids Res. 20, p4671-4672. (1992)

URA cassette
Pop-In Pop-Out URA3 Cassette


Two-dimensional agarose gel electrophoresis of DNA topoisomers.
Ryo Hanai and Joaquim Roca.
Methods in Molecular Biology. Vol 94. DNA Topoisomerase Protocols. Humana Press Inc. Totowa, NJ. Ch 4, 19-27 (1999)


Two-Dimensional Agarose Gel Electrophoresis of DNA Topoisomers.
Joaquim Roca.
Methods in Molecular Biology  582, pp 27-37  (2009)
2D gel method
Effect of DNA intercalators in 2D gel electrophoresis of DNA Lk topoisomers

 


Filter binding assays for topoisomerase-DNA complexes. 
Joaquim Roca.
Methods in Molecular Biology. Vol 95. DNA Topoisomerase Protocols. Humana Press Inc. Totowa, NJ. Ch 8, 75-80 (2001)
Filter binding method
Device for filter binding assays of Protein-DNA complexes

 


Novel display of knotted DNA molecules by two-dimensional gel electrophoresis.
Sonia Trigueros,  Javier Arsuaga,  Maria E. Vazquez, De Witt Sumners and Joaquim Roca.
Nucleic. Acids. Res.  29, 13 - e67  (2001)
Filter binding method
High resolution 2D gel electrophoresis of Knotted DNA Topoisomers

 


Genome-wide analysis of chromosomal DNA topology by means of psoralen-DNA photobinding.
Ignacio Bermúdez, José García-Martínez, José Pérez-Ortín, Joaquim Roca.
Nucleic Acids Res  38, e182  (2010)
Psoralen maps
Genome-wide profiles of in vivo Psoralen-DNA Photobinding

 

 

QuantitaQuantitative disclosure of DNA knot chirality by high-resolution 2D-gel electrophoresis.
Antonio Valdés, Belén Martínez-García, Joana Segura, Sílvia Dyson, Ofelia Díaz-Ingelmo and Joaquim Roca.
Nucleic Acids Res. Published: 15 January 2019
Psoralen maps
Resoltion of DNA trefoil chirality by high resolution 2d gel electrophoresis


TOPO-SEQ:  The first procedure to determine the topology of large libraries of circular DNA molecules.

Introduced in:
Nucleosomal DNA has topological memory.
Segura, Díaz-Ingelmo, Martínez-García, Ayats-Fraile, Nikolaou and Roca
bioRxiv DOI:10.1101/2023.05.21.541612.
Nature Com -under review- (2024)
Psoralen maps
Resoltion of DNA trefoil chirality by high resolution 2d gel electrophoresis



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