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	RESEARCH LINES & CONTRIBUTIONS TO KNOWLEDGE

	Early intuitions and insights A DNA tracking complex that does not turn relative to the double helix will produce concomitant unwinding and rewinding of DNA. J. Roca (hand-drawn), 13th International Congress of Biochemistry - Amsterdam (1985) J. Roca (hand-drawn), Doctoral Thesis - University of Barcelona (1988) Two-gate mechanism of a type II topoisomerase. J. Roca (hand-drawn), 1st Congress of the Spanish Society of Cell Biology - Madrid (1985) J. Roca (hand-drawn), Doctoral Thesis - University of Barcelona (1988) Regulation of Topo I by a factor that blocks swiveling of the duplex within the cleaved DNA complex. J. Roca (hand-drawn), Doctoral Thesis - University of Barcelona (1988) Extrusion of a DNA loop by sliding of the captured T-segment within a type-2 topoisomerase bound to the G-segment. J. Roca (hand-drawn), Doctoral Thesis - University of Barcelona (1988)

	DNA transport mechanism of type-2A topoisomerases The "three-gate" mechanism 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 71, 833-840. (1992). 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). 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). 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. M., Andoh T, and Wang C. Proc. Natl. Acad. Sci. USA 91, 1781-1785. (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).	Type-2 topoisomerases "invert" DNA-crossings A G-segment is trasiently gated to allow passage of a T-segment T-segments traverse the entire dimer interface of type-2A topoiosomerases by crossing the entry N-gate, the DNA-gate and the exit C-gate The T-segment can be hold on the central cavity of the topo II closed clamp

	DNA transport preferences of topoisomerase II The geometry of 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 Cells 1, 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) TOPO II proof-reads DNA topology before completing DNA transport 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)	DNA juxtaposition angles and loop contours in (+) and (-) supercoils Topo interaction with triple DNA crossings and DNA transport outcome (example of a single catenane within a - and + supercoiled ring ) The C-gate enables proofreading DNA topology before T-segmetn transport is completed

	Topoisomerase II inhibitors & poisons The mechanism of bisdioxopiperazines (ICRF-193) 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) Cloning, functional analysis and post-transcriptional regulation of a type II DNA Topoisomerase from Leishmania infantum. A new potential target for anti-parasite drugs.Tobias Hanke, María J.Ramiro, Sonia Trigueros, Joaquim Roca and Vicente Larraga. Nucleic. Acids. Res. 31, 4917-4928 (2003)	====> Poisoning topoisomerase II (From Roca, Thesis 1988)

	Topoisomerase activity at eukarytic chromatin 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) Tunning 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)	TOPO II-mediated relaxation of (+) DNA supercoils allows proper elongation of long gene transcripts Distintive promoter architecture of genes regulated by TOPO II The "cross-inversion" mechanism of TOPO II relaxes chromatin more efficiently than the "strand rotation" mechanism of TOPO I Chromatin conformation modulates TOPO I & TOPO II efficiency, which results in an unbalanced relaxation of (-) and (+) torsional stress

	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) DNA knotting 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	Distinct DNA paths produce distinct knot spectrums DNA packaging in viral capsids produce mainly toroidal knots Intracellular clusters of 20 nucleosomes have DNA knot probability 0.02 Topo II produces and removes the knots that block RNA Pol-II

	TOPOLOMICS 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) 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)	Psoralen-DNA phobinding reveals domains of distinct superhelical tension across intracellular chromosomes Torsional stress generated across chromosomal DNA can dissipate by telomeres
	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)	Yeast point-centromeres contraint a positive supercoil (Wr +0.6) Solution to the "Linling number paradox": ∆Lk = ∆Tw + ∆Wr

	SMCs: Mechanism and functions 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) 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 (2022)	Detection of DNA knots via 2D-gel electrophoresis (left top) led to discover that inactivation of condensin increases largely the knotting probability of DNA in vivo (left bottom). DNA entanglements occur often within and across nearby DNA loops in the cell nucleus. Condensin tightens these entanglements and so enforces their dissolution by topoisomerase (right).

	Methods Developed & Novel Approaches Separation of rooster spermatogenic nuclei by means of centrifugal elutriation. Jacint Boix and Joaquim Roca. Cytometry 14, 465-467. (1993) 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) 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) 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) 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) Two-Dimensional Agarose Gel Electrophoresis of DNA Topoisomers. Joaquim Roca. Methods in Molecular Biology 582, pp 27-37 (2009) 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) 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	Principle for 2D-gel electrophoresis of Lk topoisomers Glass fiber binding device to retain protein-DNA complexes Display of DNA knot sprectum by 2D gel electrophoresis Psoralen-DNA phobinding maps DNA helical tension genome-wide Separation of (+) and (-) noded DNA trefoils by high-resolution 2D-gel electrophoresis

	Reviews & Comments Joaquim Roca. The mechanism of DNA topoisomerases. TiBS 20, 133 - 168. (1995) Joaquim Roca. Topoisomerase II: A fitted mechanism for the chromatin landscape Nucleic. Acids. Res. . vol 37: 721-730 (2009) Joaquim Roca. Transcriptional inhibition by DNA torsional stress Transcription 2, 82-85 (2011) Joaquim Roca. The torsional state of DNA within the chromosome Chromosoma 120: 323-34 (2011) Joaquim Roca. In silico, In vitro and In vivo Imageries of Type II Topoisomerases Phys Life Rev Jul 8. pii: S1571-0645(16)30060-4 (2016) Joaquim Roca, Silvia Dyson, Joana Segura, Antonio Valdés, Belén Martínez-García Keeping intracellular DNA untangled: A new role for condensin? BioEssays* Volume 44, Issue 1 (2022)