A change of view: homologous recombination at single-molecule resolution (original) (raw)

• Dunham, M. A., Neumann, A. A., Fasching, C. L. & Reddel, R. R. Telomere maintenance by recombination in human cells. Nat. Genet. 26, 447–450 (2000).
  CAS PubMed Google Scholar
• Jasin, M. & Rothstein, R. Repair of strand breaks by homologous recombination. Cold Spring Harb. Perspect.Biol. 5, a012740 (2013).
  PubMed PubMed Central Google Scholar
• Symington, L. S., Rothstein, R. & Lisby, M. Mechanisms and regulation of mitotic recombination in Saccharomyces cerevisiae. Genetics 198, 795–835 (2014).
  CAS PubMed PubMed Central Google Scholar
• San Filippo, J., Sung, P. & Klein, H. Mechanism of eukaryotic homologous recombination. Annu. Rev. Biochem. 77, 229–257 (2008).
  CAS PubMed Google Scholar
• Szostak, J. W., Orr-Weaver, T. L., Rothstein, R. J. & Stahl, F. W. The double-strand-break repair model for recombination. Cell 33, 25–35 (1983).
  CAS PubMed Google Scholar
• Chanet, R., Heude, M., Adjiri, A., Maloisel, L. & Fabre, F. Semidominant mutations in the yeast Rad51 protein and their relationships with the Srs2 helicase. Mol. Cell. Biol. 16, 4782–4789 (1996).
  CAS PubMed PubMed Central Google Scholar
• Kass, E. M., Moynahan, M. E. & Jasin, M. When genome maintenance goes badly awry. Mol. Cell 62, 777–787 (2016).
  CAS PubMed PubMed Central Google Scholar
• Moynahan, M. E. & Jasin, M. Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nat. Rev. Mol. Cell Biol. 11, 196–207 (2010).
  CAS PubMed PubMed Central Google Scholar
• Prakash, R., Zhang, Y., Feng, W. & Jasin, M. Homologous recombination and human health: the roles of BRCA1, BRCA2, and associated proteins. Cold Spring Harb. Perspect.Biol. 7, a016600 (2015).
  PubMed PubMed Central Google Scholar
• Holliday, R. A mechanism for gene conversion in fungi [Reprinted]. Genet. Res. 89, 285–307 (2007).
  PubMed Google Scholar
• Meselson, M. S. & Radding, C. M. A general model for genetic recombination. Proc. Natl Acad. Sci. USA 72, 358–361 (1975).
  CAS PubMed Google Scholar
• Nassif, N., Penney, J., Pal, S., Engels, W. R. & Gloor, G. B. Efficient copying of nonhomologous sequences from ectopic sites via P-element-induced gap repair. Mol. Cell. Biol. 14, 1613–1625 (1994).
  CAS PubMed PubMed Central Google Scholar
• Mehta, A. & Haber, J. E. Sources of DNA double-strand breaks and models of recombinational DNA repair. Cold Spring Harb. Perspect.Biol. 6, a016428 (2014).
  PubMed PubMed Central Google Scholar
• Paques, F. & Haber, J. E. Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63, 349–404 (1999).
  CAS PubMed PubMed Central Google Scholar
• Kowalczykowski, S. C. An overview of the molecular mechanisms of recombinational DNA repair. Cold Spring Harbor Perspect. Biol. 7, a016410 (2015).
  Google Scholar
• Heyer, W. D., Ehmsen, K. T. & Liu, J. Regulation of homologous recombination in eukaryotes. Annu. Rev. Genet. 44, 113–139 (2010).
  CAS PubMed PubMed Central Google Scholar
• Morrical, S. W. DNA-pairing and annealing processes in homologous recombination and homology-directed repair. Cold Spring Harbor Perspect. Biol. 7, a016444 (2015).
  Google Scholar
• Prentiss, M., Prevost, C. & Danilowicz, C. Structure/function relationships in RecA protein-mediated homology recognition and strand exchange. Crit. Rev. Biochem. Mol. Biol. 50, 453–476 (2015).
  CAS PubMed Google Scholar
• Joo, C., Balci, H., Ishitsuka, Y., Buranachai, C. & Ha, T. Advances in single-molecule fluorescence methods for molecular biology. Annu. Rev. Biochem. 77, 51–76 (2008).
  CAS PubMed Google Scholar
• Monachino, E., Spenkelink, L. M. & van Oijen, A. M. Watching cellular machinery in action, one molecule at a time. J. Cell Biol. 216, 41–51 (2017).
  CAS PubMed PubMed Central Google Scholar
• Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nat. Methods 5, 507–516 (2008).
  CAS PubMed PubMed Central Google Scholar
• Walter, N. G., Huang, C. Y., Manzo, A. J. & Sobhy, M. A. Do-it-yourself guide: how to use the modern single-molecule toolkit. Nat. Methods 5, 475–489 (2008).
  CAS PubMed PubMed Central Google Scholar
• Bell, J. C. & Kowalczykowski, S. C. Mechanics and single-molecule interrogation of DNA recombination. Annu. Rev. Biochem. 85, 193–226 (2016).
  CAS PubMed Google Scholar
• van Mameren, J., Peterman, E. J. & Wuite, G. J. See me, feel me: methods to concurrently visualize and manipulate single DNA molecules and associated proteins. Nucleic Acids Res. 36, 4381–4389 (2008).
  CAS PubMed PubMed Central Google Scholar
• Axelrod, D. Total internal reflection fluorescence microscopy. Methods Cell Biol. 30, 245–270 (1989).
  CAS PubMed Google Scholar
• Reck-Peterson, S. L., Derr, N. D. & Stuurman, N. Imaging single molecules using total internal reflection fluorescence microscopy (TIRFM. Cold Spring Harb. Protoc. 2010, pdb.top73 (2010).
  Google Scholar
• Hoffman, M. T., Sheung, J. & Selvin, P. R. Fluorescence imaging with one nanometer accuracy: in vitro and in vivo studies of molecular motors. Methods Mol. Biol. 778, 33–56 (2011).
  CAS PubMed Google Scholar
• Stryer, L. Fluorescence energy transfer as a spectroscopic ruler. Annu. Rev. Biochem. 47, 819–846 (1978).
  CAS PubMed Google Scholar
• Lakowicz, J. Principles of Fluorescence Spectroscopy 3rd edn, (Springer, 2011).
  Google Scholar
• Ha, T. et al. Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism. Proc. Natl Acad. Sci. USA 96, 893–898 (1999).
  CAS PubMed Google Scholar
• Ma, C. J., Steinfeld, J. B. & Greene, E. C. Single-stranded DNA curtains for studying homologous recombination. Methods Enzymol. 582, 193–219 (2017).
  CAS PubMed Google Scholar
• Greene, E. C., Wind, S., Fazio, T., Gorman, J. & Visnapuu, M. L. DNA curtains for high-throughput single-molecule optical imaging. Methods Enzymol. 472, 293–315 (2010).
  CAS PubMed Google Scholar
• Lohman, T. M., Tomko, E. J. & Wu, C. G. Non-hexameric DNA helicases and translocases: mechanisms and regulation. Nat. Rev. Mol. Cell Biol. 9, 391–401 (2008).
  CAS PubMed Google Scholar
• Singleton, M. R., Dillingham, M. S. & Wigley, D. B. Structure and mechanism of helicases and nucleic acid translocases. Annu. Rev. Biochem. 76, 23–50 (2007).
  CAS PubMed Google Scholar
• Pyle, A. M. Translocation and unwinding mechanisms of RNA and DNA helicases. Annu. Rev. Biophys. 37, 317–336 (2008).
  CAS PubMed Google Scholar
• Bernstein, K. A., Gangloff, S. & Rothstein, R. The RecQ DNA helicases in DNA repair. Annu. Rev. Genet. 44, 393–417 (2010).
  CAS PubMed PubMed Central Google Scholar
• Brosh, R. M. Jr. DNA helicases involved in DNA repair and their roles in cancer. Nat. Rev. Cancer 13, 542–558 (2013).
  CAS PubMed PubMed Central Google Scholar
• Croteau, D. L., Popuri, V., Opresko, P. L. & Bohr, V. A. Human RecQ helicases in DNA repair, recombination, and replication. Annu. Rev. Biochem. 83, 519–552 (2014).
  CAS PubMed PubMed Central Google Scholar
• Niu, H. & Klein, H. L. Multifunctional roles of Saccharomyces cerevisiae Srs2 protein in replication, recombination and repair. FEMS Yeast Res. 17, fow11 (2017).
  Google Scholar
• Vannier, J. B., Sarek, G. & Boulton, S. J. RTEL1: functions of a disease-associated helicase. Trends Cell Biol. 24, 416–425 (2014).
  CAS PubMed Google Scholar
• Branzei, D. & Foiani, M. RecQ helicases queuing with Srs2 to disrupt Rad51 filaments and suppress recombination. Genes Dev. 21, 3019–3026 (2007).
  CAS PubMed Google Scholar
• Branzei, D. & Szakal, B. Building up and breaking down: mechanisms controlling recombination during replication. Crit. Rev. Biochem. Mol. Biol. 52, 381–394 (2017).
  CAS PubMed Google Scholar
• Marini, V. & Krejci, L. Srs2: the “Odd-Job Man” in DNA repair. DNA Repair 9, 268–275 (2010).
  CAS PubMed PubMed Central Google Scholar
• Symington, L. S. & Heyer, W. D. Some disassembly required: role of DNA translocases in the disruption of recombination intermediates and dead-end complexes. Genes Dev. 20, 2479–2486 (2006).
  CAS PubMed Google Scholar
• Dillingham, M. S. & Kowalczykowski, S. C. RecBCD enzyme and the repair of double-stranded DNA breaks. Microbiol. Mol. Biol. Rev. 72, 642–671 (2008).
  CAS PubMed PubMed Central Google Scholar
• Smith, G. R. How RecBCD enzyme and Chi promote DNA break repair and recombination: a molecular biologist's view. Microbiol. Mol. Biol. Rev. 76, 217–228 (2012).
  CAS PubMed PubMed Central Google Scholar
• Wigley, D. B. Bacterial DNA repair: recent insights into the mechanism of RecBCD, AddAB and AdnAB. Nat. Rev. Microbiol. 11, 9–13 (2013).
  CAS PubMed Google Scholar
• Bianco, P. R. et al. Processive translocation and DNA unwinding by individual RecBCD enzyme molecules. Nature 409, 374–378 (2001). This paper presents the earliest example of the application of SM methods to the DNA-end-processing molecular motor complex RecBCD.
  CAS PubMed Google Scholar
• Spies, M. et al. A molecular throttle: the recombination hotspot chi controls DNA translocation by the RecBCD helicase. Cell 114, 647–654 (2003). This paper describes the first SM study to demonstrate that chi recognition causes a change in the velocity of RecBCD that corresponds to a switch in the RecD and RecB motor activities.
  CAS PubMed Google Scholar
• Ha, T. et al. Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase. Nature 419, 638–641 (2002).
  CAS PubMed Google Scholar
• Myong, S., Rasnik, I., Joo, C., Lohman, T. M. & Ha, T. Repetitive shuttling of a motor protein on DNA. Nature 437, 1321–1325 (2005).
  CAS PubMed Google Scholar
• Qiu, Y. et al. Srs2 prevents Rad51 filament formation by repetitive motion on DNA. Nat. Commun. 4, 2281 (2013).
  PubMed PubMed Central Google Scholar
• Krejci, L. et al. DNA helicase Srs2 disrupts the Rad51 presynaptic filament. Nature 423, 305–309 (2003).
  CAS PubMed Google Scholar
• Veaute, X. et al. The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments. Nature 423, 309–312 (2003).
  CAS PubMed Google Scholar
• Park, J. et al. PcrA helicase dismantles RecA filaments by reeling in DNA in uniform steps. Cell 142, 544–555 (2010). This paper describes the use of smFRET experiments to study how the helicase PcrA removes RecA from ssDNA.
  CAS PubMed PubMed Central Google Scholar
• Petrova, V. et al. Active displacement of RecA filaments by UvrD translocase activity. Nucleic Acids Res. 43, 4133–4149 (2015).
  CAS PubMed PubMed Central Google Scholar
• Anand, S. P., Zheng, H., Bianco, P. R., Leuba, S. H. & Khan, S. A. DNA helicase activity of PcrA is not required for the displacement of RecA protein from DNA or inhibition of RecA-mediated strand exchange. J. Bacteriol. 189, 4502–4509 (2007).
  CAS PubMed PubMed Central Google Scholar
• Fagerburg, M. V. et al. PcrA-mediated disruption of RecA nucleoprotein filaments — essential role of the ATPase activity of RecA. Nucleic Acids Res. 40, 8416–8424 (2012).
  CAS PubMed PubMed Central Google Scholar
• Arslan, S., Khafizov, R., Thomas, C. D., Chemla, Y. R. & Ha, T. Engineering of a superhelicase through conformational control. Science 348, 344–347 (2015). In this study, researchers engineer Rep and PcrA 'superhelicases' that are 'locked' into highly active conformational states and use them to define the relationship between their structures and their functions.
  CAS PubMed PubMed Central Google Scholar
• Comstock, M. J. et al. Direct observation of structure-function relationship in a nucleic acid-processing enzyme. Science 348, 352–354 (2015). In this study, researchers use smFRET in conjunction with DNA force measurements to directly visualize the helicase UvrD acting on DNA. The findings indicate how protein conformational changes and changes in oligomeric state can influence helicase activities.
  CAS PubMed PubMed Central Google Scholar
• Korolev, S., Hsieh, J., Gauss, G. H., Lohman, T. M. & Waksman, G. Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ADP. Cell 90, 635–647 (1997).
  CAS PubMed Google Scholar
• Lee, J. Y. & Yang, W. UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke. Cell 127, 1349–1360 (2006).
  CAS PubMed PubMed Central Google Scholar
• Velankar, S. S., Soultanas, P., Dillingham, M. S., Subramanya, H. S. & Wigley, D. B. Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism. Cell 97, 75–84 (1999).
  CAS PubMed Google Scholar
• Wold, M. S. Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Annu. Rev. Biochem. 66, 61–92 (1997).
  CAS PubMed Google Scholar
• Meyer, R. R. & Laine, P. S. The single-stranded DNA-binding protein of Escherichia coli. Microbiol. Rev. 54, 342–380 (1990).
  CAS PubMed PubMed Central Google Scholar
• Bianco, P. R. The tale of SSB. Progress Biophys. Mol. Biol. 127, 111–118 (2017).
  CAS Google Scholar
• Chen, R. & Wold, M. S. Replication protein A: single-stranded DNA's first responder: dynamic DNA-interactions allow replication protein A to direct single-strand DNA intermediates into different pathways for synthesis or repair. BioEssays 36, 1156–1161 (2014).
  CAS PubMed PubMed Central Google Scholar
• Fanning, E., Klimovich, V. & Nager, A. R. A dynamic model for replication protein A (RPA) function in DNA processing pathways. Nucleic Acids Res. 34, 4126–4137 (2006).
  CAS PubMed PubMed Central Google Scholar
• Krejci, L. & Sung, P. RPA not that different from SSB. Structure 10, 601–602 (2002).
  CAS PubMed Google Scholar
• Shereda, R. D., Kozlov, A. G., Lohman, T. M., Cox, M. M. & Keck, J. L. SSB as an organizer/mobilizer of genome maintenance complexes. Crit. Rev. Biochem. Mol. Biol. 43, 289–318 (2008).
  CAS PubMed PubMed Central Google Scholar
• Marechal, A. & Zou, L. RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response. Cell Res. 25, 9–23 (2015).
  CAS PubMed Google Scholar
• Gibb, B., Silverstein, T. D., Finkelstein, I. J. & Greene, E. C. Single-stranded DNA curtains for real-time single-molecule visualization of protein-nucleic acid interactions. Anal. Chem. 84, 7607–7612 (2012).
  CAS PubMed PubMed Central Google Scholar
• Gibb, B. et al. Concentration-dependent exchange of replication protein A on single-stranded DNA revealed by single-molecule imaging. PLoS ONE 9, e87922 (2014).
  PubMed PubMed Central Google Scholar
• Deng, S. K., Gibb, B., de Almeida, M. J., Greene, E. C. & Symington, L. S. RPA antagonizes microhomology-mediated repair of DNA double-strand breaks. Nat. Struct. Mol. Biol. 21, 405–412 (2014).
  CAS PubMed PubMed Central Google Scholar
• Ma, C. J., Gibb, B., Kwon, Y., Sung, P. & Greene, E. C. Protein dynamics of human RPA and RAD51 on ssDNA during assembly and disassembly of the RAD51 filament. Nucleic Acids Res. 45, 749–761 (2017).
  CAS PubMed Google Scholar
• Graham, J. S., Johnson, R. C. & Marko, J. F. Concentration-dependent exchange accelerates turnover of proteins bound to double-stranded DNA. Nucleic Acids Res. 39, 2249–2259 (2011). In this paper, SM imaging is used to show that DNA-binding proteins Fis and HU and non-histone chromosomal protein 6A (Nhp6A) can undergo concentration-dependent exchange between free and bound states, thus defining a new mechanism that can contribute to protein–nucleic acid interaction dynamics.
  CAS PubMed Google Scholar
• Chen, T. Y. et al. Concentration- and chromosome-organization-dependent regulator unbinding from DNA for transcription regulation in living cells. Nat. Commun. 6, 7445 (2015).
  PubMed PubMed Central Google Scholar
• Ha, T. Single-molecule approaches embrace molecular cohorts. Cell 154, 723–726 (2013).
  CAS PubMed PubMed Central Google Scholar
• Aberg, C., Duderstadt, K. E. & van Oijen, A. M. Stability versus exchange: a paradox in DNA replication. Nucleic Acids Res. 44, 4846–4854 (2016).
  CAS PubMed PubMed Central Google Scholar
• Roy, R., Kozlov, A. G., Lohman, T. M. & Ha, T. SSB protein diffusion on single-stranded DNA stimulates RecA filament formation. Nature 461, 1092–1097 (2009).
  CAS PubMed PubMed Central Google Scholar
• Sokoloski, J. E., Kozlov, A. G., Galletto, R. & Lohman, T. M. Chemo-mechanical pushing of proteins along single-stranded DNA. Proc. Natl Acad. Sci. USA 113, 6194–6199 (2016).
  CAS PubMed Google Scholar
• Bell, J. C., Liu, B. & Kowalczykowski, S. C. Imaging and energetics of single SSB-ssDNA molecules reveal intramolecular condensation and insight into RecOR function. eLife 4, e08646 (2015).
  PubMed PubMed Central Google Scholar
• Myler, L. R. et al. Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins. Proc. Natl Acad. Sci. USA 113, E1170–E1179 (2016).
  CAS PubMed Google Scholar
• Bianco, P. R., Tracy, R. B. & Kowalczykowski, S. C. DNA strand exchange proteins: a biochemical and physical comparison. Front. Biosci. 3, D570–D603 (1998).
  CAS PubMed Google Scholar
• Joo, C. et al. Real-time observation of RecA filament dynamics with single monomer resolution. Cell 126, 515–527 (2006).
  CAS PubMed Google Scholar
• Bell, J. C. & Kowalczykowski, S. C. RecA: regulation and mechanism of a molecular search engine. Trends Biochem. Sci. 41, 491–507 (2016).
  CAS PubMed PubMed Central Google Scholar
• Galletto, R., Amitani, I., Baskin, R. J. & Kowalczykowski, S. C. Direct observation of individual RecA filaments assembling on single DNA molecules. Nature 443, 875–878 (2006). In this study, researchers use a multichannel flow chamber system to directly visualize the assembly of RecA filaments on single dsDNA molecules, providing new insight into filament nucleation and filament growth.
  CAS PubMed Google Scholar
• Bell, J. C., Plank, J. L., Dombrowski, C. C. & Kowalczykowski, S. C. Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA. Nature 491, 274–278 (2012). In this work, new insights into the presynaptic complex assembly mechanism are generated by using multicolour SM imaging to study the assembly of RecA filaments on single molecules of SSB-bound ssDNA in the presence or absence of the RecFOR proteins.
  CAS PubMed PubMed Central Google Scholar
• Morimatsu, K. & Kowalczykowski, S. C. RecFOR proteins load RecA protein onto gapped DNA to accelerate DNA strand exchange: a universal step of recombinational repair. Mol. Cell 11, 1337–1347 (2003).
  CAS PubMed Google Scholar
• Umezu, K., Chi, N. W. & Kolodner, R. D. Biochemical interaction of the Escherichia coli RecF, RecO, and RecR proteins with RecA protein and single-stranded DNA binding protein. Proc. Natl Acad. Sci. USA 90, 3875–3879 (1993).
  CAS PubMed Google Scholar
• Gibb, B. et al. Protein dynamics during presynaptic-complex assembly on individual single-stranded DNA molecules. Nat. Struct. Mol. Biol. 21, 893–900 (2014).
  CAS PubMed PubMed Central Google Scholar
• Lee, J. Y., Qi, Z. & Greene, E. C. ATP hydrolysis promotes duplex DNA release by the RecA presynaptic complex. J. Biol. Chem. 291, 22218–22230 (2016).
  CAS PubMed PubMed Central Google Scholar
• Lee, J. Y. et al. Base triplet stepping by the Rad51/RecA family of recombinases. Science 349, 977–981 (2015).
  CAS PubMed PubMed Central Google Scholar
• Qi, Z. et al. DNA sequence alignment by microhomology sampling during homologous recombination. Cell 160, 856–869 (2015).
  CAS PubMed PubMed Central Google Scholar
• Taylor, M. R. et al. A polar and nucleotide-dependent mechanism of action for RAD51 paralogs in RAD51 filament remodeling. Mol. Cell 64, 926–939 (2016).
  CAS PubMed PubMed Central Google Scholar
• Ogawa, T., Yu, X., Shinohara, A. & Egelman, E. H. Similarity of the yeast RAD51 filament to the bacterial RecA filament. Science 259, 1896–1899 (1993).
  CAS PubMed Google Scholar
• van Mameren, J. et al. Counting RAD51 proteins disassembling from nucleoprotein filaments under tension. Nature 457, 745–748 (2009).
  CAS PubMed Google Scholar
• Candelli, A., Modesti, M., Peterman, E. J. & Wuite, G. J. Single-molecule views on homologous recombination. Q. Rev. Biophys. 46, 323–348 (2013).
  CAS PubMed Google Scholar
• Candelli, A., Wuite, G. J. & Peterman, E. J. Combining optical trapping, fluorescence microscopy and micro-fluidics for single molecule studies of DNA-protein interactions. Phys. Chem. Chem. Phys. 13, 7263–7272 (2011).
  CAS PubMed Google Scholar
• Mameren, J. et al. Dissecting elastic heterogeneity along DNA molecules coated partly with Rad51 using concurrent fluorescence microscopy and optical tweezers. Biophys. J. 91, L78–L80 (2006).
  PubMed PubMed Central Google Scholar
• Greene, E. C. DNA sequence alignment during homologous recombination. J. Biol. Chem. 291, 11572–11580 (2016).
  CAS PubMed PubMed Central Google Scholar
• Renkawitz, J., Lademann, C. A. & Jentsch, S. Mechanisms and principles of homology search during recombination. Nat. Rev. Mol. Cell Biol. 15, 369–383 (2014).
  CAS PubMed Google Scholar
• Barzel, A. & Kupiec, M. Finding a match: how do homologous sequences get together for recombination? Nat. Rev. Genet. 9, 27–37 (2008).
  CAS PubMed Google Scholar
• Gonda, D. K. & Radding, C. M. The mechanism of the search for homology promoted by recA protein. Facilitated diffusion within nucleoprotein networks. J. Biol. Chem. 261, 13087–13096 (1986).
  CAS PubMed Google Scholar
• Forget, A. L. & Kowalczykowski, S. C. Single-molecule imaging of DNA pairing by RecA reveals a three-dimensional homology search. Nature 482, 423–427 (2012). In this study, a dual optical trap is used to manipulate the end-to-end length of a single dsDNA molecule while concurrently visualizing single RecA presynaptic complexes as they search the dsDNA for homology. This work demonstrates that the homology search is a 3-dimensional process, which involves multivalent contacts between the presynaptic complex and the dsDNA.
  CAS PubMed PubMed Central Google Scholar
• Ragunathan, K., Liu, C. & Ha, T. RecA filament sliding on DNA facilitates homology search. eLife 1, e00067 (2012).
  PubMed PubMed Central Google Scholar
• De Vlaminck, I. et al. Mechanism of homology recognition in DNA recombination from dual-molecule experiments. Mol. Cell 46, 616–624 (2012).
  CAS PubMed Google Scholar
• Jiang, L. & Prentiss, M. RecA-mediated sequence homology recognition as an example of how searching speed in self-assembly systems can be optimized by balancing entropic and enthalpic barriers. Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 90, 022704 (2014).
  PubMed PubMed Central Google Scholar
• Kates-Harbeck, J., Tilloy, A. & Prentiss, M. Simplified biased random walk model for RecA-protein-mediated homology recognition offers rapid and accurate self-assembly of long linear arrays of binding sites. Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 88, 012702 (2013).
  PubMed PubMed Central Google Scholar
• Thomas, C. A. Jr. Recombination of DNA molecules. Prog. Nucleic Acid Res. Mol. Biol. 5, 315–337 (1966).
  PubMed Google Scholar
• Danilowicz, C. et al. RecA homology search is promoted by mechanical stress along the scanned duplex DNA. Nucleic Acids Res. 40, 1717–1727 (2012).
  CAS PubMed Google Scholar
• Ragunathan, K., Joo, C. & Ha, T. Real-time observation of strand exchange reaction with high spatiotemporal resolution. Structure 19, 1064–1073 (2011).
  CAS PubMed PubMed Central Google Scholar
• Sahl, S. J., Hell, S. W. & Jakobs, S. Fluorescence nanoscopy in cell biology. Nat. Rev. Mol. Cell Biol. 16, 685–701 (2017).
  Google Scholar
• Liu, Z., Lavis, L. D. & Betzig, E. Imaging live-cell dynamics and structure at the single-molecule level. Mol. Cell 58, 644–659 (2015).
  CAS PubMed Google Scholar
• Schermelleh, L., Heintzmann, R. & Leonhardt, H. A guide to super-resolution fluorescence microscopy. J. Cell Biol. 190, 165–175 (2010).
  CAS PubMed PubMed Central Google Scholar
• Thompson, M. A., Lew, M. D. & Moerner, W. E. Extending microscopic resolution with single-molecule imaging and active control. Annu. Rev. Biophys. 41, 321–342 (2012).
  CAS PubMed Google Scholar
• Sengupta, P., van Engelenburg, S. B. & Lippincott-Schwartz, J. Superresolution imaging of biological systems using photoactivated localization microscopy. Chem. Rev. 114, 3189–3202 (2014).
  CAS PubMed PubMed Central Google Scholar
• Lambert, T. J. & Waters, J. C. Navigating challenges in the application of superresolution microscopy. J. Cell Biol. 216, 53–63 (2017).
  CAS PubMed PubMed Central Google Scholar
• Betzig, E. Single molecules, cells, and super-resolution optics (Nobel Lecture). Angewandte Chemie (Int. Ed.) 54, 8034–8053 (2015).
  CAS Google Scholar
• Bates, M., Huang, B., Dempsey, G. T. & Zhuang, X. Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317, 1749–1753 (2007).
  CAS PubMed PubMed Central Google Scholar
• Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).
  CAS PubMed PubMed Central Google Scholar
• Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).
  CAS PubMed PubMed Central Google Scholar
• Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).
  CAS PubMed Google Scholar
• Manley, S. et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat. Methods 5, 155–157 (2008).
  CAS PubMed Google Scholar
• Sengupta, P., Van Engelenburg, S. & Lippincott-Schwartz, J. Visualizing cell structure and function with point-localization superresolution imaging. Dev. Cell 23, 1092–1102 (2012).
  CAS PubMed PubMed Central Google Scholar
• Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994).
  CAS PubMed Google Scholar
• Klar, T. A. & Hell, S. W. Subdiffraction resolution in far-field fluorescence microscopy. Opt. Lett. 24, 954–956 (1999).
  CAS PubMed Google Scholar
• Westphal, V. et al. Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science 320, 246–249 (2008).
  CAS PubMed Google Scholar
• Gustafsson, M. G. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198, 82–87 (2000).
  CAS PubMed Google Scholar
• Gustafsson, M. G. Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl Acad. Sci. USA 102, 13081–13086 (2005).
  CAS PubMed Google Scholar
• Schermelleh, L. et al. Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320, 1332–1336 (2008).
  CAS PubMed PubMed Central Google Scholar
• Neale, M. J. & Keeney, S. Clarifying the mechanics of DNA strand exchange in meiotic recombination. Nature 442, 153–158 (2006).
  CAS PubMed PubMed Central Google Scholar
• Keeney, S., Lange, J. & Mohibullah, N. Self-organization of meiotic recombination initiation: general principles and molecular pathways. Annu. Rev. Genet. 48, 187–214 (2014).
  CAS PubMed PubMed Central Google Scholar
• Hunter, N. Meiotic recombination: the essence of heredity. Cold Spring Harb. Perspect. Biol. 7, a016618 (2015).
  PubMed PubMed Central Google Scholar
• Zickler, D. & Kleckner, N. Recombination, pairing, and synapsis of homologs during meiosis. Cold Spring Harb. Perspect. Biol. 7, a016626 (2015).
  PubMed PubMed Central Google Scholar
• Zickler, D. & Kleckner, N. Meiotic chromosomes: integrating structure and function. Annu. Rev. Genet. 33, 603–754 (1999).
  CAS PubMed Google Scholar
• Schucker, K., Holm, T., Franke, C., Sauer, M. & Benavente, R. Elucidation of synaptonemal complex organization by super-resolution imaging with isotropic resolution. Proc. Natl Acad. Sci. USA 112, 2029–2033 (2015).
  PubMed Google Scholar
• Prakash, K. et al. Superresolution imaging reveals structurally distinct periodic patterns of chromatin along pachytene chromosomes. Proc. Natl Acad. Sci. USA 112, 14635–14640 (2015).
  CAS PubMed Google Scholar
• Cahoon, C. K. et al. Superresolution expansion microscopy reveals the three-dimensional organization of the Drosophila synaptonemal complex. Proc. Natl Acad. Sci. USA 114, E6857–E6866 (2017). In this study, the 3-dimensional structure of the D. melanogaster synaptonemal complex is examined using SIM. The resulting images indicate that the synaptonemal complex is organized into two distinct layers that might represent separation of the sister chromatids in 3D space.
  CAS PubMed Google Scholar
• Kohler, S., Wojcik, M., Xu, K. & Dernburg, A. F. Superresolution microscopy reveals the three-dimensional organization of meiotic chromosome axes in intact Caenorhabditis elegans tissue. Proc. Natl Acad. Sci. USA 114, E4734–E4743 (2017). In this study, STORM and PALM are used to image protein components of the C. elegans synaptonemal complex, which provides a detailed map of the 3D positions of these proteins and also suggests how they might help to compartmentalize sister chromatids.
  CAS PubMed Google Scholar
• Brown, M. S. & Bishop, D. K. DNA strand exchange and RecA homologs in meiosis. Cold Spring Harb. Perspect. Biol. 7, a016659 (2014).
  PubMed Google Scholar
• Bishop, D. K. RecA homologs Dmc1 and Rad51 interact to form multiple nuclear complexes prior to meiotic chromosome synapsis. Cell 79, 1081–1092 (1994).
  CAS PubMed Google Scholar
• Shinohara, M., Gasior, S. L., Bishop, D. K. & Shinohara, A. Tid1/Rdh54 promotes colocalization of rad51 and dmc1 during meiotic recombination. Proc. Natl Acad. Sci. USA 97, 10814–10819 (2000).
  CAS PubMed Google Scholar
• Kurzbauer, M. T., Uanschou, C., Chen, D. & Schlogelhofer, P. The recombinases DMC1 and RAD51 are functionally and spatially separated during meiosis in Arabidopsis. Plant Cell 24, 2058–2070 (2012).
  CAS PubMed PubMed Central Google Scholar
• Bishop, D. K. et al. High copy number suppression of the meiotic arrest caused by a dmc1 mutation: REC114 imposes an early recombination block and RAD54 promotes a DMC1-independent DSB repair pathway. Genes Cells 4, 425–444 (1999).
  CAS Google Scholar
• Brown, M. S., Grubb, J., Zhang, A., Rust, M. J. & Bishop, D. K. Small Rad51 and Dmc1 complexes often co-occupy both ends of a meiotic DNA double strand break. PLoS Genet. 11, e1005653 (2015). In this study, STORM imaging is used to probe the properties of presynaptic complexes during meiosis and provides direct evidence that RAD51 and DMC1 co-occupy DNA ends and that these complexes are much smaller than had been previously anticipated.
  PubMed PubMed Central Google Scholar
• Lesterlin, C., Ball, G., Schermelleh, L. & Sherratt, D. J. RecA bundles mediate homology pairing between distant sisters during DNA break repair. Nature 506, 249–253 (2014). This study provides an important example of live-cell SIM being used to examine the properties of a recombination protein–DNA complex in E. coli , which provides new insights into how RecA might act while trying to align DNA sequences in vivo.
  CAS PubMed Google Scholar
• Lisby, M. & Rothstein, R. Cell biology of mitotic recombination. Cold Spring Harb. Perspect. Biol. 7, a016535 (2015).
  PubMed PubMed Central Google Scholar
• Reindl, J. et al. Chromatin organization revealed by nanostructure of irradiation induced γH2AX, 53BP1 and Rad51 foci. Sci. Rep. 7, 40616 (2017).
  CAS PubMed PubMed Central Google Scholar
• Liu, B., Baskin, R. J. & Kowalczykowski, S. C. DNA unwinding heterogeneity by RecBCD results from static molecules able to equilibrate. Nature 500, 482–485 (2013).
  CAS PubMed PubMed Central Google Scholar