Abstract
DNA combing enables the quantitative analysis of DNA replication, DNA recombination, DNA–protein interaction, and DNA methylation along genomic single DNA molecules at 1 kb resolution. However, DNA combing has been restricted to short 200–500 kb long DNA fragments, which introduces significant bias in data analysis. An improved DNA combing methodology that allows to routinely image Mb-scale single DNA molecules and occasionally up to full-length fission yeast chromosomes is presented in this chapter. DNA combing of Mb-scale single DNA molecules can be applied to accurately measure the dynamic properties of DNA replication such as the rate of origin firing, replication fork velocity, fork directionality and the frequency of fork blockage. In addition, Mb-scale single DNA molecules enable the quantitative analysis of complex genomic rearrangements including gross chromosomal translocations, repetitive DNA sequences, large deletions, and duplications, which are difficult to investigate with deep sequencing strategies.
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References
Patel PK, Arcangioli B, Baker SP, Bensimon A, Rhind N (2006) DNA replication origins fire stochastically in fission yeast. Mol Biol Cell 17(1):308–316
Czajkowsky DM, Liu J, Hamlin JL, Shao Z (2008) DNA combing reveals intrinsic temporal disorder in the replication of yeast chromosome VI. J Mol Biol 375(1):12–19
Kaykov A, Nurse P (2015) The spatial and temporal organization of origin firing during the S-phase of fission yeast. Genome Res 25(3):391–401
Elowitz MB, Levine AJ, Siggia ED, Swain PS (2002) Stochastic gene expression in a single cell. Science 297(5584):1183–1186
Artyomov MN, Das J, Kardar M, Chakraborty AK (2007) Purely stochastic binary decisions in cell signaling models without underlying deterministic bistabilities. Proc Natl Acad Sci U S A 104(48):18958–18963
Di Talia S, Skotheim JM, Bean JM, Siggia ED, Cross FR (2007) The effects of molecular noise and size control on variability in the budding yeast cell cycle. Nature 448(7156):947–951
Herrick J, Bensimon A (1999b) Single molecule analysis of DNA replication. Biochimie 81(8–9):859–871
Heichinger C, Penkett CJ, Bahler J, Nurse P (2006) Genome-wide characterization of fission yeast DNA replication origins. EMBO J 25(21):5171–5179
Hayashi M, Katou Y, Itoh T, Tazumi A, Yamada Y, Takahashi T, Nakagawa T, Shirahige K, Masukata H (2007) Genome-wide localization of pre-RC sites and identification of replication origins in fission yeast. EMBO J 26(5):1327–1339
Bensimon A, Simon A, Chiffaudel A, Croquette V, Heslot F, Bensimon D (1994) Alignment and sensitive detection of DNA by a moving interface. Science 265(5181):2096–2098
Allemand JF, Bensimon D, Jullien L, Bensimon A, Croquette V (1997) pH-dependent specific binding and combing of DNA. Biophys J 73(4):2064–2070
Bensimon D, Simon AJ, Croquette VV, Bensimon A (1995) Stretching DNA with a receding meniscus: experiments and models. Phys Rev Lett 74(23):4754–4757
Grandbois M, Beyer M, Rief M, Clausen-Schaumann H, Gaub HE (1999) How strong is a covalent bond? Science 283(5408):1727–1730
Michalet X, Ekong R, Fougerousse F, Rousseaux S, Schurra C, Hornigold N, van Slegtenhorst M, Wolfe J, Povey S, Beckmann JS, Bensimon A (1997) Dynamic molecular combing: stretching the whole human genome for high-resolution studies. Science 277(5331):1518–1523
Lebofsky R, Bensimon A (2003) Single DNA molecule analysis: applications of molecular combing. Brief Funct Genomic Proteomic 1(4):385–396
Sivakumar S, Porter-Goff M, Patel PK, Benoit K, Rhind N (2004) In vivo labeling of fission yeast DNA with thymidine and thymidine analogs. Methods 33(3):213–219
Georgescu RE, Langston L, Yao NY, Yurieva O, Zhang D, Finkelstein J, Agarwal T, O'Donnell ME (2014) Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork. Nat Struct Mol Biol 21(8):664–670
Pasero P, Bensimon A, Schwob E (2002) Single-molecule analysis reveals clustering and epigenetic regulation of replication origins at the yeast rDNA locus. Genes Dev 16(19):2479–2484
Lebofsky R, Bensimon A (2005) DNA replication origin plasticity and perturbed fork progression in human inverted repeats. Mol Cell Biol 25(15):6789–6797
Cheeseman K, Rouleau E, Vannier A, Thomas A, Briaux A, Lefol C, Walrafen P, Bensimon A, Lidereau R, Conseiller E, Ceppi M (2012) A diagnostic genetic test for the physical mapping of germline rearrangements in the susceptibility breast cancer genes BRCA1 and BRCA2. Hum Mutat 33(6):998–1009
Tuduri S, Tourriere H, Pasero P (2010) Defining replication origin efficiency using DNA fiber assays. Chromosome Res 18(1):91–102
Techer H, Koundrioukoff S, Azar D, Wilhelm T, Carignon S, Brison O, Debatisse M, Le Tallec B (2013) Replication dynamics: biases and robustness of DNA fiber analysis. J Mol Biol 425(23):4845–4855
Kaykov A, Taillefumier T, Bensimon A, Nurse P (2016) Molecular combing of single DNA molecules on the 10 megabase scale. Sci Rep 6:19636
Nemeth A (2014) Methyl-combing: single-molecule analysis of DNA methylation on stretched DNA fibers. Methods Mol Biol 1094:233–241
Moreno S, Klar A, Nurse P (1991) Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol 194:795–823
Hagan IM, Grallert A, Simanis V (2016) Cell cycle synchronization of schizosaccharomyces pombe by centrifugal elutriation of small cells. Cold Spring Harb Protoc 2016(6):pdb prot091231
Bianco JN, Poli J, Saksouk J, Bacal J, Silva MJ, Yoshida K, Lin YL, Tourriere H, Lengronne A, Pasero P (2012) Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing. Methods 57(2):149–157
Beach DH (1983) Cell type switching by DNA transposition in fission yeast. Nature 305:682–688
Toda T, Nakaseko Y, Niwa O, Yanagida M (1984) Mapping of rRNA genes by integration of hybrid plasmids in Schizosaccharomyces pombe. Curr Genet 8(2):93–97
Acknowledgments
This work was supported by Irma T. Hirschl and Charles Revson postdoctoral fellowships and Wellcome Trust Grant to PN [grant number 093917] and The Breast Cancer Research Foundation.
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Kaykov, A., Nurse, P. (2018). Analysis of Fission Yeast Single DNA Molecules on the Megabase Scale Using DNA Combing. In: Singleton, T. (eds) Schizosaccharomyces pombe. Methods in Molecular Biology, vol 1721. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7546-4_2
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DOI: https://doi.org/10.1007/978-1-4939-7546-4_2
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