Chromosome length influences replication-induced topological stress

Journal name:
Nature
Volume:
471,
Pages:
392–396
Date published:
DOI:
doi:10.1038/nature09791
Received
Accepted
Published online

During chromosome duplication the parental DNA molecule becomes overwound, or positively supercoiled, in the region ahead of the advancing replication fork. To allow fork progression, this superhelical tension has to be removed by topoisomerases, which operate by introducing transient DNA breaks1. Positive supercoiling can also be diminished if the advancing fork rotates along the DNA helix, but then sister chromatid intertwinings form in its wake1, 2. Despite these insights it remains largely unknown how replication-induced superhelical stress is dealt with on linear, eukaryotic chromosomes. Here we show that this stress increases with the length of Saccharomyces cerevisiae chromosomes. This highlights the possibility that superhelical tension is handled on a chromosome scale and not only within topologically closed chromosomal domains as the current view predicts. We found that inhibition of type I topoisomerases leads to a late replication delay of longer, but not shorter, chromosomes. This phenotype is also displayed by cells expressing mutated versions of the cohesin- and condensin-related Smc5/6 complex. The frequency of chromosomal association sites of the Smc5/6 complex increases in response to chromosome lengthening, chromosome circularization, or inactivation of topoisomerase 2, all having the potential to increase the number of sister chromatid intertwinings3. Furthermore, non-functional Smc6 reduces the accumulation of intertwined sister plasmids after one round of replication in the absence of topoisomerase 2 function. Our results demonstrate that the length of a chromosome influences the need of superhelical tension release in Saccharomyces cerevisiae, and allow us to propose a model where the Smc5/6 complex facilitates fork rotation by sequestering nascent chromatid intertwinings that form behind the replication machinery.

At a glance

Figures

  1. Top1, Top3 and Smc5/6 are required for timely completion of replication on long chromosomes.
    Figure 1: Top1, Top3 and Smc5/6 are required for timely completion of replication on long chromosomes.

    a, Release of replication-induced superhelical tension by topoisomerases or fork rotation. Arrow, replication direction; red/blue arrowheads, topoisomerase 1/2; yellow arrow, fork rotation; (+) Sc, positive supercoil; SCI, sister chromatid intertwinings. See also Supplementary Fig. 1. b, Illustration of chromosomal BrdU labelling during S phase. c, Immunodetection of BrdU incorporation after chromosome separation by PFGE. Chromosomes IV and III are highlighted. d, e, Quantification of BrdU-labelled chromosomes. The signals for Chr IV and III were normalized to total BrdU incorporation. Standard deviations and P-values (t-test, ***P<10−3) are based on n = 27 (wild type) and n = 3 (mutants).

  2. Inactivation of Top2, but not Top1, increases the frequency of Smc6 chromosomal interactions.
    Figure 2: Inactivation of Top2, but not Top1, increases the frequency of Smc6 chromosomal interactions.

    ac, Chromosomal association of Flag-tagged Smc6 in wild-type (a), top2-4 (b) and wild-type CPT-treated (c) cells. Orange peaks, significant chromosomal binding sites; blue horizontal lines, open reading frames. The y axis shows log2 of signal strength; the x axis shows chromosomal coordinates in kilobases. Vertical red lines indicate replication origins. d, Correlation between the number of Smc6 binding sites per kb and chromosome length. Red squares, wild type; black diamonds, top2-4.

  3. The Smc5/6 complex senses chromosome length and circularization of Chr III.
    Figure 3: The Smc5/6 complex senses chromosome length and circularization of Chr III.

    a, b, d, Chromosomal association of Flag-tagged Smc6. Annotation as in Fig. 2. a, b, Normal or fragmented Chr IV; the area highlighted in red shows the chromosomal region displayed in the localization map. d, Linear or circular Chr III. c, Correlation between the number of Smc6 binding sites per kb and chromosome length. Black diamonds, Chr II–III and Chr V–XVI; green triangle, normal Chr IV (~1 532kb); red squares, fragmented Chr IV (~500 and ~1,032kb); cc, correlation coefficient.

  4. The Smc5/6 complex facilitates catenation of an episomal plasmid.
    Figure 4: The Smc5/6 complex facilitates catenation of an episomal plasmid.

    a, Southern blot analysis of pRS316 from indicated cells. Controls for relaxed, supercoiled and linear monomers are shown in the two rightmost lanes. Nicked and supercoiled catenated dimers, as well as supercoiled monomers, are indicated (see Supplementary Fig. 11 and ref. 28). b, c, Percentage of catenated dimers (b) and supercoiled monomers (c) in the G2/M-arrested samples. Standard deviations and P values (t-test, ****P<10−5) are based on results from n = 4 experiments.

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Gene Expression Omnibus

Sequence Read Archive

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Author information

Affiliations

  1. Karolinska Institutet, Department of Cell and Molecular Biology, von Eulers väg 3, 171 77 Stockholm, Sweden

    • Andreas Kegel,
    • Hanna Betts-Lindroos,
    • Takaharu Kanno,
    • Kristian Jeppsson,
    • Lena Ström &
    • Camilla Sjögren
  2. Laboratory of In Silico Functional Genomics, Graduate School of Bioscience, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan

    • Yuki Katou &
    • Takehiko Itoh
  3. Research Center for Epigenetic Disease, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan

    • Katsuhiko Shirahige

Contributions

A.K. performed the PFGE-based assays; A.K. and T.K. the plasmid assays; A.K., H.B.-L. and K.J. the ChIP-on-chip; K.J., Y.K. and K.S. the ChIP sequencing. T.I. and K.S. carried out the computational analysis and C.S. the segregation experiment. H.B.-L., L.S., K.S. and C.S. initiated the study, A.K., K.S. and C.S. continued and finalized its design. A.K. and C.S. wrote the paper. All authors analysed data, discussed the results and commented on the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Original data files from ChIP-sequencing experiments can be found at http://trace.ncbi.nlm.nih.gov/Traces/sra/, accession number SRP004920, and from ChIP-on-chip experiments at http://www.ncbi.nlm.nih.gov/geo/, accession number GSE26263.

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  1. Supplementary Information (1.6M)

    The file contains Supplementary Text, Supplementary Tables 1-2, Supplementary Figures 1-11 and additional references.

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