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Visualization of recombination-mediated damage bypass by template switching

Nature Structural & Molecular Biology volume 21, pages 884–892 (2014)Cite this article

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Abstract

Template switching (TS) mediates damage bypass via a recombination-related mechanism involving PCNA polyubiquitination and polymerase δ–dependent DNA synthesis. Using two-dimensional gel electrophoresis and EM, here we characterize TS intermediates arising in Saccharomyces cerevisiae at a defined chromosome locus, identifying five major families of intermediates. Single-stranded DNA gaps of 150–200 nt, and not DNA ends, initiate TS by strand invasion. This causes reannealing of the parental strands and exposure of the nondamaged newly synthesized chromatid, which serves as a replication template for the other blocked nascent strand. Structures resembling double Holliday junctions, postulated to be central double-strand break–repair intermediates but so far visualized only in meiosis, mediate late stages of TS before being processed to hemicatenanes. Our results reveal the DNA transitions accounting for recombination-mediated DNA-damage tolerance in mitotic cells and replication under conditions of genotoxic stress.

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Figure 1: TS intermediates formed on YLpFAT7.1 minichromosomes.
Figure 2: X molecule–intermediate isolation and purification.
Figure 3: Representative EM pictures of F1 and F2 families.
Figure 4: Representative EM pictures of F3, F4 and F5 families.
Figure 5: Branch migration assays and asymmetric families of JMs.
Figure 6: Observation of F3 molecules by denaturing spreading and biochemical identification of paranemic pairing.
Figure 7: Hypothetical model of TS.

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Acknowledgements

This work was supported by European Research Council (ERC), Associazione Italiana per la Ricerca sul Cancro (AIRC) and Fondazione Telethon grants to D.B., Swiss National Science Foundation grants PP00P3_135292 and 31003A_146924 to K.Z., C.F. and M.L., and AIRC and Fondazione Telethon grants to M.F. We thank the Center for Microscopy and Image Analysis of the University of Zürich for technical assistance with the EM experiments, I. Psakhye for critical reading of the manuscript, W. Carotenuto at IFOM for initial assistance in drawing the model, members of our laboratories for helpful discussions and FIRC for various support.

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  1. Cindy Follonier

    Present address: Present address: Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA.,

Authors and Affiliations

  1. Istituto Fondazione Italiana per la Ricerca sul Cancro (FIRC) di Oncologia Molecolare (IFOM), Milan, Italy

    Michele Giannattasio, Marco Foiani & Dana Branzei

  2. Dipartimento di Bioscienze, Università degli Studi di Milano, Milan, Italy

    Michele Giannattasio & Marco Foiani

  3. Institute of Molecular Cancer Research, University of Zürich, Zürich, Switzerland

    Katharina Zwicky, Cindy Follonier & Massimo Lopes

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Contributions

M.G. designed and executed the experiments, acquired the EM images, analyzed the data and made the figures. K.Z. and C.F. acquired a subset of EM images and helped with EM data analysis. M.F. conceived the project and discussed the results. M.L. conceived the project, supervised the EM part, analyzed the EM data and commented on the manuscript. D.B. conceived and supervised the project, designed the experiments, analyzed the data and wrote the paper.

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Correspondence to Dana Branzei.

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Integrated supplementary information

Supplementary Figure 1 Features of the minichromosome YLpFAT7.1 and minichromosome-derived template switch–intermediate families.

(a) Schematic representation of the minichromosome YLpFAT7.1 with the location of the leu2d probe. (b) Undigested or BglII digested genomic DNA samples from wild-type (wt, CY11340), sgs1Δ (CY11357), rad51Δ (CY12388), sgs1Δ rad51Δ (CY12390) strains carrying the minichromosome YLpFAT7.1 were analyzed by gel electrophoresis. The minichromosome was visualized by Southern blotting using the leu2d probe. (c) Genomic DNA from wt (CY11340) and sgs1Δ (CY11357) strains was digested with a restriction enzymes mix containing PacI, XhoI, SgrAI, BssHII, SpeI, BclI and PmlI, and an aliquot of the digestion mix was examined on a first dimension gel (0.35% agarose TBE 1X). After ethidium bromide staining, the minichromosome band is visible at the expected molecular weight and the endogenous genomic DNA is cleaved to an average length smaller than 5kb. (d) Number and percentage of X-shaped DNA structures found in the wt and sgs1Δ samples that were assigned as minichromosome-derived structures. (e) Box plot showing the distribution of the ssDNA lengths in F1*-F1-F2 molecules in wt and sgs1Δ cells. Center line, median; box limits, 25th and 75th percentiles; whiskers, 10th and 90th percentiles; dots, outliers; (n), number of samples (molecules) in the data set. The median (M) and average (A) values derived from calculations are displayed. NS, P> 0.05 by t-test with two tails (f) Chart representing the distribution of the JM families in wt and sgs1Δ cells.

Supplementary Figure 2 Representative EM pictures of F1 family of molecules.

(a-c) Total views (left panels) of representative F1 family DNA structures and enlarged views (right panels) of the junction points. Branch sizes until the junction point are reported in kilobases. Two colors, grey and black, in which the length values are displayed, mark the “equal” arms. White arrows mark the junction points and red arrows the ssDNA discontinuities on one branch in immediate proximity to the junction point. Schematic representations of the junction points are shown with a black/grey code to indicate the contributions of the DNA duplexes to the junction and with a black/red code to indicate the ssDNA regions in red and dsDNA regions in black. Scale bars are shown.

Supplementary Figure 3 Representative EM pictures of F2 family of molecules.

(a-c) Total views (left panels) of representative F2 family of DNA structures and enlarged views (right panels) of the junction points. Branch sizes until the junction point are reported in kilobases. Two colors, grey and black, in which the length values are displayed, mark the “equal” arms. White arrows mark the junction points, which have an extended region of homology (double Y-like structures). Black arrows mark the thick DNA filament at the junction point that we infer to be three-stranded for the reasons explained in the main text. Red arrows mark the ssDNA discontinuity on one or two branches of the joint molecule in immediate proximity to the junction point. Black asterisks mark random crosses between DNA filaments. Schematic representations of the junction points are shown with black/grey and black/red codes as in Supplementary Figure 2. Scale bars are shown.

Supplementary Figure 4 Representative EM pictures of F3 family of molecules.

(a-c) Total views (left panels) of representative F3 family of DNA structures and enlarged views (right panels) of the junction points. Branch sizes until the junction point are reported in kilobases. Two colors, grey and black, in which the length values are displayed, mark the “equal” arms. White arrows mark the junction points, which have an extended region of homology (bubble-like structures). Lengths of the dsDNA filaments at the junction points of F3 molecules are reported in white in kilobases. Black asterisks mark random crosses between DNA filaments. Schematic representations of the junction points are shown with black/grey and black/red codes as in Supplementary Figure 2. Scale bars are shown.

Supplementary Figure 5 Representative EM pictures of F3* family of molecules.

(a-c) Total views (left panels) of representative F3* family of DNA structures and enlarged views (right panels) of the junction points. Branch sizes until the junction point are reported in kilobases. Two colors, grey and black, in which the length values are displayed, mark the “equal” arms. White arrows mark the junction points, which have an extended region of homology (bubble-like structures), and red arrows mark the ssDNA discontinuity on one of the dsDNA filaments present at the junction point. Lengths of the DNA filaments at the junction points are reported in white in kilobases. Schematic representations of the junction points are shown with black/grey and black/red codes as in Supplementary Figure 2. Scale bars are shown.

Supplementary Figure 6 Representative EM pictures of F4 family of molecules.

(a-c) Total views (left panels) of representative F4 family of DNA structures and enlarged views (right panels) of the junction points. Branch sizes until the junction point are reported in kilobases. Two colors, grey and black, in which the length values are displayed, mark the “equal” arms. White arrows mark the junction points, which have an extended region of homology (double Y-like structures). Schematic representations of the junction points are shown with black/grey color code as in Supplementary Figure 2. Scale bars are shown.

Supplementary Figure 7 Representative EM pictures of F5 family of molecules.

(a-c) Total views (left panels) of representative F5 family of DNA structures and enlarged views (right panels) of the junction points. Branch sizes until the junction point are reported in kilobases. Two colors, grey and black, in which the length values are displayed, mark the “equal” arms. White arrows mark the junction points and red arrows the ssDNA regions. Schematic representations of the junction points are shown with black/grey and black/red codes as in Supplementary Figure 2. Scale bars are shown.

Supplementary Figure 8 Representative EM pictures of single Holliday junctions and asymmetric F5* and F1* types of molecules.

(a) A representative EM picture of a single HJ-like molecule (sHJ) with a total view of the DNA structure and an enlarged view of the junction point with a schematic representation of the ssDNA regions reported in red is shown. Branch sizes until the junction point are reported in black in kilobases. Schematic representation of the junction point is also shown with a black/grey code to indicate the contributions of two DNA duplexes to the junction. Scale bars are shown. (b-c) Total views of X-shaped DNA structures of F5* family (b) or F1* family (c) and enlarged views of the junction points with schematic representations of the ssDNA regions in red. Branch sizes until the junction point are reported in kilobases. Two colors, blue and black, used to indicate the length values mark the arms belonging to the same DNA duplex involved in the junction. Schematic representations of the junction points are shown with black/grey and black/red codes as in Supplementary Figure 2. Scale bars are shown.

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Giannattasio, M., Zwicky, K., Follonier, C. et al. Visualization of recombination-mediated damage bypass by template switching. Nat Struct Mol Biol 21, 884–892 (2014). https://doi.org/10.1038/nsmb.2888

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