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Dna2 nuclease deficiency results in large and complex DNA insertions at chromosomal breaks

Nature volume 564, pages 287–290 (2018)Cite this article

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Abstract

Insertions of mobile elements1,2,3,4, mitochondrial DNA5 and fragments of nuclear chromosomes6 at DNA double-strand breaks (DSBs) threaten genome integrity and are common in cancer7,8,9. Insertions of chromosome fragments at V(D)J recombination loci can stimulate antibody diversification10. The origin of insertions of chromosomal fragments and the mechanisms that prevent such insertions remain unknown. Here we reveal a yeast mutant, lacking evolutionarily conserved Dna2 nuclease, that shows frequent insertions of sequences between approximately 0.1 and 1.5 kb in length into DSBs, with many insertions involving multiple joined DNA fragments. Sequencing of around 500 DNA inserts reveals that they originate from Ty retrotransposons (8%), ribosomal DNA (rDNA) (15%) and from throughout the genome, with preference for fragile regions such as origins of replication, R-loops, centromeres, telomeres or replication fork barriers. Inserted fragments are not lost from their original loci and therefore represent duplications. These duplications depend on nonhomologous end-joining (NHEJ) and Pol4. We propose a model in which alternative processing of DNA structures arising in Dna2-deficient cells can result in the release of DNA fragments and their capture at DSBs. Similar DNA insertions at DSBs are expected to occur in any cells with linear extrachromosomal DNA fragments.

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Fig. 1: Dna2 inhibits large insertions at DSBs.
Fig. 2: Dna2 limits mobility of transposable elements.
Fig. 3: Origin of inserted DNA at DSBs.
Fig. 4: Insertions originate from regions where replication forks stall.

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Data availability

All data supporting the findings of this study are available within the Letter. Sequences of all inserted DNA and sequences of the junctions analysed are provided in Supplementary Table 1. Source gel images are presented in Supplementary Figure 1.

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Acknowledgements

We thank A. Gabriel, D. J. Garfinkel, J. Haber, M. G. Blanco and F. Storici for the gifts of strains and plasmids, and J. Haber and P. Hastings for critical reading of the manuscript. This work was funded by grants from the US National Institutes of Health (GM080600 and GM125650 to G.I., GM125632 and HL133254 to K.C.) and the Cancer Prevention Research Institute of Texas (RP140456 to G.I. and G.P., RP150611 to K.C.).

Reviewer information

Nature thanks P. Cejka, L. Symington and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Author notes

  1. These authors contributed equally: Yang Yu, Nhung Pham, Bo Xia

Authors and Affiliations

  1. Baylor College of Medicine, Department of Molecular and Human Genetics, Houston, TX, USA

    Yang Yu, Nhung Pham, Alma Papusha, Zhenxin Yan & Grzegorz Ira

  2. Center for Bioinformatics and Computational Biology, Department of Cardiovascular Sciences, Houston Methodist Research Institute, Houston, TX, USA

    Bo Xia, Guangyu Wang & Kaifu Chen

  3. Institute for Academic Medicine, Houston Methodist Hospital, Houston, TX, USA

    Bo Xia, Guangyu Wang & Kaifu Chen

  4. Weill Cornell Medical College, Cornell University, Department of Cardiothoracic Surgery, New York, NY, USA

    Bo Xia, Guangyu Wang & Kaifu Chen

  5. Department of Clinical Cancer Prevention, University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Guang Peng

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Contributions

Y.Y., N.P. and B.X. contributed equally to this work. Y.Y., N.P. and Z.Y. constructed strains; Y.Y. and A.P. performed all experiments related to insertions at DNA breaks; N.P. carried out experiments on transposition; B.X., G.W. and K.C. designed, performed and described bioinformatics analysis. G.I., Y.Y. and G.P. designed the experiments, discussed the data and wrote the manuscript.

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Correspondence to Kaifu Chen or Grzegorz Ira.

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Extended data figures and tables

Extended Data Fig. 1 Insertion analysis at MATa, URA3 and LYS2 loci.

a, Experimental system to study insertions at DSBs and PCR analysis of MATa locus after DSB repair in wild-type and pif1-m2 dna2 cells. Analysis was repeated more than 10 times (for gel source data, see Supplementary Figure 1). b, Analysis of change of DSB ends among insertion events. c, Schematic showing experimental system. A HO break is generated at an ACT1 intron integrated in the URA3 gene. Insertion of a DNA fragment or large deletion interferes with splicing and generates uracil auxotrophs. d, Analysis of insertions by PCR and agarose gel electrophoresis at URA3. The experiment was repeated more than three times with similar results. For gel source data, see Supplementary Figure 1. e, Percentage of insertions among 5-FOA resistant colonies. Data are mean ± s.d.; n = 3 independent experiments; two-tailed t-test. f, Analysis of origin of DNA inserted at DSB at URA3 locus in indicated mutants, n represents number of independent insertions from indicated mutants. g, Percentage of insertions among 5-FOA resistant colonies after transient induction of HO break in rad51 pif1-m2 dna2. Data are mean ± s.d.; n = 3 independent experiments. h, Schematic showing experimental system to follow insertions at CRISPR–Cas9-induced DSBs within the LYS2 locus. Below, percentage of insertions among cells maintaining CRISPR–Cas9 and analysis of origin of DNA inserted at LYS2. n represents number of independent insertions sequenced in pif1-m2 dna2 cells. The experiment was repeated four times with similar results.

Extended Data Fig. 2 Origin of inserted DNA at DSBs.

a, Each triangle indicates a single insertion donor DNA; hotspots of insertion donor DNA are marked in red. b, Scatter plot of chromosome size and insertion number. n = 468 independent insertions. Correlation coefficients were calculated based on the Spearman method. c, Contact analysis between MAT locus on chromosome III and loci from which DNA was inserted. For each replicate, 1,000 random sets of DNAs (equal size and number) are compared to experimental inserted DNA. P values are determined by two-tailed Wilcoxon test; n = 358 independent inserted DNAs used for contact analysis.

Extended Data Fig. 3 Analysis of transposon cDNA stability and Ty1 expression.

a, Analysis of Ty1 cDNA stability. The experiment was repeated four times with similar results. b, Analysis of Ty1 expression and its quantification. Data are mean ± s.d. from three independent experiments. For gel source data, see Supplementary Figure 1.

Extended Data Fig. 4 Hotspots of origin of inserted DNAs.

Position of DNAs inserted within DSBs is indicated in red. Blue boxes, origins of replication; yellow circles, centromeres; green boxes, telomeres; open boxes, genes. Hotspots were defined as loci that are the source of at least two inserted DNA fragments separated from each other by no more than 3 kb.

Extended Data Fig. 5 Genetic interactions between Dna2 and Rad52, Yen1 and Mus81.

a, Overall frequency and analysis of origin of DNA inserted at DSB in indicated mutants. n represents the number of independent insertions analysed by sequencing. b, Origin of insertions in rad52Δ mutant cells. c, Active Yen1 rescues non-viability of dna2Δ cells. Tetrad dissection of PIF1/pif1-m2 YEN1/yen1ON DNA2/dna2Δ triple heterozygotes is shown. The experiment was repeated twice. d, Analysis of complex insertions (2 or more DNA fragments inserted at DSB) in Dna2-deficient mutants. Sample size, defined as the number of independent insertions analysed for each mutant is presented in Extended Data Table 1. χ2 test is used to determine the P value. e, DNA damage sensitivity analysis (spot assay, 5× dilution) in indicated mutants. The experiment was repeated twice.

Extended Data Fig. 6 Model of large insertions at DSBs in Dna2-deficient cells.

a, Unprocessed 5′ flaps are processed by alternative nuclease or displaced by synthesis leading to release of over-replicated DNA fragments. b, Stalled and reversed forks, when approached by a converging fork, leave over-replicated DNA that can be released by processing by other nucleases. c, ssDNA can be inserted into DSBs by NHEJ and Pol4.

Extended Data Fig. 7 Analysis of insertions of transformed DNA at DSBs and analysis of free, short DNA in cells.

a, Analysis of insertions of transformed DNA at DSBs in wild-type and indicated mutant cells. Schematic of the experiment (left) and percentage of cells carrying insertion (right). χ2 test was used to determine the P values; n = 160 for dsDNA and n = 320 for ssDNA, and represents the number of colonies tested for the presence of insertion. b, c, Analysis of inserted DNA after transformation of dsDNA (b) and ssDNA (c). d, Quantitative PCR analysis of short free DNA in indicated mutants. Data are mean ± s.d.; n = 3 independent experiments. Position of the primers used is shown at the top and fold change in DNA amount is shown on the bottom.

Extended Data Table 1 Analysis of NHEJ efficiency and insertion frequency
Full size table
Extended Data Table 2 Sequence analysis of DSB repair survivors in wild type and indicated mutants that do not carry a large insertion
Full size table

Supplementary information

Supplementary Information

This file contains Supplementary Figure 1, which presents gel source data and Supplementary Table 2, which includes the names and genotypes of all strains used in this study.

Reporting Summary

Supplementary Table 1

Sequences of all inserted DNA. For every insertion case, the following information is included: case number, name of the strain where insertion was identified, insertion sequence, insertion size, number of donors (complex events have more than one source of inserted DNA), donor chromosome, general feature of donor DNA (e.g. rDNA, Ty1), SGD link to the sequence inserted, end points nucleotide numbers of the inserted DNA, microhomology and its size at both junctions, changes of DSB ends, microhomology between donors for complex events, and the DNA sequences surrounding donor sequence at its original locus (15 nts upstream and downstream).

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Yu, Y., Pham, N., Xia, B. et al. Dna2 nuclease deficiency results in large and complex DNA insertions at chromosomal breaks. Nature 564, 287–290 (2018). https://doi.org/10.1038/s41586-018-0769-8

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