Double Holliday junctions are intermediates of DNA break repair - PubMed (original) (raw)

. 2010 Apr 8;464(7290):937-41.

doi: 10.1038/nature08868. Epub 2010 Mar 28.

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Double Holliday junctions are intermediates of DNA break repair

Malgorzata Bzymek et al. Nature. 2010.

Abstract

Repair of DNA double-strand breaks (DSBs) by homologous recombination is crucial for cell proliferation and tumour suppression. However, despite its importance, the molecular intermediates of mitotic DSB repair remain undefined. The double Holliday junction (DHJ), presupposed to be the central intermediate for more than 25 years, has only been identified during meiotic recombination. Moreover, evidence has accumulated for alternative, DHJ-independent mechanisms, raising the possibility that DHJs are not formed during DSB repair in mitotically cycling cells. Here we identify intermediates of DSB repair by using a budding-yeast assay system designed to mimic physiological DSB repair. This system uses diploid cells and provides the possibility for allelic recombination either between sister chromatids or between homologues, as well as direct comparison with meiotic recombination at the same locus. In mitotically cycling cells, we detect inter-homologue joint molecule (JM) intermediates whose strand composition and size are identical to those of the canonical DHJ structures observed in meiosis. However, in contrast to meiosis, JMs between sister chromatids form in preference to those between homologues. Moreover, JMs seem to represent a minor pathway of DSB repair in mitotic cells, being detected at about tenfold lower levels (per DSB) than during meiotic recombination. Thus, although DHJs are identified as intermediates of DSB-promoted recombination in both mitotic and meiotic cells, their formation is distinctly regulated according to the specific dictates of the two cellular programs.

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Figures

Figure. 1

Figure. 1. Inducible DSB-repair system

a, Map of the HIS4LEU2-SceI locus showing diagnostic restriction sites and probe positions. DNA species detected following Southern hybridization are shown below. Lollipops indicate _Xho_I restriction site polymorphisms. Diploid strains contain the I-Sce I recognition site on either the Mom or the Dad homolog. IS-JM, inter-sister joint molecule; IH-JM, inter-homolog joint molecule. b, Image of one-dimensional gel hybridized with Probe 4 showing the DNA species detailed in (a). JMs are detected using two-dimensional gel analysis (see Fig. 2). c, Time course analysis of DSB formation and repair in cycling diploid cells following DSB-induction. Sampling intervals are 15 minutes. Cell cycle stage was assessed microscopically.

Fig. 2

Fig. 2. Detection of Joint Molecules during DSB-repair

a, Images of native/native 2D gels hybridized with Probe 4. Panels on the right-hand side show magnifications of the JM region. Black and white arrows highlight the two JM species. b–d, JM signals are dependent on DSB-induction, the Rad51 protein and stabilization by psoralen crosslinking.

Fig. 3

Fig. 3. Analysis of Joint Molecule composition

a, 2D gel analysis of JMs during DSB-repair in cycling haploid cells. b, 2D gel analysis of JMs in diploid cells carrying the I-Sce I recognition site on the Dad homolog. The smaller Dad+Dad intersister JM is highlighted by a caret. The asterisk highlights a high molecular weight species of unknown identity. c, Successive hybridizations of 2D gels with Probe 4 and homolog-specific probes. Mom+Mom intersister JMs and Mom+Dad interhomolog JMs are highlighted by black and white arrows, respectively. d, Native/denaturing 2D gel analysis of the strand composition of JMs. The corresponding native/native 2D gel is shown in the top panel to align JM species with their component strands in the lower panels. The same JM sample was analyzed using two different agarose concentrations in the second dimension. Component strands are more sharply resolved in the 1.2% gel and show that interhomolog JMs comprise primarily parental length strands. The lower detection limit for this experiment is estimated to be 28% recombinant strands, i.e. 28% of interhomolog-JMs could be sHJs and go undetected by this assay.

Fig. 4

Fig. 4. Temporal analysis of JMs in wild-type and sgs1 cells

a and b, 2D analysis of JM formation over the time-course of DSB-repair in wild-type and sgs1 strains. Intersister JMs and interhomolog JMs are highlighted by black and white arrows, respectively. c, Quantitation of DSBs, JMs and recombinants (Recs). DSBs and Recs were quantitated from 1D gel analysis. JMs were quantitated from 2D gels. See Supplemental Figs. S2 and S3 for additional analysis and interpretation of recombinants in this system.

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