Replicon dynamics, dormant origin firing, and terminal fork integrity after double-strand break formation

Abstract

In response to replication stress, the Mec1/ATR and SUMO pathways control stalled- and damaged-fork stability. We investigated the S phase response at forks encountering a broken template (termed the terminal fork). We show that double-strand break (DSB) formation can locally trigger dormant origin firing. Irreversible fork resolution at the break does not impede progression of the other fork in the same replicon (termed the sister fork). The Mre11-Tel1/ATM response acts at terminal forks, preventing accumulation of cruciform DNA intermediates that tether sister chromatids and can undergo nucleolytic processing. We conclude that sister forks can be uncoupled during replication and that, after DSB-induced fork termination, replication is rescued by dormant origin firing or adjacent replicons. We have uncovered a Tel1/ATM- and Mre11-dependent response controlling terminal fork integrity. Our findings have implications for those genome instability syndromes that accumulate DNA breaks during S phase and for forks encountering eroding telomeres.

Figure 1. Cell cycle progression, checkpoint activation and DSB processing in response to DSB formation

HO (CY7184) and HO-inc (CY7382) cells were arrested in G1 with α-Factor. HO was induced in G1 for 1 hour. Cells were released in S-phase in the presence of glucose. (A) Cell cycle progression analysis by FACS. (B) Western blot of Rad53. (C) Top, Bsu36I restriction digestion strategy. The ARS305 probe is indicated as a dashed line. Bottom, southern blot with the ARS305 probe. As a loading control the filter was re-hybridized with the R315 probe recognizing a fragment ~200 kb distant on the right of ARS305.

Figure 2. Origin activation in proximity of a DSB

The experimental procedure is the one described in figure 1. (A) Genomic DNA from strains (HO) CY7184 and (HO-inc) CY7382 was purified at the indicated time points, digested with HindIII and analyzed by 2D-gels with the ARS305 probe (dashed line). (B) Genomic DNA was purified from strains (HO) CY6914 and (HO-inc) CY6965, digested with NcoI and analyzed by 2D-gel with the ARS313 probe (dashed line). Arrows indicate termination signal. The genomic locations of the HO cut sites are indicated on top of panels A and B.

Figure 3. Replicon dynamics in the presence of a DSB

The experimental procedure is the one described in figure 1. (A) The numbers indicate the relative distance of the center of each restriction fragment from the ARS305 origin. Genomic DNA from strains (HO) CY7184 and (HO-inc) CY7382 was purified and digested with BamHI or EcoRI prior to 2D gel analysis. (B) The numbers indicate the relative distance of each restriction fragment and of ARS306 from the HOcs. Genomic DNA from strains (HO) CY7184 and (HO-inc) CY7382 was purified and digested with HindIII prior to 2D gel analysis. Arrows indicate resection products that result in ssDNA formation at the second HindIII site on the right of ARS305. As a control of the timing of accumulation of replication intermediates the filters were re-hybridized with the R315 probe recognizing a fragment ~200 kb distant on the right of ARS305. Quantifications of the Y-signal are indicated.

Figure 4. Abnormal transitions of replication forks at the DSB in mre11Δ, tel1Δ and sae2Δ mutants

The experimental procedure is the one described in figure 1. Genomic DNA from wt (CY7184) and mre11Δ (CY7385) (A), wt (CY7184) and tel1Δ (CY8286) (B), wt, sae2Δ (CY8551), mre11Δ and sae2Δmre11Δ (CY8643) (C) strains, was analyzed as in figure 2A. Arrows indicate cruciform and asterisks indicate Y-like intermediates. (D) Filters in C were re-hybridized with a probe recognizing the fragment R1 as in figure 3B. (E) Possible interpretation of the 2D-gel profiles observed in the mutants described above. (E1), cruciform intermediates may result as a consequence of fork reversal (RF). (E2), the cleavage of one arm of RF would generate small Y-like intermediates (red) that would initially migrate in proximity of the monomer spot and following branch migration of the extruded arm, would give rise to bigger Y-like structures (E3). (E4) 2D-gel profile of mre11Δ as an example.

Figure 5

(A) A model for physiological and pathological (dashed rectangles) replication fork transitions and replicon dynamics following DSB formation. The yellow squares indicate the hypothetical structures accumulating by 2D-gels in the mutants. (B) Regulatory pathways controlling the integrity of stalled, damaged or terminal forks.