Break dosage, cell cycle stage and DNA replication influence DNA double strand break response - PubMed (original) (raw)

Break dosage, cell cycle stage and DNA replication influence DNA double strand break response

Christian Zierhut et al. EMBO J. 2008.

Abstract

DNA double strand breaks (DSBs) can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HR). HR requires nucleolytic degradation of 5' DNA ends to generate tracts of single-stranded DNA (ssDNA), which are also important for the activation of DNA damage checkpoints. Here we describe a quantitative analysis of DSB processing in the budding yeast Saccharomyces cerevisiae. We show that resection of an HO endonuclease-induced DSB is less extensive than previously estimated and provide evidence for significant instability of the 3' ssDNA tails. We show that both DSB resection and checkpoint activation are dose-dependent, especially during the G1 phase of the cell cycle. During G1, processing near the break is inhibited by competition with NHEJ, but extensive resection is regulated by an NHEJ-independent mechanism. DSB processing and checkpoint activation are more efficient in G2/M than in G1 phase, but are most efficient at breaks encountered by DNA replication forks during S phase. Our findings identify unexpected complexity of DSB processing and its regulation, and provide a framework for further mechanistic insights.

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Figures

Figure 1

Figure 1

Overview of the assay for the quantification of ssDNA and control experiments. (A) Overview of the assay. PP1–3: primer pairs 1–3. See main text for description. (B) Schematic representation of the three sites analysed for ssDNA formation at ARS607::HOcs. (C) Extensive linear range of qPCR amplification. Serial dilutions of whole-cell DNA extract of strain YCZ64 were used in qPCR of the amplicons at the indicated positions. WCE, whole cell extract. (D) No _Bst_UI activity is retained in the qPCRs and ssDNA is resistant to _Bst_UI digestion. DNA extracted from strain YCZ64 was digested with _Bst_UI and used for qPCR (column 1). Another sample of the same extract was boiled and two-thirds of this was digested with _Bst_UI whereas one-third was mock digested before use in qPCR (column 4). The digested sample was split in two and one half was extracted with phenol/chloroform before use in qPCR (columns 2 and 3). qPCR was performed for all four samples using oligonucleotides OCZ125/OCZ126/OCZ140, amplifying the region 0.3 kb from the HOcs. (E) Analysis of in vitro resection using T7 exonuclease. DNA was extracted from strain YCZ64 after 1 h of HO induction. Top panel: Both strands are amplified with similar efficiencies. DNA was either digested or mock-digested with T7 exonuclease and used as template in qPCR. The graphs show the difference in _C_t values between the two reactions. Middle panel: _Bst_UI digestion of dsDNA interferes with subsequent PCR amplification. DNA was mock-digested with T7 exonuclease and subsequently digested or mock-digested with _Bst_UI. Graphs represent comparisons of _C_t values. Bottom panel: ssDNA is resistant to _Bst_UI digestion. DNA was digested with T7 exonuclease and subsequently either digested or mock-digested with _Bst_UI. DNA was then used in qPCRs. The graphs show comparisons of the _C_t values obtained in the two reactions.

Figure 2

Figure 2

Analysis of DSB processing and checkpoint activation. Cells of strains YCZ101 (1cs, ARS607::_HOcs matHOcs_Δ, panels B and C), YCZ70 (1cs, ARS607::_HOcs matHOcs_Δ DDC2-GFP, panel H) and YHHD180 (2cs, ARS607::HOcs, panels D–G) were used. (A) Overview of the strain used. (B) Analysis of ssDNA formation. (C) Immunoblot, Rad53 autokinase assay and ARS607::HOcs DSB assay by Southern blot analysis. Note that in the Southern blot panel, the disappearance of the band corresponding to the cut locus is due to DSB processing. (D, E) Analysis of DSB processing in a longer G2/M experiment: (D) quantification of DSB processing; (E) immunoblot and DSB assay of the experiment shown in panel D. (F, G) Denaturing slot blot analysis confirming the instability of both strands at a DSB: (F) results from one representative experiment; (G) quantifications of the results. (H) Analysis of Ddc2–GFP focus formation.

Figure 3

Figure 3

Checkpoint activation and DSB resection are dose-dependent processes. Cells of strains YCZ173 (1cs, ARS607::_HOcs matHOcs_Δ), YCZ64 (2cs, ARS607::HOcs MATHOcs), YCZ172 (4cs, ARS607::_HOcs trp1_Δ::_HOcs leu2_Δ::HOcs MATHOcs) were used. 1cs, one HO cut site; 2cs, two HO cut sites; 4cs, four HO cut sites. (A) Overview of the 4HOcs strain. (B, C) Immunoblot, Rad53 autokinase and DSB assays. (D) Analysis of DSB processing.

Figure 4

Figure 4

DSB resection is regulated by both NHEJ-dependent and NHEJ-independent mechanisms in G1. Cells of strains YCZ101 (1cs, ARS607::_HOcs matHOcs_Δ), YCZ136 (1cs, ARS607::_HOcs matHOcs_Δ _dnl4_Δ) and YCZ202 (2cs, ARS607::_HOcs MATHOcs dnl4_Δ) were used. 1cs, one HO cut site. (A) Analysis of DSB processing. (B, C) Immunoblot, Rad53 autokinase and DSB assays.

Figure 5

Figure 5

DNA replication is required for efficient checkpoint activation and stimulates DSB processing. Strains YCZ158 (1cs, ARS607::_HOcs matHOcs_Δ), YCZ151 (2cs, ARS607::HOcs MATHOcs), YCZ166 (1cs, ARS607::_HOcs matHOcs_Δ cdc45-td) and YCZ171 (2cs, ARS607::HOcs MATHOcs cdc45-td) were used. 1cs, one HO cut site. Strains were released from G1 arrest into media containing nocodazole to prevent entry into a second cell cycle. (A) Flow cytometry. (B) Budding index. (C) Immunoblot, Rad53 autokinase and DSB assays. (D) Analysis of DSB processing.

Figure 6

Figure 6

The increased checkpoint activation by DSBs during DNA replication is dependent on Rad9 but not on Mrc1. Cultures of strains YCZ158 (ARS607::_HOcs matHOcs_Δ), YCZ200 (ARS607::_HOcs matHOcs_Δ _mrc1_Δ) and YCZ201 (ARS607::_HOcs matHOcs_Δ _mrc1_Δ) were grown in YPRaff and arrested in G1. HO was induced by shifting the culture to YPGal for 1 h, and cells were subsequently released from the G1 arrest. (A) Immunoblot and Rad53 autokinase analysis. (B) Flow cytometry analysis of DNA content.

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