A Postincision-Deficient TFIIH Causes Replication Fork Breakage and Uncovers Alternative Rad51- or Pol32-Mediated Restart Mechanisms  María Moriel-Carretero, Andrés Aguilera  Molecular Cell  Volume 37, Issue 5, Pages 690-701 (March 2010) DOI: 10.1016/j.molcel.2010.02.008 Copyright © 2010 Elsevier Inc. Terms and Conditions

Molecular Cell 2010 37, 690-701DOI: (10.1016/j.molcel.2010.02.008) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 rad3-102 Cell Response to UV Light (A) Survival curves after exposure to UV-C. (B) Chromosome VII species revealed by hybridization with ADE5,7 in pulsed-field gel electrophoresis (PFGE) of DNA from cells synchronized in G1 and UV-irradiated with 40 J/m2 prior to release. Samples were taken every 20 min after release. Nonlinear chromosomes (NLC), which include replication intermediates, correspond to the signal coming from the gel well. The NLC signal was quantified with respect to the total signal of each lane. FACS profiles are shown. FLC, full-length linear chromosomes. (C) Recombination frequency using the direct-repeat system leu2-k::ADE2-URA3::leu2-k. Error bars indicate SD of three independent experiments. (D) Recombination frequency in response to UV using the plasmid-chromosome system based on leu2-k and leu2-HO and the direct-repeat system his3-Δ5′-his3-Δ3′. Spontaneous recombination is shown by bars to highlight the net increase caused by UV. Error bars indicate SD of three independent experiments. (E) Rad52 foci accumulation in response to UV. Mid-log cultures of cells carrying the RAD52-YFP construct were either directly inspected for foci formation or irradiated and counted 2 hr later. Only cells in S and G2 contained foci and were considered. Error bars indicate SD of three independent experiments. See also Figure S1. Molecular Cell 2010 37, 690-701DOI: (10.1016/j.molcel.2010.02.008) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Analysis of Genetic Interactions of rad3-102 (A) Synthetic lethality combinations of rad3-102 cells with rad52Δ, rad50Δ, mre11Δ, xrs2Δ, and cdc44-8. Tetrads dissected on rich medium are shown. Δ indicates double mutants, which fail to grow. (B) Sensitivity to HU and 4-NQO of different rad3-102 single- and double-mutant combinations. Serial dilutions (10-fold) of exponentially growing cultures are shown. ∗ indicates that 100 mM HU was used. (C) Survival curves after exposure to UV-C of rad3-102 with or without the rad51Δ mutation. Error bars are the SD from three different experiments. Molecular Cell 2010 37, 690-701DOI: (10.1016/j.molcel.2010.02.008) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 Genetic and Physical Evidence of DSBs in rad3-102 Cells (A) UV sensitivity assayed by 10-fold serial dilutions of different mutant combinations of rad3-102, pol32Δ, and rad51Δ on YPAD after different UV-C doses. (B) Tetrad analysis of rad51Δ rad3-102 pol32Δ cross. Squares indicate triple mutants, which fail to grow. (C) Cell cycle profile of the conditional mutant rad3-102 rad52Δ in glucose as compared to simple mutants rad3-102 and rad52Δ. Cells with a GAL1p-RAD52-containing plasmid were allowed to grow in galactose and then transferred for 15 hr into a glucose-containing medium. (D) FACS profiles of cells from mid-log cultures that were transferred to fresh SC medium with 40 mM HU. Samples were taken at the indicated times. (E) Western blot against the phosphorylated form of histone H2A, used as a marker of DSBs, with and without 10 J/m2 UV dose in WT, rad3-102, and rad3-2 cells. Ponceau staining is shown as a loading control. (F) Western blot as in (E) after cells were irradiated with increasing UV doses. Molecular Cell 2010 37, 690-701DOI: (10.1016/j.molcel.2010.02.008) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Characterization of TFIIH and Its Action in rad3-102 Cells (A) FACS profiles of cells from mid-log cultures transferred to fresh SC medium with 40 mM HU. Samples were taken at the indicated times. (B) Effect of RAD2 overexpression in rad3-102 cells. The indicated strains were transformed with a plasmid bearing a GAL1p-RAD2 construction, and 10-fold serial dilutions were plated on SC supplemented with either glucose or galactose. (C) Detection of thymine dimers in DNA from cells irradiated with 10 J/m2 UV. Cells were recovered immediately after irradiation and every 20 min. The membrane was hybridized against total DNA as a loading control. Mean and SD of three different experiments are shown. (D) ChIP of Tfb4-TAP at different times after 80 J/m2 UV irradiation. Inputs and precipitates were normalized with respect to CEN14. Error bars indicate SD. See also Figure S2. Molecular Cell 2010 37, 690-701DOI: (10.1016/j.molcel.2010.02.008) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Analysis of Replication Intermediates by 2D Gel Electrophoresis (A) Replication intermediates monitored at early origin ARS305 and region C in WT and rad3-102 with or without 40 mM HU. Cells were synchronized in G1 with α factor and monitored at different times after release. A scheme of the chromosomal region studied is shown (drawn to scale). Relevant probes are indicated in gray, and restriction sites are indicated in bp. FACS patterns are displayed at the bottom. Accumulation of X-molecules is indicated by asterisks. (B) Quantification of different replication intermediates at region C. Amplified images of WT and rad3-102 at 40 min without HU from (A) are shown. A diagram depicting the expected intermediates at their approximate migration positions within cones A and B is drawn. Signal quantification is shown as a histogram. Molecules in cone B were quantified relative to all molecules extruding from the Y arc. The fraction of cone B molecules not overlapping with cone A is indicated by black bars. Mean and SD of three consecutive time points are shown for each strain and condition. See also Figure S3. Molecular Cell 2010 37, 690-701DOI: (10.1016/j.molcel.2010.02.008) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 Dependence of Aberrant Replication Intermediates on Rad51 Replication intermediates monitored at early origin ARS305 and region C in rad3-102 rad51Δ cells. FACS patterns are displayed at the bottom. Details are as in Figure 5. See also Figure S4. Molecular Cell 2010 37, 690-701DOI: (10.1016/j.molcel.2010.02.008) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 7 Model of Replication Fork Breakage in rad3-102 TFIIH is recruited to the DNA adduct and creates a bubble around it, allowing subsequent cleavage by Rad2/XPG and Rad1-10/XPF-ERCC1. Prolonged attachment of TFIIH at the site of damage inhibits the filling reaction that restores the double helix (see also Figure S5). The abortive ssDNA nicks or gaps give rise to one-ended DSBs during replication. DSBs completely require MRX, Rad52, and Rfc1 for its repair with the sister chromatid, which can proceed via either Rad51-mediated recombination or Pol32-dependent synthesis to rescue the fork breakage and promote replication restart, causing branched intermediates. If another fork arrives from the opposite side, complex structures combining X and Ys would be created. Eventually, topological stress may lead to fork reversal. Molecular Cell 2010 37, 690-701DOI: (10.1016/j.molcel.2010.02.008) Copyright © 2010 Elsevier Inc. Terms and Conditions