Multiple Rad5 Activities Mediate Sister Chromatid Recombination to Bypass DNA Damage at Stalled Replication Forks  Eugen C. Minca, David Kowalski  Molecular Cell  Volume 38, Issue 5, Pages 649-661 (June 2010) DOI: 10.1016/j.molcel.2010.03.020 Copyright © 2010 Elsevier Inc. Terms and Conditions

Molecular Cell 2010 38, 649-661DOI: (10.1016/j.molcel.2010.03.020) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 Incomplete Replication of Damaged Chromosomal DNA in the Absence of Rad5 (A) Experimental outline. G1-synchronized cells were released into S phase in the presence of 1 μM adozelesin for 1 hr and allowed to recover in drug-free medium for 18 hr. (B) Ethidium-bromide-stained pulsed-field gels show fully replicated chromosomal DNA during recovery from adozelesin exposure in WT but not in rad5Δ cells. Chromosomes are labeled with Roman numerals. (C–E) rad5Δ cells fail to enter mitosis and undergo nuclear and cellular division after DNA damage, as observed by flow-cytometry measurement of DNA content (C), microscopy (D), and cell counting (E). In (D), pictures of representative cells are an overlay of DAPI fluorescence and bright field. See also Figure S1. Molecular Cell 2010 38, 649-661DOI: (10.1016/j.molcel.2010.03.020) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Role of Rad5 in X-DNA Structure Formation during Replication Fork-Stalling Damage (A) Map of the ORI305 DNA fragment analyzed by 2D-gel electrophoresis of replication intermediates (B, C). (B and C) Rad5 mediates the formation of X-DNA intermediates at active ORI305 during adozelesin exposure in S phase. G1-synchronized WT and rad5Δ cells were released into S phase in drug-free medium (B) or in the presence of 1 μM adozelesin (C) and subjected to 2D-gel analysis at indicated time points. Relevant replication intermediates observed in WT cells are schematically represented in the cartoons. For rad5Δ samples, abnormal Y-shaped replication intermediates are indicated by stars. Relative quantification of the bubble arc, late-Y arc, and X signals is shown in the graphs. Molecular Cell 2010 38, 649-661DOI: (10.1016/j.molcel.2010.03.020) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 Differential Requirement of Rad5 during DNA-Damage Recovery at Early and Late Replication Origins (A and B) Rad5 is required for fork restart at early but not at late replication origins. WT and rad5Δ cells were processed as described in Figure 1A. Samples were taken at the indicated recovery time points for 2D-gel analysis at early-firing ORI305 (A) and late-firing ORI1412 (B). For rad5Δ samples, abnormal Y-shaped replication intermediates at ORI305 are indicated by brackets and bubbled structures created by origin firing at ORI1412 are indicated by arrows. See also Figure S2. Molecular Cell 2010 38, 649-661DOI: (10.1016/j.molcel.2010.03.020) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Rad5-Mediated X-DNA Structures Induced by MMS and Adozelesin during S Phase Contain Holliday Junctions (A) Rad5-dependent branch-migrating X-DNA forms at active ORI305 during MMS exposure in S phase. G1-synchronized WT and rad5Δ cells were released into S phase in the presence of 0.033% MMS for 30 and 45 min and 2D-gel analyzed. Relative quantification of the bubble arc, late-Y arc, and X signal at ORI305 is shown in the graph. A duplicate WT-45 min sample was incubated in branch-migrating conditions between first- and second-dimension electrophoresis. The arrow indicates linearized DNA molecules (1N). (B) The X-DNA induced by adozelesin contains Holliday junctions. G1-synchronized WT cells were released into S phase in the presence of 1 μM adozelesin for 45 min. Four identical samples were 2D-gel analyzed with: no further modifications (Control), incubation in branch migrating conditions between first and second dimension (Branch migration), RuvC resolvase treatment between first and second dimension (RuvC), or mung bean nuclease treatment prior to first dimension (Mung bean nuclease), respectively. The arrow indicates linearized DNA molecules (1N). See also Figure S3. Molecular Cell 2010 38, 649-661DOI: (10.1016/j.molcel.2010.03.020) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Recombination Factors Are Required for X-DNA Formation and for Completion of Chromosomal DNA Replication following Fork-Stalling Damage (A) X-DNA intermediates are not induced by adozelesin in rad51Δ and rad52Δ mutants at active ORI305. Cells were processed and analyzed as in Figure 2C. The graph shows a relative quantification of the bubble arc, late-Y arc and X signal. Abnormal Y-shaped replication intermediates are indicated by stars. See also Figure S4. (B) rad51Δ and rad52Δ mutants fail to complete chromosomal DNA replication and to undergo mitosis after adozelesin exposure in S phase. Samples were analyzed by PFGE and flow cytometry as described in Figure 1. Molecular Cell 2010 38, 649-661DOI: (10.1016/j.molcel.2010.03.020) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 Role of Rad5 Functional Domains in DNA Damage Bypass (A) Schematic representation of the Rad5 domain structure and the point mutation sites used in this study. (B) Rad5 ATPase and ubiquitin-ligase activities are independently required for adozelesin tolerance. G1-synchronized cells were released in S phase with adozelesin for 60 min. Dilutions were spread on YPD plates and colonies were counted after 3 days of growth. Error bars represent standard deviation. (C) rad5 point mutants are deficient in X-DNA formation at stalled forks. Replication intermediates were 2D-gel analyzed at ORI305 and compared with WT samples described in Figure 2C. Relative quantification of the bubble arc, late-Y arc, and X signal is shown in the graphs. For rad5 mutant samples, abnormal Y-shaped replication intermediates are indicated by stars. See also Figure S5. Molecular Cell 2010 38, 649-661DOI: (10.1016/j.molcel.2010.03.020) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 7 Model for Rad5-Dependent Template-Switch DNA Damage Bypass Replication forks are stalled by DNA lesions (filled squares) on the leading strand. Uncoupling between lagging and leading strand synthesis creates limited ssDNA and allows the Rad5-dependent recombinational template-switch bypass, the absence of which results in abnormal replication fork structures. Both Rad5 ATPase and E3-ligase activities, as well as recombination factors, coordinately mediate a template switch at the stalled fork through the formation of Holliday junctions between sister chromatids. The sister chromatid template-switch allows the error-free bypass of lesions, which may be repaired during the process or following replication completion and Holliday-junction resolution (filled triangles). Molecular Cell 2010 38, 649-661DOI: (10.1016/j.molcel.2010.03.020) Copyright © 2010 Elsevier Inc. Terms and Conditions