Lcd1p Recruits Mec1p to DNA Lesions In Vitro and In Vivo John Rouse, Stephen P. Jackson  Molecular Cell  Volume 9, Issue 4, Pages 857-869 (April 2002) DOI: 10.1016/S1097-2765(02)00507-5

Figure 1 Retrieval of the Lcd1p/Mec1p Complex from Cell Extracts with dsDNA (A) Cell lysates were analyzed by Western blotting with anti-MYC antibodies. (B–E) Cell extracts (2 mg protein) were incubated with P1/P2 dsDNA beads (10 μl) at 4°C and binding assays done as described in Experimental Procedures. Analysis was by SDS-PAGE and Western blotting with anti-MYC antibodies (B and D) or anti-HA antibodies (C and E). Ethidium bromide (EtBr) was included where indicated at a final concentration of 50 μg/ml. Where indicated, oligonucleotide P1/P2 was treated with 10,000 Jm−2 of ultraviolet light (254 nm). Strains: (A) W303; JR56 (MYC(13)-MEC1); JR55 (MYC(13)LCD1). (B) W303 (MEC1) and JR56 (MYC(13)-MEC1). (C) W303 (lane 1); YLL476.34/2C (wild-type [wt] HA(9)-MEC1; lanes 2 and 7) and YLL593.1.3 (kinase dead [kd] HA(9)-MEC1, lanes 3–6 and 8). (D) W303 (LCD1); JR55 (MYC(13)-LCD1). (E) chk1Δ cells transformed with pRS415 (WT; lane 7) or pRS415-HA-CHK1 (HA; lanes 1–6). Inputs in each experiment represent 80% of the extract used in each DNA pull-down experiment. Molecular Cell 2002 9, 857-869DOI: (10.1016/S1097-2765(02)00507-5)

Figure 2 Specificity of Interaction of the Lcd1p/Mec1p Complex with DNA DNA binding in cell extracts (2 mg protein) was analyzed as in Figure 1, and Western blot analysis was performed using anti-MYC antibodies (A, C, and D) or antibodies against Rfa1p (B). (A and B) ds72 and ds35 denote dsDNA formed by annealing oligonucleotides P1 and P2 or P4 and P5, respectively, while ss72 denotes oligonucleotide P1 alone. ds72* denotes DNA formed by annealing oligonucleotides P1* and P2*. (C) Increasing amounts of closed circular pRS416 (supercoiled) or EcoRI-digested pRS416 (linear), corresponding to 2-, 5-, and 10-fold molar excess over dsDNA on beads, were included in the DNA binding reaction. (D) Lanes 1 and 2 are duplicates, as are lanes 3 and 4. Strains: (A and C) JR55 (MYC(13)-LCD1) and JR56 (MYC(13)-MEC1). (B) JR55. (D) JR55 (W303 MYC(13)-LCD1); JR56 (W303 MYC(13)-MEC1); RI59 transformed with pNML1 (ku80Δ MYC(13)-LCD1); and JR68 (ku80Δ MYC(13)-MEC1). Molecular Cell 2002 9, 857-869DOI: (10.1016/S1097-2765(02)00507-5)

Figure 3 Lcd1p Loads Mec1p onto DNA In Vitro Independently of Rad9p/Rad17p/Rad24p DNA binding in cell extracts was analyzed as described in Figure 1 followed by Western blotting with anti-MYC antibodies. Where indicated, DNA was treated with 10,000 Jm−2 UV-C. The right panels in (A) and (B) and the bottom panel in (C) show Western blots of cell extracts before DNA binding. Strains: (A) W303 transformed with pBAD79 ([RNR3]); JR58 (lcd1Δ [pRNR3]); JR58 transformed with pLCD1. (B) JR66 (sml1Δ MYC(13)-LCD1); JR67 (mec1Δsml1Δ MYC(13)-MEC1); JR67 transformed with YCp-MEC1. (C) JR55 (W303 MYC(13)-LCD1), JR62 (A364a MYC(13)-LCD1), and JR61 (A364a rad9Δ/rad17Δ/rad24Δ/mec3Δ MYC(13)-LCD1). Molecular Cell 2002 9, 857-869DOI: (10.1016/S1097-2765(02)00507-5)

Figure 4 Mutations in the Lcd1p Basic Motif Abrogate DNA Binding by Lcd1p/Mec1p (A) 10-fold serial dilutions of Δlcd1 cells (strain JR57) carrying empty vector (pRS416), pLCD1 (LCD1-WT), pNML1 (MYC(13)-LCD1-WT), pNML-K178A, or pNML-R180A were spotted on synthetic complete medium lacking uracil (SC–URA) in the presence or absence of 50 mM HU, or irradiated with 50 Jm−2 UV-C before incubation at 30°C for 3 days. (B and C) Extracts from lcd1Δ cells expressing HA(9)-Mec1p (strain JR64) carrying empty vector (pRS416), pNML1, or pNML1-K178A or R180A mutants were immunoprecipitated with anti-MYC antibodies, then analyzed by Western blotting with anti-MYC antibodies (top panel) or anti-HA antibodies (bottom panel). The last three lanes in each panel of (C) show protein levels in whole-cell extracts. (D) Affinity-purified recombinant Lcd1p (see Supplemental Data at http://www.molecule.org/cgi/content/full/9/4/857/DC1) was analyzed by SDS-PAGE, followed by silver staining or Western blotting with anti-GST antibodies as indicated. (E) DNA binding to P1/P2 DNA was analyzed in extracts from lcd1Δ cells expressing MYC(13)-Mec1p (strain JR58) as in Figure 1, after incubation for 30 min at 4°C in the presence of 100 ng GST, or 1, 4, 12, or 20 ng of GST-Lcd1-His6 followed by Western blot analysis with anti-MYC antibodies. The first lane shows similar DNA binding analysis of MYC(13)-Mec1p from LCD1 cells (strain JR56). (F) Purified GST-Lcd1 (wild-type)-His6, GST-Lcd1 (K180A)-His6 (referred to as KA), or GST-Lcd1 (R180A)-His6 (referred to as RA) (10 ng each) was subjected to DNA binding analysis using P1/P2 dsDNA or P1 ssDNA, followed by Western blotting with anti-GST antibodies. The last three lanes show 100% of each protein input. (G) Purified GST-Lcd1-His6 (15 ng; upper panel) or GST alone (lower panel) was incubated with 5 μg BSA (as carrier protein; lanes 1–3) or extract (1 mg) from lcd1Δ cells (lanes 4–6) and subjected to DNA binding analysis, using P1/P2 dsDNA or P1 ssDNA, followed by Western blotting with anti-hexahistidine antibodies. Lane 7 shows 100% of input. Molecular Cell 2002 9, 857-869DOI: (10.1016/S1097-2765(02)00507-5)

Figure 5 Lcd1p Is Recruited to cdc13-1-Induced DNA Damage In Vivo (A and C) Cells were released from G1 at 23°C or 37°C for 30 or 60 min, as indicated (A) or for 60 min (C), before ChIP analysis of anti-MYC immunoprecipitates. Serial dilutions of DNA were analyzed by dot blots with radioactively labeled telomeric DNA ([A], upper panel, and [B] and [C]), or a GAL10 probe ([A], lower panel). (B) Cells were released at 23°C into medium containing 0.15 mg/ml nocodazole for 2.5 hr until large budded cells accounted for at least 95% of the cell population. At this point, cells were either maintained at 23°C or shifted to 37°C for a further 60 min, before ChIP analysis as above. (D) Cells were released from G1 at 23°C or 37°C, as indicated, for 120 min. After cell lysis, extracts were analyzed for Rad53p kinase activity in situ (upper panel) or by Western blotting with antibodies against 3-phosphoglycerate kinase (3-PGK) as loading control (lower panel). (E) Cells were released from G1 at 23°C or 37°C, for the indicated times. After cell lysis, extracts were analyzed for Rad53p kinase activity in situ (upper panel) or by Western blotting with antibodies against Rad53p (lower panel). Strains: (A) cdc13-1 lcd1Δ cells (strain JR69) transformed with pLCD1 or pNML1 (MYC(13)-LCD1). (B) cdc13-1 lcd1Δ cells (strain JR69) transformed with pNML1. (C) cdc13-1 lcd1Δ cells (strain JR69) transformed with pLCD1, pNML1, pNML-K178A, or pNML-R180A. (D) cdc13-1 LCD1 cells (strain DDX); cdc13-1 lcd1Δ cells (strain JR69) transformed with pRS416 (vector), pNML1 (WT), pNML-K178A, or pNML-R180A. (E) Strain DDX. Molecular Cell 2002 9, 857-869DOI: (10.1016/S1097-2765(02)00507-5)

Figure 6 Lcd1p Is Targeted to cdc13-1-Induced DNA Damage In Vivo Independently of Mec1p, Rad9p, and Rad24p (A and B) Cells were released from G1 at the permissive (23°C) or restrictive (37°C) temperature for cdc13-1 for 60 min before ChIP analysis of anti-MYC immunoprecipitates. Serial dilutions of DNA were analyzed by dot blot with radioactively labeled telomeric DNA. (C) Cells from (A) and (B) were released from G1 at the indicated temperature for 60 min, and extracts were analyzed for Rad53p in situ kinase activity. Strains: (A) cdc13-1 lcd1Δ cells (strain JR69; denoted MEC1) or cdc13-1 lcd1Δ mec1Δ (strain JR70; denoted mec1Δ), both transformed with pNML1 (MYC(13)-LCD1). (B) cdc13-1 RAD9RAD24 cells (strain DLY408) or cdc13-1 rad9Δrad24Δ cells (strain DLY411) both transformed with pNML1 (MYC(13)-LCD1). Molecular Cell 2002 9, 857-869DOI: (10.1016/S1097-2765(02)00507-5)

Figure 7 Recruitment of Mec1p to Sites of DNA Damage In Vivo Requires Lcd1p Cells were released from G1 at 23°C or 37°C for 30 min (A) or 60 min (A–C), before ChIP analysis of anti-MYC immunoprecipitates. Serial dilutions of DNA were analyzed by dot blot with radioactively labeled telomeric DNA ([A], upper panel, and [B] and [C]) or GAL10 probe ([A], lower panel). Strains: (A) cdc13-1 cells (DDX, denoted MEC1) and cdc13-1 MYC(13)-MEC1 cells (strain JR51). (B) cdc13-1 MYC(13)-MEC1 cells (strain JR51) transformed with pBAD79 (RNR3); strain JR52 (cdc13-1 MYC(13)-MEC1 lcd1Δ); strain JR52 transformed with pLCD1. (C) cdc13-1 MYC(13)-MEC1 lcd1Δ cells (strain JR52) transformed with pRS416 (vector), pLCD1, pLCD1-K178A, or pLCD1-R180A. (D) Strains JR71 and JR72 were grown to mid-log phase, and glucose or galactose was added to 2% (w/v) for the indicated times. Cells were then subjected to crosslinking and ChIP analysis with anti-MYC antibodies. PCR analysis was performed with two sets of primers in each reaction, corresponding to a region near the HO restriction site at the MATα locus or a region of the SML1 gene. PCR products were electrophoresed on TBE/5% polyacrylamide gels and stained with EtBr. Molecular Cell 2002 9, 857-869DOI: (10.1016/S1097-2765(02)00507-5)