Hector et al. Supplemental Material

SUPPLEMENTAL MATERIAL for Hector et al., Mec1p Associates with Functionally-Compromised Telomeres

Supplemental Table S1. Saccharomyces cerevisiae strains used in this study
Strain / Genotype / Reference
JMY73 / MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 sml1::HIS3 / (Mallory and Petes 2000)
KRY22 / MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 TEL1-HA / (Mallory and Petes 2000)
KYR22 yku70 / MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 TEL1-HA yku70::URA3 / (Hector et al. 2007)
KYR22 yku70 exo1∆ / MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 TEL1-HA yku70::URA3 exo1∆::TRP1 / this study
MD78 / MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 sml1::HIS3

MEC1-HA

/ (Mallory and Petes 2000)
MD78 est2 / MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 sml1::HIS3

MEC1-HA est2::TRP1 [pVL291, i.e. YCplac33-EST2-URA3]

/ this study
MD78 yku70 / MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 sml1::HIS3

MEC1-HA yku70::URA3

/ this study
MD78 yku70 exo1∆ / MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 sml1::HIS3

MEC1-HA yku70::URA3 exo1∆::TRP1

/ this study
MD78 exo1∆ / MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 sml1::HIS3

MEC1-HA exo1∆::TRP1

/ this study
MD78 yku70 bar1∆ / MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 sml1::HIS3

MEC1-HA yku70::LEU2 bar1∆:URA3

/ this study
Supplemental Table S2. Oligonucleotides used in this study
Name
/
Sequence

PCR primers for ChIP

ARO1 / CAGCAATTAGTCCTGGAATTCAAGG
ARO1AS / GTCAATTGATTGGTAACCTTGACAT
GAL10S / ACCTCATAGAAGGGAATGTGATGC
GAL10AS / CCGTACTTCAATATAGCAATGAGCAG
VI-R70S / ACGTTTAGCTGAGTTTAACGGTG
VI-R70AS / CATGACCAGTCCTCATTTCCATC
XV-L-26AS / TAACCCTGTCCAACCTGTCT
XV-L-383S / ATACTATAGCATCCGTGGGC
HO1(S) / CCAGATTTGTATTAGACGAGGGACGGAGTGA
HO1(AS) / AGAGGGTCACAGCACTAATACAGCTCGTAAT
HO2(S) / GGTGTCCTCTGTAAGGTTTAGTACTTTTGT
HO2(AS) / TTTATACAGTTTCCCCGAAAGAACAAAAATCA
Telomere PCR primers
TELVIR-88 / AAATGAGGACTGGGTCATGG
dG18-BamHI / CGGGATCCG18
Gene Deletion (dS + dAS) and gene deletion confirmation PCR primers (ES + FAS)
EST2dS / GGAATGAAAAAAAGTAAAGCATGGCAATGAATGACACAAGTGAAATAGAAAGATTGTACTGAGAGTGCAC
EST2dAS / TCCTTATCAGCATCATAAGCTGTCAGTATTTCATGTATTATTAGTACTAACTGTGCGGTATTTCACACCG
EST2ES / CCTTTAGATGAGCTATTGGTGA
EST2FAS / GCTTATTACAAAGTTTGCGGCAA
Yku70dS / tgttaagtgactctaagcctgattttaaaacgggaatattagattgtactgagagtgcac
Yku70dAS / TTGTATGTAACGTTATAGATATGAAGGATTTCAATCGTCTCTGTGCGGTATTTCACACCG
Yku70ES / GGTAGCTACCAAATGACATTC
Yku70FAS / CATCAAATACCCTACCCTACC
BAR1dS / ATCGCCTAAAATCATACCAAAATAAAAAGAGTGTCTAGAAGGGTCATATAAGATTGTACTGAGAGTGCAC
BAR1dAS / TATTTATATGCTATAAAGAAATTGTACTCCAGATTTCTTAATATGTTGGTCTGTGCGGTATTTCACACCG
BAR1ES / TTCTTTAATGATCTTCGCGTGAT
BAR1FAS / GCTTTCCATGTATTAAAAATGACTA
EST2dS / GGAATGAAAAAAAGTAAAGCATGGCAATGAATGACACAAGTGAAATAGAAAGATTGTACTGAGAGTGCAC
EST2dAS / TCCTTATCAGCATCATAAGCTGTCAGTATTTCATGTATTATTAGTACTAACTGTGCGGTATTTCACACCG
EST2ES / CCTTTAGATGAGCTATTGGTGA
EST2FAS / GCTTATTACAAAGTTTGCGGCAA
Exo1d-pRS(S) / ACCACATTAAAATAAAAGGAGCTCGAAAAAACTGAAAGGCGTAGAAAGGAAGATTGTACTGAGAGTGCAC
Exo1d-pRS(AS) / ATTTTCATTTGAAAAATATACCTCCGATATGAAACGTGCAGTACTTAACTCTGTGCGGTATTTCACACCG
EXO1-ES / CTTCAGGTATATCTATATGCTCT
EXO1-FAS / AGGCTTCTTACTCCAACCGTA

PCR primers for confirming the presence of the 3 HA tags in the MEC1-HA allele

Mec1T-S / ACACCATATCAATTACTATCGCC
Mec1T-AS / AAGGGTTTCCAAGATATCCAAAG

Fig. S1. Mec1-HAp retains functional binding to a DNA double-strand break. As a different epitope-tagged version of Mec1p was previously shown to associate with DNA double-strand breaks (DSBs)(Kondo et al. 2001; Rouse and Jackson 2002), we determined whether the epitope-tagged Mec1-HAp used in this work also retained this property. A DSB was induced in Mec1-HAp cells following a previously established method (Kondo et al. 2001) and Mec1-HAp association was monitored by ChIP. Cells were transformed with a centromere plasmid bearing the gene for the yeast HO site-specific endonuclease under the control of the GAL1 promoter (a gift from George Sprague), and grown to early log phase (OD660 of 0.2) on medium that selects for this plasmid but does not induce the promoter (i.e. medium lacking uracil with 2% sucrose). Galactose was then added to a final concentration of 2% (0 hr), cells were grown to a final density of OD660 0.5 (6 hr) and samples were taken for ChIP. A) A schematic of the HO endonuclease site and the 2 sets of PCR primers (Table S2) used to detect association of Mec1-HAp with the induced DSB. ARO1, an internal locus on a separate chromosome (Fig. 1), was used as a negative control for a sequence not associated with the induced DSB. B) The analysis of two independent ChIP experiments shows that Mec1-HAp association increases after induction of the HO endonuclease. The DSB enrichment values are calculated as the ratio of the intensities of the HO1 product relative to ARO1 product in the ChIP experiments with anti-HA Ab, normalized to the same ratio from the ChIP experiments without Ab (as described in the Materials and Methods). The results for the HO2 product were similar to those of HO1. The relative amplifications are similar to previous reports (Kondo et al. 2001; Rouse and Jackson 2002), demonstrating that the Mec1-HAp used in this work is functional for binding to DSBs.

Fig. S2. Recombinationin est2∆ cells occurs at Y’ elements in chromosomal telomeres. To determine whether recombination occurred at chromosomal telomeres other than the VI-R telomere analyzed in Fig. 1, multiple chromosome ends were examined. A) A schematic of the sequence elements at yeast chromosome ends. Yeast telomeric regions contain an X element followed by 0 to 4 repeats of a Y’ element (gray box), with short stretches of TG1-3 (open boxes) present in-between the X and Y’ elements (reviewed in (Louis 1995)). The majority of yeast telomeres in the strains used in this work have a Y’ middle-repetitive element adjacent to the terminal TG1-3 repeats, and a conserved Xho I site (Zakian 1996). The Y’ elements occur in two well-conserved 5.2 and 6.7 kb size variants, and digestion with Xho I releases fragments corresponding to these sizes in telomeres where the Y’ elements are present at 2 or more copies. To analyze recombination at the Y’ elements, DNA from the same culture samples used in the ChIP experiments in Fig. 1 were cleaved with Xho I and monitored by Southern blotting using 32P-labeled TG1-3 sequences as probe (the black boxes, performed as in (Ray and Runge 1998)). B) Southern blot of the MEC1-HA est2∆ cells from different population doublings after loss of the EST2 gene. PD70 cells, which had a subpopulation of rearranged VI-R telomeres (Fig. 1), also show evidence of Y’ telomere recombination. At this population doubling, an increase in intensity of the 5.2 and 6.7 kb bands was observed, as well as a background smear of material that hybridizes to the TG1-3 probe. This hybridization pattern is characteristic of cells entering the survivor pathway (Lundblad and Blackburn 1993; Teng and Zakian 1999)(reviewed in (McEachern and Haber 2006; Runge 2006)) due to an increase in recombination between telomeres that leads to an increase in the number of Y’ repeats at chromosome ends. These results support the conclusion from Fig. 1 that Mec1-HAp telomere association occurs coincident with the appearance of cells in the population that are losing telomere function.

Fig. S3. Representative gels for the ChIP analyses of yku70∆ cells expressing Mec1-HAp or Tel1-HAp. Shown are gels for representative time courses that were used to produce the data in Figure 3. Wild type and yku70∆ cells were grown at 25°C (0 hr) or at 37°C for the lengths of time shown and processed for ChIP with anti-HA antibody as described in the main text. The Input dilutions are, from left to right, 1:3,000, 1:9,000, 1:27,000, 1:81,000 and 1:243,000 of DNA isolated from the Input chromatin that was subjected to the same procedures as the ChIP DNA. All of ChIP samples are within the linear range of amplification of the Input DNA dilution. A) The results from cells expressing Mec1-HAp. VI-R enrichment values at the bottom of the lanes are the values for this experiment, while those shown in Figure 3C are derived from this experiment and a completely separate repeat of this experiment. B) The results from cells expressing Tel1-HAp, presented as in panel A. The Tel1-HAp data in Figure 3E are a result of this experiment and an independent time course.

Supplemental Figure S1.

Supplemental Figure S2.

Supplemental Figure S3.

Supplemental References

Hector RE, Shtofman RL, Ray A, Chen BR, Nyun T, Berkner KL and Runge KW (2007) Tel1p preferentially associates with short telomeres to stimulate their elongation. Mol. Cell 27:851-858.

Kondo T, Wakayama T, Naiki T, Matsumoto K and Sugimoto K (2001) Recruitment of Mec1 and Ddc1 checkpoint proteins to double-strand breaks through distinct mechanisms. Science 294:867-870.

Louis EJ (1995) The chromosome ends of Saccharomyces cerevisiae. Yeast 11:1553-1573.

Lundblad V and Blackburn EH (1993) An alternative pathway for yeast telomere maintenance rescues est1-senescence. Cell 73:347-360.

Mallory JC and Petes TD (2000) Protein kinase activity of Tel1p and Mec1p, two Saccharomyces cerevisiae proteins related to the human ATM protein kinase. Proc. Natl. Acad. Sci. U S A 97:13749-13754.

McEachern MJ and Haber JE (2006) Break-induced replication and recombinational telomere elongation in yeast. Annu. Rev. Biochem. 75:111-135.

Rouse J and Jackson SP (2002) Lcd1p recruits Mec1p to DNA lesions in vitro and in vivo. Mol. Cell 9:857-869.

Runge KW (2006) Telomeres and Aging in the Yeast Model System. In: Conn PM (ed) Handbook of Models for Human Aging, Elsevier Academic Press, New York pp 191-206

Teng SC and Zakian VA (1999) Telomere-telomere recombination is an efficient bypass pathway for telomere maintenance in Saccharomyces cerevisiae. Mol. Cell. Biol. 19:8083-8093.

Zakian VA (1996) Structure, function and replication of Saccharomyces cerevisiae telomeres. Annu. Rev. Genet. 30:141-172.

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