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Supplementary Figure Legends:
Figure 1.
SUA5, KAE1 and MTR10 deletion mutants of BY4742 yeast strain are
viable
The SUA5, KAE1 and MTR10 heterozygote cells were sporulated and dissected
on YPD plate.
Photographs were taken when spores were grown for 3 days.
The
deletion mutants were viable and grew slowly.
Figure 2.
Shortened telomeres in sua5 cells are not by-products
(A) Ten-fold serial dilutions of wild type and sua5 cells were dotted on YPD
(dextrose), YPE (ethanol), YPG (glycerol) and YPL (lactate acid) plates.
The
mitochondrion-defective mutant cannot grow on non-fermentable media (YPE,
YPG and YPL).
The sua5 cells were dotted firstly and cultureed for 2 days
The photographs were taken when sua5
before the wild type cells were dotted.
cells were grown for 4 days, and the WT cells were grown for 2 days.
(B) Shortened telomeres in sua5 cells are not by-products of the mitochondrial
defect.
Loss of mitochondrion DNA (0) does not affect the telomere length (left
panel).
Deletion of CYC1 whose expression level is decreased in sua5 cells
does not affect telomere length either.
(C) SUA5 is not involved in nonsense mRNA decay (NMD) pathway. The total
RNAs of the corresponding strains were separated on a denatured-agarose gel,
transferred to nylon membrane, and hybridized with a CYH2 probe.
CYH2
pre-mRNA accumulated in the nmd2 cells, serving as a positive control (Cui et
al, 1995).
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(D) SUA5 does not affect rRNA maturation.
The schematic diagram (left panel)
presents the maturation processes of the rRNA in budding yeast.
The numbers
on top indicates the relative position for the five primer sets that were used to
examine the amount of rRNA processing products (right panel).
The PCR
products of the primer set 1 represent the total rRNA (including the intermediates
and matured), whereas the PCR products of the other sets of primers represent the
different processing products as indicated. Nsr1 is a known rRNA maturation
factor, and the RNA levels in nsr1 cells serve as positive controls (Lee et al,
1992).
Figure 3.
The left telomere of chromosome I (Telomere I-L) in the sua5
mutant progressively shortens with the increasing division cycles
(A) Single telomere shortens during early passages in sua5 cells.
Telomere I-L was
amplified with telomere PCR (Forstemann et al, 2000; Lee et al, 2007). The
telomere PCR products are bracketed, and two independent clones are shown.
(B) Analysis of the sequenced telomeres. The sequences of Tel01L of the first- and
fifth-restreaked sua5 cells are displayed and analyzed as in Figure 2A. The
sequences from two independent clones corresponding to (A) are shown.
Figure 4.
Sua5p is not a telomere capping factor
(A) The SUA5 deletion cells do not show temperature sensitivity. Ten-fold serial
dilutions of wild-type and sua5 cells were dotted on YPD plate and cultured at
30 or 37 oC.
The sua5 cells were dotted firstly and cultureed for 2 days before
the wild type cells were dotted.
The photographs were taken when sua5 cells
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were grown for 4 days, and the WT cells were grown for 2 days.
(B) The single-stranded telomeric DNA does not accumulate in the sua5 cells.
Quantitative amplification of single-stranded DNA (QAOS) method (Booth et al,
2001) was applied to measure the telomeric single-stranded DNA in sua5 cells.
The single-stranded TG DNA at “YER186C” locus which lies 15 kb away from
Telomere V-R was examined. The cdc13-1 cells which were cultured at the
permissive and restrictive temperatures serves as positive controls.
(C) Deletion of EXO1 does not restore the shortened telomeres of sua5 cells. The
genomic DNA from the isogenic strains (labeled on top) was digested with XhoI,
and hybridized with a TG probe.
Figure 5.
Over-expression TLC1 RNA in wild-type and sua5 cells.
TLC1 RNA was over-expressed under the control of a GPD promoter in both
wild type and sua5 cells.
Northern blot was performed to examine TLC1
expression levels from the indicated strains with TLC1 and snoRNA probes.
Figure 6.
Sua5 is conserved among Fungi
(A) Alignment
of
S.
cerevisiae
Sua5p
Schizosaccharomyces pombe homologs.
with
Candida
albicans
and
The alignment was performed with
Clustal X and edited with Genedoc software.
(B) S. pombe Sua5p and C. albicans Sua5p complement the depletion of S. cerevisiae
Sua5p. The SpSUA5 and CaSUA5 were over-expressed under control of a TEF2
promoter.
Figure 7.
The purified recombinant Sua5p specificaly binds single-stranded
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telomeric DNA in vitro
(A) Sua5p does not bind single-stranded C1-3A DNA.
The CA20 probe
(5’-CACCACACCCACACACACCA-3’) was subjected to the same reactions
described in Figure 5D.
The protein-DNA complex wasn’t detected.
(B) Sua5p does not bind double-stranded telomeric DNA.
A double-stranded
telomere probe (TG20/CA20) was subjected to the gel shift reactions, and the
protein-DNA complex wasn’t detected.
Rap1p which binds double-stranded
telomeric DNA serves as a positive control (right panel).
Figure 8.
Sua5p three-dimensional structure modeling and structure-directed
mutagenesis
(A) Alignment of S. cerevisiae Sua5p with Sulfolobus tokodaii Sua5p, Escherichia
coli yrdC, and Pyrococcus horikoshii SUA5 domain.
latter three proteins are list in the figure:
The PDB codes of the
Sulfolobus tokodaii Sua5p (2EQA);
Escherichia.coli yrdC (1HRU); Pyrococcus horikoshii SUA5 domain (2YV4).
The alignment was performed with Clustal X and edited with Genedoc software.
(B) Structural representation of sua5 mutant.
Yellow residues indicate non-essential
mutations, and red residues show loss-of-function sua5 mutations.
Figure 9.
The recombinant fungal Sua5 proteins do not show ATPase or
GTPase activity
(A) ScSua5p, SpSua5p and CaSua5p were purified in E.coli.
The GST-fusion
proteins were over-expressed, and purified with the Glutathione-affinity column.
The GST-tag was cleaved by PreScission protease. The proteins were further
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purified with Q-sepharose and Superdex-200 columns.
The purified
recombinant proteins are shown by Coomassie blue stained gel.
(B) Proteins were added in the ATPase reactions (25 mM HEPES, pH 7.6; 5 mM
MgCl2, 2 mM ATP, 1 mM DTT, 100 g/ml bovine serum albumin (BSA), 0.5 Ci
of [-32P]ATP) at 37 ºC for 30 min.
About 100 ng of hPif1p (Zhang et al, 2006)
was used as a positive control and 1g of Sua5p was added in the reactions.
About 200 g/ml single-stranded telomeric DNA or RNA were added in the
reaction.
(C) GTPase assay was carried out as (B) except GTP and [-32P]GTP were used
instead of ATP and [-32P]ATP.
Reference:
Booth C, Griffith E, Brady G, Lydall D (2001) Quantitative amplification of single-stranded DNA
(QAOS) demonstrates that cdc13-1 mutants generate ssDNA in a telomere to centromere direction.
Nucleic Acids Res 29: 4414-4422
Cui Y, Hagan KW, Zhang S, Peltz SW (1995) Identification and characterization of genes that are
required for the accelerated degradation of mRNAs containing a premature translational termination
codon. Genes Dev 9: 423-436
Forstemann K, Hoss M, Lingner J (2000) Telomerase-dependent repeat divergence at the 3' ends of
yeast telomeres. Nucleic Acids Res 28: 2690-2694
Lee JY, Kozak M, Martin JD, Pennock E, Johnson FB (2007) Evidence that a RecQ helicase slows
senescence by resolving recombining telomeres. PLoS Biol 5: e160
Lee WC, Zabetakis D, Melese T (1992) NSR1 is required for pre-rRNA processing and for the proper
maintenance of steady-state levels of ribosomal subunits. Mol Cell Biol 12: 3865-3871
Zhang DH, Zhou B, Huang Y, Xu LX, Zhou JQ (2006) The human Pif1 helicase, a potential
Escherichia coli RecD homologue, inhibits telomerase activity. Nucleic Acids Res 34: 1393-1404
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