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J. Biol. Chem., Vol. 275, Issue 32, 24921-24927, August 11, 2000
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From the Institute for Biochemistry, University of Munich
(LMU), Feodor-Lynen-Str. 25, D-81377 Munich, Germany,
§ Département de Microbiologie et Infectiologie,
Faculté de Médecine, Université de Sherbrooke,
Sherbrooke, Quebec J1H 5N4, Canada, and
Received for publication, March 27, 2000, and in revised form, May 11, 2000
The Yku heterodimer from Saccharomyces
cerevisiae, comprising Yku70p and Yku80p, is involved in the
maintenance of a normal telomeric DNA end structure and is an essential
component of nonhomologous end joining (NHEJ). To investigate the role
of the Yku70p subunit in these two different pathways, we generated
C-terminal deletions of the Yku70 protein and examined their ability to
complement the phenotypes of a yku70 The human Ku heterodimer, composed of two subunits of 70 and 86 kDa, binds specifically to
dsDNA1 ends and DNA
discontinuities such as gaps and nicks (1, 2). Human Ku together with a
465-kDa catalytic subunit forms the DNA-dependent protein
kinase (3). Mutations of any of these three
DNA-dependent protein kinase subunits cause severe
sensitivity to ionizing irradiation and a defect in V(D)J
recombination, indicating a role in DNA double-stranded break repair
and recombinational events in higher eukaryotes (4, 5).
While DNA double-stranded breaks are repaired mainly by nonhomologous
end joining (NHEJ) in higher eukaryotes, in the yeast Saccharomyces cerevisiae double-stranded breaks are
predominantly repaired by homologous recombination (6). Therefore,
factors involved in NHEJ in S. cerevisiae eluded detection
for a long time. 6 years ago, we identified a DNA-binding protein in
yeast sharing significant functional and sequence homology with the human Ku heterodimer. This protein complex, Yku (formerly known as
Hdfp), is a heterodimer composed of a subunit of 70 kDa, Yku70p (formerly Hdf1p), and one of 80 kDa, Yku80p (formerly Hdf2p) (7, 8).
Yku binds specifically to DNA ends in a sequence-independent manner
(7). Like human Ku, the Yku heterodimer is involved in DNA repair
processes. Recently, it has been shown that the Yku protein is an
essential part of the NHEJ pathway in S. cerevisiae (9-11).
Yeast cells expressing a functional Yku heterodimer can precisely join
cohesive ends of a transformed linearized plasmid, but cells deficient
for one Yku subunit display reduced recircularization efficiency.
Additionally, in Yku A second important role of Yku within the cell is maintenance of
telomere integrity. Disruption of one Yku subunit results in a dramatic
decrease of telomeric repeat lengths (11, 17). In fact, the Yku
heterodimer binds directly to telomeric DNA, as demonstrated by
in vivo cross-linking experiments (18). Furthermore, probing
the DNA arrangements at chromosome ends, an S phase-specific overhang
of the guanosine-rich strand occurs in wild-type cells (19, 20). This
overhang is present throughout the entire cell cycle in
Yku We were thus interested to know whether there are separate domains that
would be implicated specifically in different functions of the Yku
heterodimer. Stepwise increased deletions of C-terminal amino acids of
Yku70p were generated and expressed in a yeast strain carrying a
deletion of YKU70. Taking care that at least the same amount
of the truncated protein was expressed as normally wild-type protein,
we tested whether these proteins could complement the characteristic
phenotypes of the strains lacking Yku. We find that the extreme C
terminus of the Yku70p subunit is important for DNA binding, telomere
maintenance, and NHEJ. Moreover, the data suggest that the last 25 C-terminal amino acids form a small domain essential for telomere
stability but not for DNA binding and NHEJ.
S. cerevisiae Strains, Media, Growth Conditions, and
Transformation--
Strains used in this study are W303-1A, W303aL,
and W303aLh2 as described elsewhere (7, 8). Yeast cell culturing was performed as described elsewhere (8). Yeast transformation was
performed by the lithium acetate method (31).
Gel Retardation Assay--
Gel retardation assays were performed
as described elsewhere (7).
Construction of Mutated YKU70 Genes--
C-terminal truncations
were generated by Bal31 digestion of an
EcoRI-linearized plasmid pGEM4ZHDF1. The deleted fragments were subcloned into pGEM4Z containing a stop codon in all reading frames and sequenced. This cloning strategy resulted in the addition of
several new amino acids to the truncated cDNA. To
YKU70-c09, YKU70-c20, and YKU70-c25
the amino acid sequence GYRALDIN has been added, and GNQL has been
added to YKU70-c30. A PstI/EcoRI fragment, containing the truncated C-terminal part of the
YKU70 gene, and a XhoI/PstI fragment
isolated from the plasmid pRS316HDF1 containing the promoter region and
the N-terminal part of the YKU70 gene were cloned into the
plasmid pRS316 (25). For overexpression experiments, the wild-type or
mutated YKU70 genes were cloned under control of the
ADH1 promoter into the plasmid pAU (URA3 marker)
(8). The wild-type YKU80 gene was cloned under control of
the ADH1 promoter into the plasmid pAH (HIS3
marker) (8).
End-joining Assay--
To generate the plasmids pRS313-kan and
pRS314-kan used for end-joining assays, we isolated the
KanMX gene from the plasmid pUG-6 (32) and cloned it into
the multiple cloning site of the plasmid pRS313 and pRS314,
respectively (25). Plasmids pRS313-kan and pRS314-kan were
digested with the restriction enzyme NcoI to completion as
determined by gel electrophoresis. The restriction enzyme was
inactivated by treatment at 65 °C for 20 min. End-joining assays
were performed as described elsewhere (9). Cells were plated onto SD
medium lacking histidine or tryptophan and containing Geneticin, G418,
(300 µg/ml) for selection of accurately repaired plasmids. The
averages from three independent experiments are given.
Yeast DNA Extraction and Analysis of Telomeric DNA--
Genomic
DNA was isolated from 5-7-ml overnight cultures of yeast cells
expressing a mutated YKU70 gene as indicated using the
commercially available kit from Amersham Pharmacia Biotech. Telomere
blots were performed as described elsewhere (13). Nondenaturing in gel
hybridizations, including the single-stranded and double-stranded DNA
controls, were performed as described previously (33).
Production of Rat Anti-YKU70 Monoclonal Antibodies--
A
GST-Yku70 fusion protein was used for immunization. A DNA fragment
encoding residues 1-207 of YKU70 was cloned into plasmid pGEX-3X (Amersham Pharmacia Biotech) and transformed into E. coli XL1 Blue. Protein expression and purification on
glutathione-Sepharose 4B (Amersham Pharmacia Biotech) were carried out
as described elsewhere (34).
GST-Yku70 fusion protein was injected into Lou/C rats. After a 4-week
interval, a boost was given 3 days before fusion of the rat spleen
cells with the murine myeloma cell line P3 × 63-Ag8.653 (35).
Hybridoma supernatants were tested in an enzyme-linked immunosorbent
assay using bacterial extracts from E. coli expressing either the GST-Yku70 fusion protein or an irrelevant fusion protein. Monoclonal antibodies reacting with Yku70 fusion protein and not with
an irrelevant fusion protein were analyzed in Western blotting. HDF-5F2
(rat IgG2a) was selected for the reaction pattern in Western blot.
Electrophoresis and Immunoblot Detection of Yku70p--
40 µg
of whole cell extract were incubated with Laemmli loading buffer for 2 min at 95 °C, separated on a 12% SDS gel, and analyzed by Western
blotting. The polyvinylidene difluoride membranes (NEN Life Science
Products) were first blocked using TBST (10 mM Tris-HCl, pH
8.0, 150 mM NaCl, 0.05% Tween 20) plus 10% FCS, incubated
with the anti-Yku70 monoclonal antibody HDF-5F2, washed three times
with TBST, and incubated with a second antibody (goat anti-rat IgG and
IgM, peroxidase-conjugated (Dianova, Hamburg, Germany), diluted 1:5000
in TBST plus 10% fetal calf serum). For detection, the Western blot
Chemiluminescence Reagent Plus (NEN Life Science Products) was used.
Generation of YKU70 Deletion Mutants--
The Yku heterodimer
binds to dsDNA ends with high affinity. Gene disruption of either
subunit results in loss of Yku DNA end binding activity in crude
extracts (7). To generate C-terminally truncated Yku70 proteins, we
digested the coding sequence beginning at the 3'-end, using
Bal31 nuclease (see "Materials and Methods"). This
procedure deleted 30-235 amino acids from the C terminus. We then
expressed the truncated proteins under control of the YKU70
promoter from a yeast single copy plasmid in a yku70
deletion strain and measured DNA end binding activity in crude extracts.
In this first set of experiments, even the shortest truncation of only
30 amino acids (Yku70-c30p) abolished Ykup DNA end binding activity
(see below; Fig. 3).
Therefore, the C-terminal 30 amino acids of Yku70p seem to be essential
for function of the Yku heterodimer (see data below). To further
characterize this small C-terminal domain of the Yku70 protein, we
generated three additional mutants (see "Materials and Methods"),
removing nine (Yku70-c09p), 20 (Yku70-c20p), and 25 (Yku70-c25p), amino
acids from the C terminus (Fig. 1).
C-terminal Deletions Influence Stability of Yku70p--
Truncation
of a protein may have an influence on its stability. Therefore, we
tested expression of the various truncated Yku70p subunits using a
monoclonal antibody (HDF-5F2, rat IgG2a) directed against the
N-terminal part of the protein by Western blots. The antibody
recognizes the Yku70p subunit in yeast crude extracts, and there is no
significant cross-reactivity in the vicinity of 70 kDa (Fig.
2A, lanes
1 and 2). Protein levels of Yku70-c09p and
Yku70-c20p, when expressed from the native YKU70 promoter, were slightly reduced compared with the expression level of the full-length protein. However, in these conditions, the amount of
Yku70-c25p is clearly reduced, and Yku70-c30p is not detectable by
Western blot (Fig. 2A, lanes
3-7).
In order to avoid likely influences of reduced protein levels on
phenotype characteristics (see below), we cloned the mutated YKU70 genes under control of the ADH1 promoter.
The expression level of the full-length protein from this promoter is
at least 10-fold higher as compared with the expression from the
YKU70 promoter (Fig. 2B, lanes
2 and 7). Expression levels of Yku70-c09p, Yku70-c20p, and Yku70-c25p are slightly reduced as compared with the
overexpressed full-length protein, but protein levels are still
significantly higher as when they are expressed from the wild-type
promoter (Fig. 2B, lanes 3-5 and
7). Yku70-c30p is detectable under these overexpression
conditions, the amount of protein being comparable with the amount of
wild-type Yku70p expressed from its own promoter (Fig. 2B,
lanes 6 and 7).
In addition, we co-overexpressed both subunits, Yku80p and the
truncated Yku70 proteins, respectively, under control of the ADH1 promoter in a yku70/yku80 double
mutant strain. Analysis of protein extracts by Western blot using the
anti-Yku70p-specific antibody indicates that in this situation, the
level of full-length Yku70 protein now is at least 20-fold higher than
when expressed alone from its own promoter, indicating a stabilization
of the Yku70p subunit by the Yku80p subunit (Fig. 2C,
lanes 1 and 3). Yku70-c09p,
Yku70-c20p, and Yku70-c25p also are overexpressed at levels that are
very comparable with the full-length protein (Fig. 2C,
lanes 4-6). However, even in these conditions,
Yku70-c30p expression is drastically reduced when compared with the
other proteins, but the expression level still is significantly higher than Yku70p expression from its own promoter (Fig. 2C,
compare lanes 1 and 7).
Deletion of 30 C-terminal Amino Acids Abolishes Ykup DNA Binding
Activity--
We then tested mutated versions of the Yku70 protein for
their ability to restore the Yku heterodimer DNA end binding activity in a yku70 deletion strain.
Truncation of up to 25 C-terminal amino acids of Yku70p appears not to
affect DNA binding of the Yku heterodimer. We found no reduction in DNA
end binding activities in crude extracts of a yku70 mutant
strain expressing Yku70-c09p or Yku70-c20p as compared with expressing
the full-length protein (Fig.
3A, lanes
2-6). Although there is a significant reduction in
Yku70-c25p protein amounts in these cells (see Fig. 2A), DNA
end binding activity as measured in this assay is still very comparable
with expressing the wild-type protein (Fig. 3A,
lane 7). However, we cannot detect any
corresponding Yku-DNA complex after expression of Yku70-c30p (Fig.
3A, lane 8). From these results, we
conclude that the C-terminal 25 amino acids of Yku70p are not essential
for DNA binding. Since the DNA binding activity depends completely upon
the formation of the Yku heterodimer (7, 8), these results also
demonstrate that these amino acids are expendable for formation of the
heterodimer.
To verify these results, we measured Yku DNA binding activity in
strains overexpressing the truncated Yku70p versions under the
ADH1 promoter. We did not detect any changes in DNA binding activity of all C-terminal truncated Yku70 proteins compared with the
expression under control of the YKU70 promoter (data not
shown). Although Yku70-c30p is detectable by Western blot analysis in these conditions (Fig. 2B, lane 6),
again no DNA binding activity corresponding to the Yku70-c30p/Yku80p
heterodimer was observed for this protein (data not shown).
Furthermore, we co-overexpressed both subunits, Yku80p and the
truncated Yku70 proteins, respectively, under control of the ADH1 promoter in a yku70/yku80 double
mutant strain. Using the full-length Yku70p in such experiments yielded
a Yku DNA binding activity that is increased at least 20-fold over
expressing Yku70p alone under its native promoter (Fig. 3B,
lanes 2-4). Overexpression of Yku70-c09p,
Yku70-c20p, and Yku70-c25p, respectively, in this setup resulted in no
qualitative changes of DNA binding activity as compared with expression
under control of the YKU70 promoter (Fig. 3A).
However, the DNA binding activity of the active mutant proteins was
increased to a level comparable with the overexpressed wild-type
protein (Fig. 3B, lanes 4-8). Even in
these co-overexpression experiments, Yku70-c30p displayed no DNA
binding activity (Fig. 3B, lane 9),
although significant amounts of the protein were expressed (Fig.
2C, lane 7). These co-overexpression
experiments of the truncated proteins with Yku80p thus confirm the
results obtained by expressing them under the control of the native
YKU70 promoter.
Telomeres Shorten Gradually with Truncation of the Yku70p C
Terminus--
Telomeric repeats are dramatically shortened in strains
deficient for the Yku heterodimer (11, 17). To investigate the effect
of C-terminal deletions of Yku70p on the lengths of telomeric repeats,
we tested the effects of an expression of these mutant proteins under
control of the YKU70 promoter or when they are overexpressed
on telomere length. Yeast telomeres end in about 300 base pairs of a
heterogeneous sequence that can be abbreviated C1-3A/TG1-3. Most telomeres also
contain a conserved subtelomeric element, called Y', just proximal to
these terminal repeats (24). Due to a conserved XhoI site in
this Y' element, most terminal restriction fragments derived from
telomeres of wild-type cells will be about 1.3 kilobase pairs in length
(Fig. 4A, lane
2, bracketed). There are, however, non-Y'-derived
terminal restriction fragments that are of larger sizes, depending of
where the next internal XhoI site is located (Fig.
4A, lane 2, arrows).
Expressing Yku70-c09p in yku70 cells under its own promoter
yielded slightly shortened telomeric repeat tracts (Fig. 4A,
lane 3), although protein levels appear not to be
affected in this experiment (Fig. 2A). This shortening,
however, clearly is not as severe as observed in a yku70
deletion strain transformed with a vector control (Fig. 4A,
lane 1). In strains expressing Yku70-c20p and
Yku70-c25p, respectively, the shortening is increased in a stepwise
fashion (Fig. 4A, lanes 4 and
5), and the telomeric repeat tracts are about as short as in
the deletion strain, when Yku70-c30p is expressed (Fig. 4A,
lane 6).
When the various mutant proteins were overexpressed from the
ADH1 promoter (Fig.
5B), a slight decrease in
telomere length could be detected, and again, overexpression of
Yku70-c30p resulted in telomere lengths comparable with a
yku70 strain (data not shown). However, this pattern did
change in cells in which both one of the various Yku70p constructs and
Yku80p were simultaneously overexpressed (Fig. 4B). In this
case, no telomere shortening is detectable in a strain expressing
either Yku70-c09p, Yku70-c20p, or Yku70-c25p (Fig. 4B,
lanes 3-5). In contrast, overexpression of
Yku70-c30p together with Yku80p cannot prevent telomere shortening even
at these protein levels that are higher than those found for Yku70p in
wild-type cells (Fig. 4B, lane 6).
We therefore conclude that the C-terminal amino acids of Yku70p are
important for telomere length maintenance and that the DNA binding
activity of the Yku heterodimer seems to be essential for telomere
length control.
C-terminal Deletion Mutants of Yku70p Exhibit ssDNA Overhang at the
Telomeres--
It has been shown that yeast chromosome ends acquire a
transient, single-stranded overhang of the G-rich strand (G-tails) specifically during S phase (19, 20). In Yku
In cells co-overexpressing Yku80p and Yku70-c09p, no G-tail signals
were detectable (Fig. 6A,
lanes 5 and 6), while cells overexpressing Yku70-c25p together with Yku80p displayed weak but
clearly perceptible G-tail signals (Fig. 6A,
lanes 7 and 8). Co-overexpression of
Yku70-c30p and Yku80p resulted in G-tail signals nearly as strong as
found in yku70 cells (Fig. 6A, lanes 3, 4, 9, and 10). These
results confirm our finding that the DNA binding activity of Ykup,
abolished in a Yku-c30p mutant, is essential for maintenance of
telomere integrity. Deletion of 9 amino acids from the C terminus has
only a minor effect on telomere length and single-strandedness of the
chromosome end, and these effects can be suppressed by overexpressing
the mutated Yku heterodimer. Deletion of 20 or 25 amino acids from the
C terminus results in a significant increase in single-stranded
G-tails, and overexpression of Yku70-c25p together with Yku80p does not
restore the chromosomal end structure to wild-type level.
The C-terminal Domain of Yku70p Is Not Involved in End
Joining--
Yeast cells normally repair DNA double-stranded breaks by
homologous recombination. In addition, a second pathway exists in yeast, where nonhomologous DNA bearing cohesive ends can be
precisely joined without a loss or addition of nucleotides. It has been shown that this NHEJ pathway in S. cerevisiae depends
on the presence of a functional Yku heterodimer (9). To investigate the
influence of our Yku70p mutations on NHEJ, we generated pRS313-kan and
pRS314-kan plasmids in which the KanMX gene was cloned into
the CEN/ARS plasmid pRS313 and pRS314, respectively (25).
The respective plasmids were linearized using NcoI, a
restriction enzyme cutting inside the start codon of the
KanMX gene. Yeast cells are not able to replicate such
linear plasmids, and recircularization is an essential step for colony
formation on selective medium. After transformation of linearized and
supercoiled control plasmids into yeast strains expressing the
different yku70 mutants under control of the
YKU70 promoter, the cells were plated onto His
Strains expressing Yku70-c09p and Yku70-c20p, respectively, under
control of the YKU70 promoter, on average formed the same number of colonies on G418 medium as a wild-type strain in this end-joining assay, indicating that these truncations did not influence the end-joining capability of Yku (Table
I). The Yku70-c25p-expressing cells
displayed a decrease in accurate end joining to 60% of the wild-type
(Table I). However, since Yku70-c25p is present at reduced levels in
these cells (Fig. 2A), this reduction in NHEJ could simply
reflect a reduced availability of Yku heterodimer for NHEJ.
Overexpression of Yku70-c25p from the ADH1 promoter restores
the protein levels for this truncation to at least wild-type levels
(Fig. 2B), and NHEJ efficiency now is also restored to a
wild-type level (Table I). The strain overexpressing Yku70-c30p formed
no colonies on G418 and behaved in this assay like a
yku70-deficient strain carrying the vector control.
From these data, we conclude that deletion of the C-terminal 25 amino
acids from Yku70p does not significantly influence the end-joining
activity of the Yku heterodimer. DNA binding activity, however, appears
essential for NHEJ, since our construct that abolishes DNA binding
activity, Yku70-c30p, also abolishes end joining.
The Yku heterodimer is a DNA end-binding protein involved in at
least two different pathways: maintenance of a proper chromosomal end
structure and NHEJ. Here we used C-terminal truncation mutants of
the Yku70 subunit to identify and characterize separate domains involved in these functions.
Our data indicate that DNA binding activity is essential for all
functions of the Yku heterodimer. The one mutation, a deletion of 30 C-terminal amino acids in Yku70-c30p, that abolishes DNA binding
activity in all conditions tested, causes a Yku70 On the other hand, the extreme C terminus of Yku70p does appear to
define at least one domain involved especially in maintenance of the
telomere. This conclusion is mainly based on our observations made
using cells expressing a version of Yku70p with the C-terminal 25 amino
acids deleted (Yku70-c25p). When this protein is overexpressed from a
strong promoter, detectable protein levels are at least as high as
those observed for the wild-type protein expressed from its native
promoter (Fig. 2). Using these conditions, our data show that DNA
binding capacity conferred by this protein is indistinguishable from
the wild-type protein (Fig. 3), and NHEJ is fully functional (Table I).
However, these same cells still display shortened telomeres (Fig.
5B) and clearly detectable ssDNA overhangs of the 3'-ends
(Fig. 5A). These data indicate that the extreme C-terminal
domain of Yku70p is specifically involved in maintaining telomere
integrity, while this domain is dispensable for NHEJ, provided that the
protein is expressed at levels comparable with the wild-type protein.
Therefore, for the first time, we were able to separate Ykup functions
in telomere maintenance from functions related to NHEJ.
Simultaneous overexpression of Yku70-c25p and Yku80p results in a
dramatic increase of active protein able to bind to DNA (Fig.
3B). Nevertheless, telomeres of these cells still display weak G-tail signals (Fig. 6A). These results indicate that
Yku70-c25p-associated phenotypes cannot simply be complemented by
increasing the amount of active protein above the levels found for the
wild-type protein.
In extracts derived from Yku70-c30p-expressing cells, DNA end binding
activity is completely abolished, while in extracts derived from
Yku70-c25p-expressing cells, DNA binding seems not to be affected.
Thus, deleting only 5 amino acids more from the Yku70-c25p construct
completely abolishes DNA binding of the Yku complex and renders it
nonfunctional. A previous study examining the human Ku70 subunit has
identified nine domains conserved between human, mouse,
Drosophila, and S. cerevisiae, respectively (see Fig. 1) (26). Deletion of the 159 C-terminal amino acids of human
Ku70p, including domains 8 and 9, abolished DNA binding activity (26).
Another study has shown that deletion of 89 amino acids from the C
terminus of human Ku70p, thereby truncating domain 9 by 7 amino acids,
does not influence DNA binding activity, but DNA repair capacity of the
Ku heterodimer appears to be affected (27). Deletion of 67 amino acids
from the C terminus of human Ku70p abolishes DNA binding activity
analyzed by gel shift assay but not in an immunoprecipitation assay
(28). In the yeast Ku70 protein, the conserved domain 9 is located
closer to the C terminus than in human or mouse Ku70p. Therefore,
deletion of 30 amino acids truncates domain 9 by 9 amino acids
(Yku70-c30p, Fig. 1). This deletion abolishes the DNA binding activity
of the Yku heterodimer as measured in our assays, while truncation of
domain 9 by 4 amino acids in Yku70-25p does not influence DNA binding
activity. These results indicate that this domain 9 is essential for
DNA binding of the Yku heterodimer, since only the last 4 amino acids
of this domain may be dispensable for DNA binding.
It has been shown that DNA binding activity of Ku depends on the
formation of the Ku70-Ku80 heterodimer. For the human protein, heterodimerization seems to be blocked if domains 8 and 9 are deleted
(26). Two dimerization regions, amino acids 1-115 and 430-482,
respectively, have been identified in human Ku70p (28). Consistent with
these results, pull-down assays using human Ku70p deletions fused to
glutathione S-transferase have indicated that there is a
weak Ku70p-Ku80p interaction domain in the N-terminal part (amino acids
1-136) of human Ku70p and a strong one located in the C-terminal part
(amino acids 449-578) (29). In addition, it has been shown that the
stability of human Ku subunits not assembled in the heterodimeric
complex is clearly reduced (30). While we do not know whether a
heterodimerization domain is affected in Yku70-c30p, a lack of
heterodimerization might lead to a decreased stability of the protein,
as observed for the human Ku. The stabilities of the truncated Yku70
proteins generated in the present study are only slightly reduced for
Yku70-c09p and Yku70-c20p, while Yku70-c25p protein levels are
significantly reduced when it is expressed from its native promoter,
and Yku70-c30p is virtually nondetectable in such an experiment (Fig.
2A). Nevertheless, using co-overexpression of Yku70-c30p
with Yku80p, we could achieve Yku70-c30p protein levels that are
comparable with Yku70p in wild-type cells (Fig. 2C, compare
lanes 1 and 7), although Yku70-c30p
levels still are drastically reduced when compared with when Yku70p was co-expressed with Yku80p (Fig. 2C, compare lanes
3 and 7). This instability of Yku70-c30p may thus
be an indication for a diminished heterodimerization followed by
degradation of the free Yku70-c30 protein. Further experiments are
required to determine whether interaction with Yku80p is prevented in
the Yku70-c30p mutant, thereby abolishing Yku function, or whether the
effect is due to a lack of a combination of both dimerization and DNA binding.
Shorter stepwise deletions of C-terminal amino acids did result in
slight, but detectable, effects on telomere structure but did not
interfere with NHEJ. Thus, as mentioned above, the terminal 25 amino
acids of Yku70p appear to form a special domain involved only in
telomere stability but not in DNA binding or end-joining activity of
Ykup. Interestingly, these 25 amino acids contain 8 lysine residues
(Fig. 1). Deletion of 3 of these lysine residues in the mutant
Yku70-c09p causes a weak decrease in telomere length when compared with
wild-type cells. This decrease is more pronounced in Yku70-c20p, where
6 of the 8 lysine residues are deleted. All 8 lysine residues are
removed in the Yku70-c25p mutant, and shortening of telomeres is almost
as severe as observed in a yku70-deficient strain. Lysine
residues may be important for interacting with yet unidentified
protein(s). However, since full NHEJ activity can be achieved in cells
expressing Yku70-c25p at levels comparable with the wild-type protein,
this lysine-rich domain at the C terminus does not appear to be
essential for the end-joining activity of the Yku heterodimer.
Stepwise truncation of the C terminus additionally induces a stepwise
increase in temperature sensitivity for growth (data not shown), which
appears to roughly correlate with telomere shortening and an increase
in ssDNA detectable at the telomeres. However, again, proficiency for
NHEJ clearly does not correlate with these phenotypes. For instance,
while cells expressing the Yku70-c20p display no decrease in NHEJ
(Table I), they are detectably temperature-sensitive (data not shown),
telomeres in these cells are clearly shortened (Fig. 4A),
and ssDNA of the G-rich telomeric strands is detectable (Fig.
5A). These data are consistent with the hypothesis that the
temperature-sensitive growth defect observed in Yku We thank B. Meier, M. Jurk, H. Ibelgaufts, K. Marbach, and S. Gravel for helpful discussions and critical advice.
*
This work was supported by Deutsche Forschungsgemeinschaft
Grant Wi 319/11-3, Project 7, and by Canadian Medical Research Council
Grant MT12616 (to R.J.W.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
A chercheur-boursier of the Fonds de la Recherche en
Santé du Québec.
**
To whom correspondence should be addressed: Institut für
Biochemie der Universität München, Feodor-Lynen-Str.
25, D-81377 München, Germany. Tel.: 49-89-2180-6962; Fax:
49-89-2180-6999; E-mail: fmann@lmb.uni-muenchen.de.
Published, JBC Papers in Press, May 18, 2000, DOI 10.1074/jbc.M002588200
The abbreviations used are:
dsDNA, double-stranded DNA;
ssDNA, single-stranded DNA;
NHEJ, nonhomologous
end joining.
A Short C-terminal Domain of Yku70p Is Essential for Telomere
Maintenance*
,
, GSF-National
Research Center for Environment and Health, Institute of Molecular
Immunology, Marchioninistr. 25, D-81377 Munich, Germany
ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
strain.
Deleting only the 30 C-terminal amino acids of Yku70p abolishes Yku DNA
binding activity and causes a yku phenotype; telomeres
are shortened, and NHEJ is impaired. Using conditions in which at least
as much mutant protein as full-length protein is normally detectable in
cell extracts, deleting only 25 C-terminal amino acids of Yku70p
results in no measurable effect on DNA binding of the Yku protein, and
the cells are fully proficient for NHEJ. Nevertheless, these cells
display considerably shortened telomeres, and significant amounts of
single-stranded overhangs of the telomeric guanosine-rich strands are
observed. Co-overexpression of this protein with Yku80p could rescue
some but not all of the telomere-related phenotypes. Therefore, the
C-terminal domain in Yku70p defines at least one domain that is
especially involved in telomere maintenance but not in NHEJ.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
cells, plasmids are not repaired
accurately but in an error-prone way, yielding molecules that underwent
losses of up to several hundred base pairs at the joining site (9-11).
Several other components of the yeast NHEJ pathway have been
identified. Lig4p, a newly discovered yeast DNA ligase is essential for
end joining and displays significant amino acid sequence homology with
human DNA ligase IV (12). Moreover, Rad50p, Mre11p, and Xrs2p have been
identified as important components of NHEJ (13). Finally, Sir2p, Sir3p, and Sir4p, but not Sir1p, have been suggested to be involved in end-joining processes in yeast (14). After introduction of a single
unrepairable double-stranded break, the Yku heterodimer seems to
protect free DNA ends. For instance, deletion of YKU70 results in an accelerated 5' to 3' degradation of a broken chromosome and prevents adaptation to G2/M arrest after DNA damage
(15). In addition, it has been shown that Yku is released from
telomeres in response to DNA damage and is recruited rapidly to DNA
double-stranded breaks (16).
strains (18). In addition, both Yku subunits interact
genetically with Cdc13p, a protein binding to the single-stranded part
of telomeric DNA and that is essential for telomerase activity in vivo (18, 21). Deletion of Yku also affects the transcriptional silencing of genes in close proximity to telomeres, a process called
telomere position effect (22). Telomere position effect is severely
diminished in Yku cells, although the repression of the
silent mating type loci is maintained normally in the same cells (13,
18, 21). Finally, mutations of the Yku heterodimer affect the
subnuclear organization of yeast telomeres. In Yku mutant cells, the
clustered distribution of telomeric foci is abolished, and telomeric
signals are detected in a more dispersed localization throughout the
whole nucleus (23).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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View larger version (15K):
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Fig. 1.
Protein structure of Yku70 and C-terminal
sequence of the truncated Yku70 proteins used in this study. The
bars indicate nine regions conserved between humans, mice,
Drosophila, and S. cerevisiae. The shaded
area represents ATP-binding motif A. The
striped areas represent potential Leu/Ser
zipper regions. The amino acids in parentheses were added as
a result of the cloning strategy.
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Fig. 2.
Expression levels of Yku70p mutants under
different expression conditions. Monoclonal antibody HDF-5F2 was
used to detect Yku70 proteins in Western blot analysis. The
arrow indicates the position of Yku70p. A,
expression under control of the native YKU70 promoter. 40 µg of crude extract prepared from W303-1A (wild type) or W303aL
(yku70) cells expressing the indicated truncated Yku70
proteins under the control of the YKU70 promoter were used.
B, overexpression of the various Yku70 proteins. 40 µg of
crude extract prepared from W303-1A (wild-type) or W303aL
(yku70) cells expressing the indicated truncated Yku70
proteins under the control of the ADH1 promoter were used.
C, simultaneous overexpression of Yku80p and various Yku70
proteins. 40 µg of crude extract prepared from W303-1A (wild-type)
or W303aLh2 (yku70/yku80) cells expressing Yku80p
and the indicated truncated Yku70 proteins under the control of the
ADH1 promoter were used.
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Fig. 3.
Ykup DNA end-binding assay in
yku70 deletion mutants expressing truncated Yku70
proteins. A 39-base pair dsDNA oligonucleotide, PGK12, was labeled
with Klenow polymerase and used as a probe for DNA end binding
activity. 25 fmol of the oligonucleotide were incubated with crude
extract. A, expression under control of the native
YKU70 promoter. 30 µg of crude extract prepared from
W303-1A (wild-type) or W303aL (yku70) cells expressing the
indicated truncated Yku70 proteins under the control of the
YKU70 promoter were used. B, simultaneous
overexpression of Yku80p and various Yku70 proteins. 5 µg of crude
extract prepared from W303aLh2 (yku70/yku80)
cells expressing Yku80p and the indicated truncated Yku70 proteins
under the control of the ADH1 promoter were used. 5 µg
(lane 2) and 50 µg (lane
3) of crude extract prepared from W303-1A (wild type) were
used as control.
View larger version (70K):
[in a new window]
Fig. 4.
Analysis of telomere length in yeast cells
expressing truncated Yku70 proteins. Genomic DNA was prepared as
described under "Materials and Methods." After XhoI
digestion, DNA was separated by gel electrophoresis and blotted to a
nylon membrane. Membrane was probed using a poly(GT)20
oligonucleotide specific for telomeric repeats. Telomeric repeats are
indicated by brackets and arrows. A,
DNA prepared from W303aL expressing the indicated truncated Yku70
proteins. B, DNA prepared from W303aLh2 overexpressing
Yku80p together with the indicated truncated Yku70 proteins.
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Fig. 5.
Analysis of chromosome end structure in yeast
cells overexpressing truncated Yku70 proteins. A,
genomic DNA from W303aL cells (yku70) expressing the
truncated Yku70 proteins under control of the ADH1 promoter
was prepared as described under "Materials and Methods." After
XhoI digestion, DNA was separated by gel electrophoresis and
analyzed by nondenaturing in-gel hybridization using a 22-mer
C1-3A oligonucleotide as a probe. Lane
1, control ssDNA; lane 2, control
dsDNA; lanes 3 and 4, pAU vector
control; lanes 5 and 6, pAU-YKU70;
lanes 7 and 8, pAU-YKU70-c09;
lanes 9 and 10, pAU-YKU70-c20;
lanes 11 and 12, pAU-YKU70-c25;
lane 13, marker, end-labeled 1-kilobase pair
ladder DNA. Note that there is significantly less DNA loaded in
lane 9 as compared with the other lanes (see
B); thus, the signal for ssDNA in this lane is
underrepresented. B, the same gel as in A after
denaturation of the DNA in the gel and rehybridization to the same
probe.
cells, this
ssDNA overhang is present throughout the entire cell cycle (18). To
investigate whether this increase of telomeric G-tails can be observed
in cells expressing the C-terminal truncated Yku70 proteins, we
analyzed the telomere structure in such cells by in-gel hybridization
to native DNA. To again avoid influences of reduced protein levels, we
used the yku70 mutants expressed under control of the
ADH1 promoter. For Yku70-c09p, we observed a slight increase
of detection of G-tails as compared with the wild type (Fig. 5,
lanes 5-8). Yku70-c20p- and
Yku70-c25p-expressing cells clearly displayed a significant increase of
the G-tail signals, albeit not reaching the strength observed in
yku70 cells (Fig. 5, lanes 9-12,
3, and 4).
View larger version (70K):
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Fig. 6.
Analysis of chromosome end structure in yeast
cells simultaneously overexpressing Yku80p and the truncated Yku70
proteins. A, genomic DNA from W303aL cells
(yku70) expressing Yku80p and the truncated Yku70 proteins
under control of the ADH1 promoter was prepared as described
under "Materials and Methods." After XhoI digestion, DNA
was separated by gel electrophoresis and analyzed by nondenaturing
in-gel hybridization using a 22-mer C1-3A
oligonucleotide as a probe. Lanes 1 and
2, pAU-YKU70; lanes 3 and
4, pAU vector control; lanes 5 and
6, pAU-YKU70-c09; lanes 7 and
8, pAU-YKU70-c25; lanes 9 and
10, pAU-YKU70-c30; lane 11,
C1-3A control ssDNA; lane 12,
TG1-3 control ssDNA; lane 13,
control dsDNA. B, the same gel as in A after
denaturation of the DNA in the gel and rehybridization to the same
probe.
(pRS313-kan) or Trp (pRS314-kan) medium containing G418.
Cells capable of joining the ends of the linearized plasmids in a
precise way can grow on His/G418 (Trp/G418)
plates. Cells deficient in NHEJ will often lose genetic information at the site of linearization. This results in a
nonfunctional KanMX gene, and the cells will be unable to
grow on medium containing G418.
End-joining capacity of YKU70 mutants
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
phenotype; telomeres are very short, displaying G-tails, and NHEJ is
not functional. This is not simply due to the instability of the
truncated protein, since overexpression of Yku70-c30p from the
ADH1 promoter results in protein levels that are comparable with those found for Yku70p in wild-type cells (Fig.
2B).
mutants is not associated with a lack of NHEJ but rather is due to
defects in telomere maintenance.
ACKNOWLEDGEMENTS
FOOTNOTES
Present address: Adolf-Butenandt-Institute for Cell Biology,
University of Munich (LMU), Schillerstr. 42, D-80336
München, Germany.
ABBREVIATIONS
REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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