ABSTRACT
In mammalian cells, the Ku autoantigen is an end-binding DNA protein required for the repair of DNA breaks [Troelstra, C.
and Jaspers, N. G. J. (1994)
Curr. Biol.
, 4, 1149-1151].
A yeast gene (
HDF1
) encoding a putative homologue of the 70 kDa subunit of Ku has recently been
identified [Feldmann, H. and Winnacker, E. L. (1993)
J. Biol. Chem.
,
268, 12895-12900].
We find that
hdf1
mutant strains have substantially shorter telomeres than wild-type strains. We speculate that Hdf1p may bind the natural ends of the chromosome, in addition to binding to the ends of broken DNA molecules.
Strains with both an
hdf1
mutation and a mutation in
TEL1
(a gene related to the human ataxia telangiectasia gene) have extremely short
telomeres and grow slowly.
Yeast chromosomes terminate with a simple repeated sequence
poly G
1-3
T (
3
). Although this terminal tract in wild-type strains is usually between 350 and 500 bp in length, mutant strains
with shorter or longer tracts have been identified (
4
). In one such mutant,
tel1
, the telomeric poly G
1-3
T tracts are shortened to
~50 bp (
5
). The
TEL1
-encoded protein shares homology with phosphatidylinositol (PI)/protein kinases, and is most similar to the human ataxia
telangiectasia (AT) protein (
6
,
7
).
The mammalian Ku protein includes two subunits (70 and 82 kDa) that bind to the ends of double-stranded DNA (
8
,
9
). In addition to interacting with the ends of DNA, Ku also interacts with DNA-dependent protein kinase (p350) (
10
-
12
). Since mammalian cells that are defective in Ku are X-ray sensitive and deficient in VDJ joining (
1
), Ku is involved in the repair of double-strand DNA breaks. A heterodimeric (70 and 85 kDa) DNA-binding protein has been identified in
Saccharomyces cerevisiae
and the gene encoding the 70 kDa subunit (
HDF1
, high affinity DNA-binding factor) has been cloned (
2
). The yeast protein has ~22% identity to the human Ku protein over the C-terminal half. Cells with
hdf1
null mutations fail to grow at 37oC, but grow and sporulate normally at 30oC (
2
).
Below, we show that a mutation in
HDF1
reduces the length of chromosome telomeres. In addition, the
hdf1
mutation has a synthetic interaction with
tel1
(another mutation affecting telomere length) resulting in strains that grow
slowly and have very short telomeres.
Yeast strains with the
hdf1
and/or
tel1
mutations were constructed by transformation of the wild-type strains W303a (
a ade2 his3 leu2 trp1 ura3
) or SLK29-1B (
a ade2 ura3
) using plasmids containing disruptions of these genes. Strains with the
genotype
TEL1 hdf1
were derived from SLK29-1B (derivative SPY38) and W303a (derivative W303aU) by transformation of
the parental strains with an
Ssp
I-
Hin
dIII fragment derived from plasmid pGEM4Z S-H/URA (
2
) (
hdf1
::
URA3
); strain W303aU was provided by H. Feldmann (University of Munich). SPY37 is a
spontaneous
ura3
derivative of W303aU. The
tel1 HDF1
strain PG21 was constructed by a two-step transplacement of SLK29-1B with the plasmid pPG25 (
tel1
::IS1 insertion in YIp5; ref.
7
), and the
tel1 HDF1
strain SPY40 was derived from W303a by a single-step transplacement with an
Eco
RI-
Kpn
I fragment of the plasmid pPG47 (
tel1
::
URA3
insertion in Bluescript SK
-
vector; ref.
7
). The
tel1 hdf1
strain SPY39 was derived from PG21 by transformation with the plasmid pGEM4Z S-H/URA, and the
tel1 hdf1
strain SPY41 was constructed from SPY37 by transformation with the plasmid
pPG47.
The plasmid pYT14 contains telomeric repeats and ~1 kb of the sub-telomeric Y' element (
13
). This plasmid was used as a hybridization probe.
The lengths of telomeres were examined by treating DNA with the restriction enzyme
Xho
I and examining the products by Southern analysis, using pYT14 as a
hybridization probe (details of the protocol in refs
7
,
14
). The enzyme
Xho
I cuts in the conserved Y' repeat located at the ends of most yeast chromosomes, generating
terminal restriction fragments of ~1.3 kb in wild-type strains. This fragment includes ~400 bp of the telomeric repeat poly G
1-3
T.
Cultures in the exponential phase of growth were harvested and resuspended in
water at a density of ~10
7
cells/ml. Samples were irradiated with doses of 0, 10, 50, 100 and 220 krad and
spread on plates containing rich growth medium in order to monitor cell survival (
7
). Four plates were analyzed for each X-ray dose in each experiment. The plates containing unirradiated samples
had between 100 and 500 colonies. Ninety-five percent confidence intervals on measurements were calculated using
the GraphPad INSTAT program on the Macintosh computer.
To determine whether Hdf1p, a putative homologue of the Ku protein, is involved
in the control of telomere length in yeast, we examined isogenic strains of
four types:
TEL1 HDF1
,
tel1 HDF1
,
TEL1 hdf1
, and
tel1 hdf1
. Two different genetic backgrounds were used, W303 (
2
) and SLK29-1B (
7
). Mutations were introduced into these strain backgrounds by transformation;
since the introduced DNA contain disruptions of the coding sequence of
TEL1
or
HDF1
, the mutant alleles are likely to represent null mutations.
The results described above indicate that Hdf1p has a role in telomere
metabolism. Since the
tel1 hdf1
double mutant has phenotypes that are different from the single mutants, the effect of Hdf1p on
telomere length is likely to involve a different pathway from that involving the
TEL1
gene product. Given the known end-binding activity of Hdf1p, one obvious possibility is that this protein
protects the telomeres from cellular exonucleases, and the mutant phenotype reflects an altered balance between telomere elongation
and degradation. Since Tel1p shares homology with yeast genes involved in checkpoint control of DNA damage, Tel1p may be
involved in checkpoint control of telomere length (
7
). Given the role of Hdf1p in telomere metabolism in yeast, it would be of
interest to examine the lengths of telomeres in Ku
-
mammalian cell lines.
It is also possible that Hdf1p affects telomere length without binding to the
chromosomal ends. Strains with the
hdf1
mutation are temperature-sensitive and, at the restrictive temperature, accumulate elevated amounts
of DNA (
2
). Feldmann and Winnacker (
2
) suggested, therefore, that Hdf1p may be involved in the control of the initiation of DNA replication. Although our experiments were done at the
permissive temperature for growth of
hdf1
strains, it is possible that the regulation of DNA replication is somewhat
aberrant. Thus,
hdf1
strains could have an imbalance between cycles of `normal' DNA replication and
telomere replication, leading to shortened telomeres.
As described above, the
tel1 hdf1
strains grew slowly relative to the single mutant strains. One possibility is
that this slow rate reflects chromosome loss from some cells in the population
whose chromosomal telomeres have been completely eliminated. Although the
tel1
mutation increases chromosome loss rate only 3-4-fold, the double mutant may have a stronger effect. Whatever the
cause of the slow growth, by isolating mutants that restore the wild-type growth rate, one may be able to identify other yeast gene products
that interact with Tel1p or Hdf1p.
We found that strains with
hdf1
,
tel1
or
tel1 hdf1
genotypes were not more X-ray sensitive than wild-type strains. These results confirm studies, done in other genetic
backgrounds, indicating that
tel1
(
7
) and
hdf1
(
16
) strains are not unusually X-ray sensitive. Since the double mutant also has wild-type sensitivity to X-rays, we conclude that the two gene products are not
functionally redundant with each other in the repair of X-ray damage. Interestingly, however, both gene products appear to be
functionally redundant with other gene products involved in the cellular
response to X-ray damage. Strains with the
tel1 mec1
genotype are much more sensitive to X-rays than strains with either single mutation, and the X-ray sensitivity of
mec1
strains can be complemented with a single extra dose of the
TEL1
gene product (
17
). In addition,
hdf1 rad52
genotype are more sensitive to X-ray damage than strains with either single mutation (
16
). In summary, our data and those of other investigators indicate that both the
Tel1p and Hdf1p proteins have dual roles, a unique function in telomere metabolism and a functionally
redundant role in the repair of X-ray damage.
We thank H. Feldmann and E. Winnacker for providing yeast strains and plasmids used in this study, and E. Perkins for help with the irradiation experiments. This research was supported from grants from N.I.H.
(GM52319 and GM24110).
REFERENCES
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