(Received for publication, August 10, 1995; and in revised form, January 21, 1996)
From the
The HDF1 protein of Saccharomyces cerevisiae shares biochemical properties and structural homology with the 70-kDa subunit of the human autoantigen Ku. The Ku protein, a heterodimer composed of a 70-kDa subunit and an 80-kDa subunit, has been identified as the regulatory subunit of the DNA-dependent protein kinase. This enzyme has recently been shown to be involved in DNA repair and recombination processes in mammalian cells. Here we show that hdf1-disrupted S. cerevisiae strains are strongly sensitive toward the radiomimetic antibiotic bleomycin. In addition, mating-type switching and rates of spontaneous mitotic recombination are strongly reduced. This phenotype is similar to that of mammalian cells lacking components of the DNA-dependent protein kinase holoenzyme, suggesting that HDF1 participates in and exerts equivalent functions in S. cerevisiae.
DNA double-strand breaks (DSBs) ()are intermediates
of recombination events in both pro- and eukaryotic cells. DSBs can
also be induced by treatments with ionizing radiation and with the
radiomimetic antibiotic bleomycin(1, 2, 3) .
In Saccharomyces cerevisiae, DSB repair is mediated by
activities of the RAD52 epistasis group(4) , which
includes gene products RAD50-RAD57. A prototype of RAD50-57 activities
is the product of the RAD52 gene, which has been suggested to
be required for almost all DSB events(5) . Specifically,
mating-type switching, which is initiated by a double-strand cleavage
by the HO endonuclease at the MAT locus, is highly reduced in rad52 cells(6, 7) . rad52 strains
fail to undergo proper meiotic chromosome segregation and therefore are
defective in meiotic
recombination(8, 9, 10) . Additionally, rad52 strains are defective in most mitotic recombination
events(7, 11) .
Homologues of S. cerevisiae RAD51, RAD52, and RAD54 proteins have been identified in various higher eukaryotes(12, 13, 14, 15) , suggesting that they play a similar functional role. Another activity involved in recombination events of higher eukaryotes has recently been attributed to the so-called Ku autoantigen. Ku is a heterodimeric protein composed of 70- and 80-kDa subunits that has recently been identified as the regulatory subunit of the DNA-dependent protein kinase (DNA-Pk)(16, 17) . Ku protein also binds to double-strand DNA ends, nicks, and hairpins (18, 19, 20, 21, 22, 23, 24, 25) , suggesting that Ku may be involved in DNA repair, recombination, or replication events(21, 22) . This view is supported by the observation that the 80-kDa subunit of Ku is not detectable in x-ray-sensitive xrs hamster cell lines and that these cells fail to support normal V(D)J recombination(26, 27) . Both mutant phenotypes are complemented by the human XRCC5 gene, which has been shown to be identical with the gene for the Ku p80 subunit (28, 29) . Furthermore, the catalytic subunit of DNA-Pk, p350, is a strong candidate for the afflicted gene in cells derived from severe combined immunodeficient (SCID) mice(30, 31) . Like xrs cells, SCID cells are sensitive to ionizing radiation and defective in V(D)J recombination(32) . The SCID defect appears to affect the rejoining of certain types of DNA strand breaks (33, 34, 35, 36) , revealing a significant overlap between the mechanisms of V(D)J recombination and the joining of DSBs during DNA repair(37) .
V(D)J recombination is a specific form of DNA rearrangement that does not require extensive sequence homology. Therefore, the rejoining activity for DSBs that is deficient in the SCID and in xrs mutant cells appears distinct from the recombination repair activities in S. cerevisiae and hence might represent a specialized function of higher eukaryotes(37) . On the other hand, DNA-Pk activities have been detected recently in cultured cells from mouse, hamsters, Xenopus, and Drosophila. Although these activities are approximately 10-50 times less abundant than in human cells(38) , their presence suggests that DNA-Pk is ubiquitous in eukaryotic cells and that it may have an evolutionarily conserved basal function in DSB repair and recombination.
The most compelling argument for the ubiquitous existence of DNA-Pk is the recent description of the Ku-like HDF1 protein in S. cerevisiae that binds to DNA ends and shares homology with the 70-kDa subunit of Ku(39) . Here we show that disruption of the HDF1 gene in S. cerevisiae affects DSB repair, mating-type switching, and spontaneous mitotic recombination. The phenotype of hdf1 mutants is very similar to that of rodent xrs and SCID cells. This observation, together with the analogies in biochemical properties and the structural homology between HDF1 and Ku p70, suggests a functional conservation of these activities in eukaryotic organisms.
Complementation analysis of hdf1 mutants have been performed with the single copy vector
pRS316 carrying the genomic XhoI-EcoRI fragment
of the HDF1 gene. Mitotic recombination experiments were
performed with strains or their derivatives generously provided by
Robert Malone(7) . Strains GM21 and GM28 were obtained by a
sequence of crosses: K65-3D and K49-3A-7A were first crossed with
IL993-5C (40) to remove the HO allele. The ho-negative segregants were then back-crossed three times with
RM37-5D and RM38-7D, respectively, to develop isogenic strains. RAD52 segregants out of these crosses were identified by their
ability to grow on YPD (Bacto yeast extract, 2% Bacto peptone, 2%
dextrose (Difco)) plates containing 0.02% methyl methanesulfonate (MMS)
(Sigma). Diploid GM2128 cells, resulting of crosses between GM21 and
GM28, were used to determine wild-type mitotic recombination
frequencies. Disruptions of the HDF1 gene in GM21 and GM28,
followed by crosses of the hdf1-disrupted strains GM21U
and GM28aU, resulted in diploid strains GM2128
UaU. These were
employed for the determination of spontaneous mitotic recombination
frequencies in homozygous hdf1/hdf1 strains. Strains
GM7726, GM2122, and GM5255 and hdf1/hdf1 derivatives
thereof were generated in the same way. Standard genetic techniques
were used throughout. hdf1-disrupted strains were constructed
by lithium acetate transformation (41) with plasmid pS/H-URA3 (39) digested with EcoRI and HindIII.
The rich (YPD) and minimal (SD) (0.67% yeast nitrogen base without amino acids, plus nutrients(42) , 2% dextrose (Difco)) media used in crosses and mating-type switch experiments were as described previously (42) . Assays measuring bleomycin sensitivity were carried out with YED medium containing 0.5% yeast extract (Difco) and 1% dextrose (Difco) as described in (43) . Cells were plated on YED medium solidified by 2% agar (Difco) (YEDA).
Although bleomycin affected survival rates in haploid wild-type strains only slightly, we observed a marked reduction by 4 orders of magnitude of survival rates in haploid hdf1 strains at a bleomycin concentration of 15 µg/ml (Fig. 1A). The decline is not as drastic as that observed for rad52 strains, which display the same response already at a concentration of 3.75 µg/ml, suggesting that the RAD52 activity is a somewhat stronger factor for repair of bleomycin-induced DNA damage than the HDF1 function. Bleomycin sensitivity of hdf1-deficient strains can be restored to wild-type levels by the introduction of a functional HDF1 gene expressed from a single copy vector. The observed bleomycin sensitivity of hdf1-deficient cells thus must be caused by loss of HDF1 gene function. Comparable bleomycin sensitivities were observed for diploid hdf1/hdf1 strains, whereas heterozygous hdf1/HDF1 diploids and HDF1/HDF1 wild-type diploids displayed a resistant phenotype (Fig. 1B).
Figure 1:
hdf1 deficiency results in an
increased sensitivity toward bleomycin. A, haploid wild-type
W303-1A (), isogenic hdf1-deficient W303aL (
), and
W303aL complemented with the wild-type HDF1 gene (
)
were plated on solid YEDA media containing bleomycin in concentrations
of 1.88-15 mg/ml. Surviving cells were grown up as colonies and
counted after incubation at 23 °C for 8 days. The data of rad52-deficient strain W303rad52-4D (
) are shown for
comparison. B, increased bleomycin sensitivity is detectable
also in homozygous hdf1/hdf1. Diploid wild-type
W303a
(
), heterozygous hdf1/HDF1 W303aLwt
(
), and homozygous hdf1/hdf1 W303aL
U
(
) were assayed for bleomycin sensitivity as described
above.
Because the hdf1 deficiency causes temperature-sensitive growth at 37 °C, bleomycin sensitivity assays were each performed at 23 and 30 °C. The results of these experiments demonstrate that bleomycin sensitivity was not temperature-dependent. No effects discriminating between hdf1-deficient and wild-type strains were observed when bleomycin was replaced by either streptomycin, tetracycline, erythromycin, ampicillin, or chloramphenicol at concentrations of up to 75 µg/ml. This indicates the specificity of the bleomycin effect on hdf1-deficient strains (data not shown).
Surprisingly hdf1 rad52 double mutants are significantly more sensitive to bleomycin than the isogenic rad52 single mutated clones (Fig. 2). This effect of hdf1 and rad52 deficiency on bleomycin sensitivity indicates that both gene products are involved in repair of DNA DSBs in a cumulative manner.
Figure 2:
Haploid hdf1 rad52 double mutants
exhibit hypersensitivity for bleomycin. Spore clones of complete
tetratype tetrades out of the cross of hdf1-deficient
W303U with rad52-deficient W303rad52-4D were assayed for
bleomycin sensitivity as described above. The data are shown for one
tetrade only. Equivalent results were obtained for the others.
, W3031u-17: HDF1 RAD52;
, W3031U-18: hdf1
RAD52;
, W303Lu-19: HDF1 rad52;
, W303LU-20: hdf1 rad52.
This hypersensitive phenotype of hdf1 rad52 double mutants could also be observed with the radiomimetic agent MMS, which is known to induce strand breaks in DNA (4) (data not shown). In contrast, double mutants of hdf1 with either the rad3 or the rad6 mutations do not show hypersensitivity for bleomycin or UV irradiation (data not shown). This indicates that the observed bleomycin hypersensitivity of the hdf1 rad52 double mutant is specific for the rad52 mutation.
A significant reduction of mating-type switching events to 30% or less in comparison with wild-type was observed in hdf1-deficient strains after 5 h of induction of the HO endonuclease gene (Fig. 3). This indicates that the HDF1 activity represents an important function for mating-type switching but may not be as important as the RAD52 gene product, because it has been shown by others that the rad52 mutation prevents homothallic switching altogether(7) .
Figure 3:
hdf1-deficient strains show a
significant reduction in induced mating-type switching events. Haploid
wild-type W303-1A () and isogenic hdf1-deficient
W303aL (
) were transformed with plasmid YCp50 carrying the HO endonuclease gene under the control of the galactose-inducible GAL10 promoter. Exponential cultures grown on raffinose were
washed and shifted into SD medium containing galactose as carbon
source. Aliquots were spread every hour on YPD media and incubated at
30 °C for 5 days. Mating types were determined for at least 50
colonies of each time point.
Our data show a 10-40-fold reduction of spontaneous mitotic recombination events in hdf1/hdf1 diploids with respect to wild-type strains (Table 2), whereas rad52/rad52 diploids showed reduction rates of 50-200-fold. Comparable values were obtained for four independent crosses of wild-type strains and four independent crosses of hdf1 mutants.
This paper describes experiments that try to understand and explain the phenotype of the hdf1 mutation by studying its effects on bleomycin sensitivity, mating-type switching, and mitotic recombination frequencies. Bleomycin is generally regarded as a radiomimetic antibiotic because it induces a variety of but not all types of DNA damages known to be caused by ionizing radiation(44, 47) . Bleomycin is particularly well known for its ability to generate DNA DSBs, but there are also types of DNA damages thought to be specifically induced by bleomycin. For example, bleomycin produces 5`-phosphate and 3`-phosphoglycolate termini in DNA molecules, leaving a one-nucleotide gap(48, 49, 50, 51) . Using haploid and diploid radiosensitive strains, it was shown that all those single rad mutants that are sensitive to bleomycin are also sensitive to x-ray radiation, with the exception of rad15(44) . Furthermore, six of seven complementation groups of mutants (not blm2) directly selected for bleomycin sensitivity (blm mutants) are also sensitive to ionizing radiation(52) . In conclusion, bleomycin sensitivity can be taken as an indicator for x-ray radiation sensitivity, although this may not be true for all mutations. In a strict sense, our data for bleomycin sensitivity of hdf1 mutants thus do not prove an involvement of HDF1 activity in DSB repair, although this appears very likely. Bleomycin sensitivity is less pronounced in hdf1 than in rad52 strains, whereas double mutants display a cumulative effect. This could be explained if it was assumed that the HDF1 activity is involved in the repair of bleomycin-specific DNA damages while the RAD52 activity is responsible for DSB repair. The observed effect, however, could also be due to one singular DSB repair pathway if both activities affected different steps of this pathway. Although the rad52 mutation causes drastic effects on DNA repair, homothallic mating-type switching and mitotic recombination, there still remains some residual activity (7) . In vivo, the RAD52 protein interacts physically with the RAD51 protein(53, 54) . The recA-like activity of RAD51 catalyzes homologous DNA pairing and strand exchange, which is ATP-dependent(55) . On the other hand, the functional defect in V(D)J-recombination in Ku/DNA-Pk-deficient cells seems to impair the rejoining of the free DNA ends of the coding strands (33, 34, 35, 36) . If the HDF1 protein had a comparable function in S. cerevisiae, it could act downstream of the strand exchange activity of the RAD51/RAD52 complex. Successful DSB repair and recombination in rad52 mutants would then still require the downstream acting HDF1 activity.
To investigate further phenotypic similarities between hdf1 and DNA-Pk deficient cells, we studied the effects of hdf1 deficiency on mating-type switching and spontaneous mitotic recombination. V(D)J recombination is initiated by a precise double-strand cleavage between DNA segments and their flanking heptamers(56) . Similarly, the mating-type switch is initiated by a DSB introduced by the HO endonuclease at the MAT locus(6) . Both recombination events only require a limited number of base pairs for site specificity. V(D)J recombination results in the deletion of spacing sequences, whereas the mating-type switch results in a gene conversion event maintaining the donor sequence. We used an expression system with the HO endonuclease gene under the inducible GAL10 promoter to study the effects of hdf1 deficiency on mating-type switching rates. The significant reduction of mating-type switches in hdf1 mutants indicates that the HDF1 activity elicits a phenotype similar to the rad52 mutants. Taken in conjunction with data reported for bleomycin hypersensitivity, this further suggests that HDF1 activity may be involved in processing of DSB events.
DNA-Pk has been suggested to affect rejoining of broken DNA strands (33, 34, 35, 36) . It seems reasonable to assume that during mating-type switching the HDF1 product might affect rejoining of donor and acceptor DNA after HO endonuclease cleavage and/or rejoining of DNA strands after resolution of the crossover fork, which occurs after strand exchange within the MAT locus. hdf1 deficiency should then also affect spontaneous mitotic recombination. The data presented in Table 2demonstrate that this is indeed the case. Therefore, we suggest that the HDF1 activity displays a very basic function in genetic recombination events.
The phenotype of hdf1-deficient strains strongly resembles that of rad52-deficient strains investigated so far, although the effect of hdf1 deficiency is not as pronounced with respect to bleomycin sensitivity, mating-type switching, and spontaneous mitotic recombination. It remains to be established whether this is a genuine feature of HDF1 or whether there is a second gene whose activities overlap with HDF function in S. cerevisiae. The fact that hdf1 and rad52 deficiencies differ phenotypically in respect to growth and viability parameters does not clarify the issue. hdf1 deficiency results in the inability to grow at 37 °C, whereas members of RAD50-57 group, e.g. rad51 and rad54, show only a modest reduction in growth rate and no obvious reduction in vegetative viability at the higher temperature(39, 37) . Furthermore, hdf1/hdf1 diploids have no apparent defects in sporulation, whereas rad52/rad52 fail to undergo sporulation(8, 9, 10) . Further studies are therefore required to elucidate the relationship between HDF1 and RAD50-57 activities.
The discussion above has been focussed on the
supposition that HDF1 has a ``classical'' repair function
like other RAD activities of S. cerevisiae and thus led to our
attempts to assign its function to known repair pathways. However, an
emphasis on the homology of HDF1 protein to the Ku p70 product and
hence on its function as a regulatory subunit of a putative DNA-Pk in S. cerevisiae leads to a broader, generic view of HDF1
function(57) . Specifically, in higher eukaryotic cells DNA-Pk
together with its regulatory subunit might act as a checkpoint
modulator, because it is able to phosphorylate p53 (58) . p53,
in turn, is required for activation of G checkpoint
mechanisms that arrest cell cycle progression, presumably to allow time
to repair DNA damage before DNA is replicated(59) . Cell cyle
progression from G
to S is inhibited when p53 levels are
elevated. Exposures to UV light or
-irradiation causes a transient
increase in p53 stability, which in turn increases its nuclear
concentration. Finally, mutations in Ser
of human p53,
which is phosphorylated by DNA-Pk in vitro, produces a
remarkable increase in p53 half-life(60) . By regarding HDF1
activity as one essential element of this damage recognition and
controlling machinery in S. cerevisiae, the pleiotropic
effects on repair, recombination, and viability functions become more
comprehensible. Finally, when DNA-Pk is considered as just one element
in a concerted interaction of DNA damage-induced kinases(57) ,
the intimate relationship of HDF1 to DNA-Pk might also explain the
reduced effects of hdf1 deficiency as compared with those
observed upon loss of RAD52 function.
In conclusion, the reduced ability to perform mating-type switching and a reduction in spontaneous mitotic recombination events definitively prove that HDF1 is involved in recombination processes. The sensitivity of hdf1-deficient strains for the radiomimetic drug bleomycin indicates that the activity of HDF1 is not restricted to mating-type switching and spontaneous mitotic recombination. In view of the homology of HDF1 with the 70-kDa subunit of Ku protein of mammalian cells and its Ku-like activity, i.e. its ability to bind sequence-independently to ends of double-strand DNA, we suggest an analogous molecular function for HDF1 in the processing of DSB events in S. cerevisiae. The phenotypic characteristics of hdf1 deficiency in S. cerevisiae and of Ku/DNA-Pk deficiency in mammalian cells strongly support the notion that S. cerevisiae cells contain a DNA-Pk activity involved in functions similar to those in higher eukaryotes. We also suggest that DNA-Pk activity in higher eukaryotes may not be restricted to DSB repair and V(D)J recombination and may actually affect other recombinational processes such as gene conversion and mitotic recombination.