ABSTRACT
The radiation-sensitive mutant
pso4-1
of
Saccharomyces cerevisiae
shows a pleiotropic phenotype, including sensitivity to DNA cross-linking agents, nearly blocked sporulation and reduced mutability. We have
cloned the putative yeast DNA repair gene
PSO4
from a genomic library by complementation of the blocked UV-induced mutagenesis and of sporulation in diploids homozygous for
pso4-1.
Sequence analysis revealed that gene
PSO4
consists of 1512 bp located upstream of
UBI4
on chromosome XII and encodes a putative protein of 56.7 kDa.
PSO4
is allelic to
PRP19
, a gene encoding a spliceosome-associated protein, but shares no significant homology with other yeast
genes. Gene disruption with a destroyed reading frame of our
PSO4
clone resulted in death of haploid cells, confirming the finding that
PSO4/PRP19
is an essential gene. Thus,
PSO4
is the third essential DNA repair gene found in the yeast
S.cerevisiae
.
At least 50 genes encoding proteins involved in DNA repair are known in the
yeast
Saccharomyces cerevisiae
(
1
,
2
) and, since molecular studies on many of them have revealed
a close relationship
to DNA repair enzymes found in humans and mammals, this simple eukaryote has
advanced to a model in which the complex mechanisms underlying DNA repair might
be unraveled in the near future. The many DNA damage-sensitive yeast mutants were initially grouped into three phenotypic
groups (
3
,
4
) which, by double mutant analysis that measured synergistic or epistatic
interactions, were allocated to three epistasis groups (
5
-
7
). This classification more or less holds until today, with group I comprising ~20 genes that are
RAD3
-like and encode proteins for nucleotide excision repair, group II
comprising ~12 genes that are
RAD52
-like and which seem to encode proteins involved in repair of DNA double-strand breaks via recombinational mechanisms and the largest, and by
far the most heterogeneous, group III, defined by
RAD6
, comprising ~20 genes, many of which are responsible for error-prone DNA repair (
1
,
2
,
8
). Some DNA damage-sensitive mutants, e.g. several of the yeast mutants sensitive to
photoactivated psoralens (
pso
mutants;
9
) have been allocated to more than one epistasis group, depending on the mutagen
applied and the biological end point scored in the respective double mutants (
8
). A mutant allele of gene
PSO4
, which is involved in error-prone repair and some types of recombination, i.e. gene conversion,
crossing-over and intrachromosomal recombination (
10
,
11
,
12
), was originally isolated as X-ray-sensitive mutant xs9 (
13
); it was found to be particularly sensitive to photoactivated bifunctional 8-methoxypsoralen and was thus renamed
pso4-1
(
10
). This yeast mutant is phenotypically similar to the
recA
mutant of
Escherichia coli
in that it combines mutagen and radiation sensitivity with a block in
recombination and induced mutagenesis. The pleiotropic repair phenotype caused
by the singly existing
pso4-1
mutant led to allocation of
PSO4
to more than one epistasis group (
8
,
14
).
More recent studies showed that heterologous expression of
recA
increased resistance to UV and ionizing radiation in
S.cerevisiae
wild-type
but not in
recombination-deficient
rad52-1
mutant
cells (
15
,
16
). However,
recA
-like yeast mutant
pso4-1
showed restored induced mutability after transformation with a multicopy vector
containing the
E.coli recA
gene (
17
). Biochemical analysis proved normal incision of 8-MOP + UVA-induced interstrand cross-links (ICLs), but failure of DNA strand rejoining in
pso4-1
(
16
), which seems to depend on a recombinational step. The apparent role of gene
PSO4
in DNA repair processes attributed to genes belonging to epistasis group II
(recombination) and III (error-prone) makes it a very interesting candidate for our future understanding
of interconnections between these repair processes in yeast. Progress in
elucidating the function of Pso4 protein necessitated the molecular cloning of
PSO4
, which we wish to report in this communication.
The yeast strains used are listed in Table
1
. Single copy vector pRS316 (
18
) and multicopy vector pRS426 (
19
), both containing
URA3
as selectable marker, were used for subcloning, complementation tests and for
sequencing. The original complementing isolate pMG470 is based on the multicopy
plasmid YEp24 (
20
). Hence, subcloning was performed using multicopy vector pRS426 to also allow for screening of a possible multicopy suppressor of
PSO4
(Fig.
1
A).
Diploids homozygous for mutant allele
pso4-1
show higher than wild-type UV sensitivity, lower than wild-type UV-induced mutation and mitotic recombination and sporulate very
poorly (<1% asci) on appropriate medium (
8
).
PSO4
was molecularly cloned by screening for transformants in diploid MG5128
homozygous for
pso4-1
and heterozygous for
CAN1
(Table
1
), in which inducibility of mutagenesis by UV
254
had been restored (
CAN1
->
can1
r
). Amongst 6000 transformants two were found to show wild-type-like UV-induced mutagenesis and furthermore restored sporulation (40%
in the transformants and the respective wild-type versus a maximal number of <1% asci in the mutant) and restored resistance to DNA cross-linking mutagens. Restriction analysis of the two complementing plasmids, named pMG470 and
pMG490, showed that the both passengers contained an identical 6.5 kb
overlapping fragment (depicted in Fig.
1
A). Hybridization of a
32
P-labeled 2 kb
Eco
RI fragment from this area (Fig.
1
A, pMG475) revealed that the passengers were part of chromosome XII (not shown).
Two subfragments obtained by restriction of plasmid pMG470 at the singular
Sal
I site yielded the two plasmids pMG471 and pMG473 (Fig.
1
A), both unable to restore the
PSO
phenotype (Table
2
A), indicating that
Sal
I is located within the
pso4-1
complementing open reading frame (ORF) or its promoter. We determined the DNA
sequence in this area and found a match with gene
PRP19
(
22
), containing the
Sal
I site. Further sequencing revealed that
PSO4
/
PRP19
is located ~1.5 kb upstream of
UBI4
and that it shares a promoter region of ~320 bp with another ORF (L0916, Fig.
1
A) upstream of
PRP19
encoding a hitherto uncharacterized protein containing a putative ATP/GTP
binding site. Subcloning of a 2.4 kb fragment containing only gene
PRP19
(pMG480, Fig.
1
A) revealed that this gene alone is sufficient to restore the wild-type-like phenotype when transformed into a
pso4
mutant (Fig.
1
C and Table
2
A).
We determined the size of the
PSO4/PRP19
gene to be 1512 bp, which corresponds to a 56.7 kDa protein consisting of 503
amino acids, instead of 502, as originally described for
PRP19
by Cheng
et al
. (
22
), owing to one additional proline at position 239. Sequence analysis of the
putative protein revealed one myb-like DNA binding domain (W[ST]X
2
E[DE]X
2
[LIV]) at the C-terminus of the Pso4/Prp19 protein (positions 457-465, WTKDEESAL). The retroviral oncogene v-
myb
and its cellular counterpart c-
myb
encode nuclear DNA binding proteins. In
S.cerevisiae
, the
myb
-related genes include the DNA-binding proteins
REB1
(
23
) and
BAS1
(
24
). However, the Pso4/Prp19 protein does not share any considerable homology with
the putative proteins encoded by these two other yeast genes. The nucleotide
sequence of
PSO4
will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under
the accession no. X99770.
After
in vitro
disruption of the Pso4/Prp19-encoding ORF at the
Sal
I site by inserting
HIS3
(cf. Fig.
1
), we performed one-step disruption experiments in haploid and diploid wild-type cells. While no haploid transformants with correct insertion
could be found, diploid disruptants (MG5100, Table
2
) showed only two surviving His auxotrophic ascospores upon sporulation and
tetrad analysis. The absence of any His prototrophic spores confirmed the
previously reported essentiality of the
PSO4/PRP19
gene (
22
). However, it has never been demonstrated whether this essentiality is
restricted to spore germination. Therefore, the heterozygous diploid MG5100 was
transformed with pMG480 (
URA3
;
PSO4
), resulting in four viable spores after sporulation. Plasmid loss experiments
with spores containing the
HIS3
disruption marker did not yield any viable Ura auxotrophs, while haploid His
auxotrophic non-disruptants showed plasmid loss of ~20% within 36 h. This indicates that the essentiality of
PSO4
is not restricted to spore germination, since loss of the plasmid results in
lethality of haploid disruptants. As essentiality could be verified with
another construct using
URA3
as reporter gene (not shown), the disrupted alleles will be generally named
pso4-0
for the purpose of simplification during discussion.
Besides the fact that gene
PRP19
alone was sufficient to complement the
pso4-1
mutation, final evidence for the allelism of the cloned ORF with
PSO4
was obtained by showing that null allele
prp19
::
HIS3
cannot complement any phenotype of the
pso4-1
mutant in a heteroallelic diploid which was constructed by crossing the original
pso4-1
mutant xs9 with the haploid
prp19
::
HIS3
disruptant MG5100-1C of opposite mating-type, the latter containing
PSO4
-harboring plasmid pMG480 to ensure viability of the haploid disruptant. After plasmid loss, the resulting Ura auxotrophic diploid MG5101, heteroallelic for
pso4-1/prp19
::
HIS3
, was viable and showed the pleiotropic phenotype typical of a homozygous
pso4-1
diploid, including a strong reduction in sporulation and in induced mutagenesis
together with sensitivity to several DNA cross-linking mutagens (Table
2
and Fig.
1
C). Since the
prp19-0
allele is unable to complement a
pso4-1
mutant phenotype, we have final proof that both genes are allelic. Thus the
terms
pso4
::
HIS3
(
pso4-0
) and
prp19
::
HIS3
(
prp19-0
) can be considered as synonymous.
Table 2
Although a disruption of
PSO4/PRP19
is lethal, mutants containing the
pso4-1
allele are viable but show the above-mentioned phenotype. Often, viable mutants of essential genes are isolated
as sensitve to temperatures >30oC. We therefore tested our diploids harboring different alleles of
PSO4
for possible temperature sensitivity. In fact, both diploids either homoallelic for
pso4-1
or heteroallelic (
pso4-1
/
prp19-0
) were sensitive to 36oC. Again, the
prp19-0
allele failed to complement this sensitivity, thus once more confirming
allelism of both genes (Fig.
1
B). When comparing the growth yield of our diploid strains at 30oC we found that heteroallelic
pso4-1/pso4-0
diploid MG5101 can grow at 30oC (cf. above) but has a severely retarded growth rate with a generation
time >3 h in YEPD medium, while the isogenic
PSO4/pso4-0
diploid MG5103 showed a generation time of ~1.5 h, comparable with other wild-types (not shown).
Repair of DNA double-strand breaks in
S.cerevisiae
involves genes of the
RAD52
epistasis group. The corresponding proteins are supposed to act via a
recombinational mechanism and some of them have been shown to constitute a
protein complex which has been termed a recombinosome (
25
,
26
). A
pso4-1
mutant has previously been shown to exhibit a pleiotropic phenotype, i.e. sensitivity to many DNA damaging agents, nearly blocked induced mutagenesis, lowered spontaneous and induced recombination and nearly totally
blocked pre-meiotic DNA synthesis and sporulation (
8
,
12
,
27
). We have now isolated gene
PSO4
, which is a member of the
RAD52
epistasis group by complementation of the
pso4-1
mutant's drastically reduced induced mutability and blocked sporulation.
Characterization of DNA repair gene
PSO4
showed its allelism to the yeast gene
PRP19
, which encodes a 502 amino acid spliceosome-associated protein (
22
,
28
). In contrast, our
PSO4
sequence contains one additional proline-coding CCC triplet and thus encodes a 503 amino acid protein. However,
this discrepancy may be due to a strain-specific difference and is presently most likely irrelevant for discussion
of the putative protein.
The allelism of
PSO4
and
PRP19
implicates that this yeast gene encodes a protein that has functions in RNA
splicing and in error-prone DNA repair, recombination and sporulation in this organism. It is
thus the first DNA repair gene with a function linked to the processing of RNA
and its essentiality underscores the vital function in the latter process. Next
to
RAD3
and
SSL2/RAD25
,
PSO4/PRP19
is the third DNA repair gene that is essential for growth and viability of
S.cerevisiae.
The Prp19 protein has been demonstrated to be essential for splicing of pre-mRNA (
28
). However, biochemical characterization revealed that it is not tightly associated with snRNAs, but is associated with
the spliceosome during the splicing reaction (
22
). Interestingly, Cheng
et al
. discussed the fact that Prp19 is distinct from other Prp proteins or other
spliceosomal components regarding the protein sequence and that it does not
contain any of the four motifs found in other Prp sequences. It could be
further demonstrated that the Prp19 protein is associated with a protein
complex different from the spliceosome, consisting of at least seven proteins
in addition to Prp19, itself most likely present in an oligomeric form (
29
). However, the Prp19-associated complex, itself about as big as the spliceosome, is unlikely to bind to the latter
complex (
29
). The authors suggest that the Prp protein may be released to become associated
with the splicing complex. This event was found to be concomitant with or just
after dissociation of the U4 snRNA and an ATP-dependent conformational change before formation of a functional
spliceosome, suggesting that Prp19 may function in this step of spliceosome
assembly (
30
).
What could be, on the other hand, the function of the
PRP19/PSO4
-encoded protein in recombinational and error-prone repair? All evidence gathered from genetic and biochemical
experiments using allele
pso4-1
point to a late function in repair of DNA lesions missing in the mutant strain.
The excision of 8-MOP + UVA-induced ICLs from DNA is thought to proceed normally, with
production of DNA double-strand breaks as repair intermediates which, however, are not rejoined in
pso4-1
(
14
). It has been suggested that repair of DNA lesions of the ICL type would
require two modes of repair (
31
,
32
), the first being removal of the damage by the repairosome postulated for
nucleotide excision repair (
33
) and the second another enzyme complex for repair of strand breaks, in which
the
PSO4/PRP19
encoded protein would have a function. The existence in
S.cerevisiae
of such a DNA double-strand repair-specific recombinosome, a complex containing at least proteins
encoded by
RAD51
,
RAD52
,
RAD55
and
RAD57
, has recently been suggested (
26
). We suggest that the Prp19/Pso4-associated protein complex found by Tarn and co-workers (
29
) might be the above-mentioned recombinosome.
The fact that both existing viable alleles of the essential gene
PSO4
/
PRP19
, as represented by the previously described
prp19
mutant (
22
,
34
) and by the
pso4-1
mutant respectively, exhibit a temperature sensitivity implies that the 503
amino acid protein encoded by
PSO4/PRP19
might have two or more functional domains, of which one or more would be still
active in the protein expressed by haploid and diploid
pso4-1
mutants: since there is no obvious growth retardation at 30oC in
pso4-1
haploid and homoallelic diploid mutants we must assume that RNA splicing
proceeds with an efficiency close or identical to that of the wild-type. This hypothesis is underscored by the fact that the viable
prp19
mutant accumulates unprocessed pre-mRNA at the non-permissive but not at the permissive temperature (
34
), suggesting that viability of a
pso4
/
prp19
mutant is dependent on functional splicing. Survival of a
pso4-1
mutant but not of
pso4-0
therefore suggests that the former mutant allele still expresses a partially
functional protein at 30oC; while its role in spliceosomal assembly and function would be about
normal its function in repair of DNA single- and double-strand breaks via recombinational processes would be significantly perturbed. We suggest that these
phenotypes could be due to disturbed binding of the other proteins to Pso4 in
the multienzyme complex found by Tarn
et al
. (
29
), indicating that at least one of these hitherto unidentified proteins may
belong to the
RAD52
epistasis group.
An alternative, but in our opinion highly unlikely, explanation for the repair
phenotype of the
pso4-1
mutant would be the incorrect processing of transcripts of DNA repair genes.
Amongst the many repair genes in yeast only one, namely
RAD14
, which is involved in incision of damaged DNA, is intron-containing (
35
) and hence would require splicing for proper function. Since
pso4-1
mutants are able to incise DNA (
14
), the only known splicing-dependent repair gene is functional, once again pointing to normal
splicing activity in
pso4-1
mutants at 30oC. Also, if non-functional splicing always resulted in repair deficiency phenotypes, at least some of the ~50 DNA repair genes cloned ought to encode proteins needed for
splicing, i.e. they should have turned out to be allelic to one of the many
other
PRP
genes.
The severe handicap in growth in complete medium at 30oC of heteroallelic
pso4-1/pso4-0
but not of heterozygous
pso4-1/PSO4
diploids indicates that the quantity of protein produced from one
pso4-1
mutant allele is not sufficient for normal spliceosome activity, whereas the
protein encoded in one
PSO4
wild-type allele of a normal growing
PSO4/pso4-0
diploid apparently satisfies cellular demand for the spliceosome-associated protein function. In this context the partial complementation
of the yeast
pso4-1
mutant's sensitivity to photoactivated 8-MOP + UVA by overexpression of RecA protein (
17
) has new significance. RecA protein can bind to single-and double-stranded DNA, thereby facilitating initiation of recombination (
36
). Interstrand cross-link repair in
E.coli
as well as in yeast has been shown to proceed via double-strand breaks (cf.
2
,
31
); these secondary lesions would be a substrate for RecA or RecA-like proteins which, after helical filament formation, would initiate recombinational repair. The RecA and Rad51 proteins share considerable sequence homology and there is
strong evidence that Rad51 binds to the Rad52 protein (
26
). The sensitivity of the
pso4-1
mutant to 8-MOP + UVA, its partial complementation by overexpressed RecA protein and
the epistatic interactions of the
pso4-1
mutant with
rad51
and
rad52
mutant alleles (
17
) has lead to the hypothesis that it functions in the repair of primary or
secondary induced DNA strand breaks via a recombinational mechanism. The
association with a functional protein complex, perhaps in forming a
recombinosome (
26
), could be one function of the Pso4/Prp19 protein; the other, more essential to the yeast cell, would be its role as a spliceosome-associated protein in pre-mRNA processing. Recently, a similar two-way association has been described for yeast transcription
factor TFIIH: it participates in formation of a holocomplex active in RNA
polymerase II transcription initiation and can also be found in the repairosome, which is specific for nucleotide excision repair (
37
,
38
).
We thank Dr A.C.Schenberg for the
pso4-1
mutant and Drs J.Brozmanova and W.Siede for helpful discussion. Sequencing of
the left arm of chromosome XII was supported by a grant to AD from the European
Union BIOTECH program under the coordination of Joerg Hoheisel, DKFZ,
Heidelberg. Research was supported by Qualitätsweine D.Brendel. The Brazilian-German cooperation was facilitated by CNPq-DAAD travel grants.
Strain
Relevant genotype
Plasmid
Sporulation
HN2
30oC
36oC
Mutability
(
A
)
MG5128
pso4-1/pso4-1
pRS426
<1%
S
+
-
-
pMG470
45%
R
+
+
+
pMG471
<1%
S
+
-
-
pMG473
<1%
S
+
-
-
pMG480
35%
R
+
+
+
(
B
)
MG5101
prp19-0/pso4-1
pRS426
0.1%
S
+
-
-
pMG470
40%
R
+
+
+
pMG471
0.1%
S
+
-
-
pMG473
0.1%
S
+
-
-
pMG480
35%
R
+
+
+
(
C
)
W303
PSO/PSO
None
35%
R
+
+
+
MG5100
prp19-0/PSO
None
33%
R
+
+
+
MG5128
pso4-1/pso4-1
None
<1%
S
+
-
-
MG5103
pso4-1/PSO
None
32%
R
+
+
+
MG5101
prp19-0/pso4-1
None
0.1%
S
+
-
-
REFERENCES
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