(Received for publication, December 14, 1994 )
From the
A 5`- to 3`-exonuclease of about 45 kDa has been purified
from various mammalian sources and shown to be required for the
completion of lagging strand synthesis in reconstituted DNA replication
systems. RTH1 encodes the yeast Saccharomyces cerevisiae counterpart of the mammalian enzyme. To determine the in vivo biological role of RTH1-encoded 5`- to 3`-exonuclease, we
have examined the effects of an rth1 mutation on various
cellular processes. rth1
mutants grow poorly at 30
°C, and a cessation in growth occurs upon transfer of the mutant to
37 °C. At the restrictive temperature, the rth1
mutant exhibits a terminal cell cycle morphology similar to that
of mutants defective in DNA replication, and levels of spontaneous
mitotic recombination are elevated in the rth1
mutant
even at the permissive temperature. The rth1
mutation
does not affect UV or
-ray sensitivity but enhances sensitivity to
the alkylating agent methyl methanesulfonate. The role of RTH1 in DNA replication and in repair of alkylation damage is
discussed.
In Escherichia coli, the 5`- to 3`-exonuclease activity of DNA polymerase I is involved in the removal of RNA primers attached to the 5`-end of newly replicated DNA. The E. coli polA ex1 mutant is defective in 5`- to 3`-exonuclease activity and is temperature-sensitive for growth. The joining of Okazaki fragments is retarded in the polA ex1 mutant, and as a consequence, it exhibits elevated levels of genetic recombination (1, 2) .
In eukaryotes, a 5`- to 3`-exonuclease
with a molecular size of 45 kDa has been purified from HeLa cells,
mouse, and calf thymus(3, 4, 5) . Ishimi et al.(4) reconstituted the replication of simian
virus 40 origin containing DNA using SV40 large T antigen and other
purified components (single-stranded DNA binding protein RPA, DNA
polymerase -primase complex, topoisomerase II, ribonuclease H, 5`-
to 3`-exonuclease, and DNA ligase) isolated from HeLa cells. The 5`- to
3`-exonuclease was essential for the production of replicating form I
DNA. A 5`- to 3`-exonuclease from mouse cells has also been shown to be
required in a pol
-primase-dependent replication that used
single-stranded circular DNA as a template(5) . Both of these
studies indicated that the removal of RNA primers required the combined
action of RNase H and 5`- to 3`-exonuclease. In another
study(6) , the 5`- to 3`-exonuclease has been shown to
functionally interact with DNA polymerase
,
, or
in the
completion of lagging strand DNA synthesis. Using purified proteins
from calf thymus, Turchi et al.(7) have shown that
RNase H1 cleaves the primer RNA one nucleotide 5` of the RNA-DNA
junction, and the remaining monoribonucleotide is removed by the 5`- to
3`-exonuclease activity.
The mouse and human 5`- to 3`-exonuclease genes have been cloned, and they encode highly homologous proteins of 378 and 380 amino acids, respectively(8, 9) . Both proteins share a high degree of homology with the Saccharomyces cerevisiae protein of 382 amino acids encoded by the YKL510 open reading frame(8, 9) , and the S. cerevisiae protein also has a 5`- to 3`-exonuclease activity(8) . All these exonucleases share homology with the S. cerevisiae and human nucleotide excision repair proteins RAD2 and XPG, respectively. Both the RAD2 and XPG proteins contain DNA endonuclease and 5`- to 3`-exonuclease activities(10, 11, 12) . RAD2 and XPG, however, are much larger proteins containing 1031 and 1186 residues, respectively, and the homology between RAD2/XPG and the above noted mammalian and yeast 5`- to 3`-exonucleases occurs in three regions(8, 13, 14) . Because of the homology of the S. cerevisiae YKL510 open reading frame encoded protein with RAD2, we have named the gene contained within the YKL510 DNA fragment RTH1 (RAD two homolog).
The strict
dependence of reconstituted mammalian DNA replication systems on the
5`- to 3`-exonuclease suggested that RTH1 may be an essential
gene. To define the biological role of RTH1, we have examined
the effects of a null mutation in RTH1 on viability, cell
cycle morphology, and spontaneous mitotic recombination. We find that
the rth1 mutation is conditionally lethal, and at the
restrictive temperature, mutant cells exhibit a cell cycle morphology
characteristic of mutants defective in DNA replication.
For -ray irradiation, RTH1 and rth1
strains were suspended in U-wells and
transferred to yeast extract-peptone-dextrose (YPD) (
)plates. Following irradiation with a cobalt-60 source at a
dose rate of 9 kilorads/min, plates were incubated at either 25, 30, or
34 °C for 3-4 days and examined at regular intervals.
Three 10-ml cultures were
grown to a density of 10 cells/ml at either 25, 30, or 34
°C in synthetic liquid medium lacking leucine. As nearly all of the HIS3
recombinants show a simultaneous loss of
the LEU2 gene and pBR322 sequences, HIS3
recombinants do not divide in medium lacking leucine. Therefore,
the frequency of HIS3
recombinants measures
the actual recombination rate. Log phase cultures were washed,
resuspended in 1 ml of sterile water, diluted, and plated onto
synthetic complete media to determine viability and onto synthetic
complete media lacking histidine to determine the frequency of HIS3
recombinants. Plates were incubated for
3-4 days at 25, 30, or 34 °C, and the frequency of HIS3
recombinants was determined.
Recombination rates were calculated by the method of median of Lea and
Coulson (as described previously in (19) ).
For examining terminal morphology
at 37 °C, following ethanol fixation, cells were rehydrated in 40
mM KPO, pH 6.5 buffer containing 0.5 M MgCl
and 1.2 M sorbitol, and mounted on
slides coated with 0.1% polylysine (M
400,000).
For visualization of nuclear morphology, a drop of mounting medium
containing 4`,6`-diamidino-2-phenylindole was applied to the slide, and
cells were photographed with a Leitz Laborlux D fluorescence microscope
equipped with an Olympus PM-10AD photomicrographic system.
Figure 1:
UV survival
of rth1, rad2
, and rad2
rth1
strains.
, wild type strain;
, rth1
;
, rad2
;
, rad2
rth1
. A,
wild type strain LP3041-6D and its rth1
, rad2
, and rth1
rad2
derivative mutant
strains were grown at 30 °C, and cells were plated and
UV-irradiated, followed by incubation of plates at 30 °C in the
dark. Similar results were obtained at 25 or 34 °C. B,
wild type strain EMY6 and its mutant derivatives were grown at 34
°C and following UV irradiation plates were incubated at 34 °C
in the dark. Similar results were observed at 25 or 30 °C. C, MMS sensitivity of rth1
mutant. The wild type (WT) strain EMY6 and its rth1
derivative were
grown on YPD + 0.03% MMS (+ MMS) or on YPD medium lacking MMS
(-MMS) at 30 °C. Similar results were observed for
LP3041-6D and its rth1
derivative.
The rth1 mutant stops
growth upon transfer to 37 °C (Fig. 2A). Microscopic
examination revealed that rth1
mutant cells stop division as
two large cells consisting of the mother and the daughter cell, with a
block in nuclear division. In some cases, the nucleus has migrated to
the neck between the two cells, whereas in other cases, cells exhibit
an elongated nucleus stretching between the mother and daughter cell (Fig. 2B). The cell cycle arrest phenotype of the rth1
mutant resembles that of the various S.
cerevisiae mutants known to be defective in DNA replication. For
example, cdc2 mutants with a defect in DNA polymerase
stop at the restrictive temperature with the nucleus that has migrated
to the neck of the mother cell but has not elongated, whereas the DNA
ligase defective mutant cdc9 stops cell division with an
elongated nucleus extending between the two cells(21) .
Figure 2:
Lethality of rth1 mutant at
37 °C. A, rth1
mutation inhibits growth at 37 °C.
Log phase cultures of LP3041-6D and its rth1
derivative were diluted to approximately A
= 0.05, and the cultures were then split. One half of the
culture was incubated at 30 °C and the other half at 37 °C.
, RTH1 (30 °C);
, RTH1 (37 °C);
, rth1
(30 °C);
, rth1
(37 °C). B, cell cycle morphology of rth1
strain. The terminal morphology of rth1
cells was
examined by 4`,6`-diamidino-2-phenylindole
staining.
In vitro reconstitution of the DNA replication machinery from
different mammalian sources has indicated the requirement of a 5`- to
3`-exonuclease activity in the completion of lagging strand DNA
synthesis. RTH1 encodes the S. cerevisiae counterpart
of the mammalian 5`- to 3`-exonuclease. In this study, we have
determined the in vivo biological role of RTH1 by
examining the effects of the rth1 mutation on viability,
mitotic recombination, and DNA repair. We find that RTH1 is
not an essential gene. However, growth rate is slowed very considerably
in the rth1
mutant at the permissive temperature, and the rth1
mutation is inviable at the restrictive temperature
of 37 °C. These results suggest the presence of an alternate 5`- to
3`-exonuclease activity that, at the permissive temperature, can
substitute for the activity missing in the rth1
mutant;
however, at the elevated temperature, the other 5`- to 3`-exonuclease
activity is unable to support DNA replication. Because of the homology
of RTH1-encoded protein with the S. cerevisiae RAD2
protein and the fact that RAD2 also possesses a 5`- to 3`-exonuclease
activity(10) , we determined the effect of the rad2
mutation on viability in combination with the rth1
mutation. However, the rad2
mutation has no effect
on viability or growth rate of the rth1
mutation,
indicating that RAD2 does not fulfill the role of the alternate 5`- to
3`-exonuclease in DNA replication.
As expected for a mutant
defective in DNA replication, the rth1 mutant stops
division at the restrictive temperature as two large cells with a
defect in nuclear division. At the permissive temperature, the rth1
mutation results in a reduction in growth rate, and
a further decline in growth rate occurs in the rth1
rad52
double mutant. A slowdown in the removal of RNA primers in the rth1
mutant would retard the joining of nascent DNA
fragments; subsequent channeling of these discontinuities into the RAD52 recombinational repair pathway would result in elevated
levels of spontaneous mitotic recombination observed in the rth1
mutant. Elimination of recombinational repair by the rad52
mutation would leave the DNA lesions in the rth1
mutant unrepaired, resulting in a further reduction
in growth rate of rth1
rad52
mutant strain over that
of the rth1
and rad52
single mutants. We
have shown previously that DNA ligase-deficient mutations are lethal in
combination with mutations in the RAD52 gene(23) .
We find no evidence for the involvement of RTH1 in the
repair of UV damage. The rth1 mutation, however, confers
sensitivity to MMS, suggesting a role for the RTH1 5`- to
3`-exonuclease in the repair of alkylation damage. Following removal of
the alkylated base by a DNA glycosylase and subsequent cleavage of the
phosphodiester bond at the 5`-side of the apurinic/apyrimidinic (AP)
residue by a class II AP endonuclease, the 5`- to 3`-exonuclease could
effect the release of the AP residue, resulting in a 1-nucleotide gap,
which could then be filled in by repair synthesis.