(Received for publication, November 10, 1995; and in revised form, January 17, 1996)
From the
Saccharomyces cerevisiae Apn1 and Escherichia coli endonuclease IV are homologous enzymes that initiate the repair of
abasic (AP) sites or oxidative DNA strand breaks. Yeast lacking Apn1 (apn1) are hypersensitive to simple
alkylating agents (which produce many AP sites) and to oxidants and
display an elevated spontaneous mutation rate due to endogenous
damages. We explored whether the prokaryotic repair enzyme could
substitute for its yeast counterpart. Plasmid constructs were generated
that expressed endonuclease IV at 1/20 to 10-fold the AP endonuclease
activity of wild-type yeast; some of these plasmids expressed hybrid
forms of endonuclease IV equipped with the C-terminal nuclear
localization signal of Apn1. Although hybrid endonuclease IV-Apn1 (but
not native endonuclease IV) was selectively localized to the yeast
nucleus, expression of this chimeric protein at 25% of the normal Apn1
level did not restore alkylation or oxidant resistance to apn1
yeast, but it did partially counteract
the mutator phenotype of apn1
yeast.
Expression of either the hybrid protein or native endonuclease IV at
10 times wild-type Apn1 levels restored wild-type resistance to
methyl methanesulfonate and near-wild-type H
0
resistance. High level expression of native endonuclease IV also
restored the normal spontaneous mutation rate to apn1
yeast. These data place limits on the
amounts of AP endonuclease activity necessary for repair of DNA damages
caused by both endogenous and environmental agents and point to a
direct role of spontaneous AP sites as potentially mutagenic lesions.
Ionizing radiation, chemical oxidants, and aerobic metabolism
produce toxic oxygen derivatives such as superoxide, hydrogen peroxide,
and hydroxyl radical (von Sonntag, 1987; Imlay and Linn, 1988). These
reactive oxygen species have deleterious effects, in part by producing
oxidative DNA strand breaks bearing blocked 3`-termini and various
types of oxidized abasic (AP) ()sites (Hutchinson, 1985; von
Sonntag, 1987; Demple et al., 1986). Nonoxidized AP sites are
also formed by simple alkylating agents and spontaneous base hydrolysis
(Lindahl, 1993). Cells must repair oxidative strand breaks to allow
survival (Demple et al., 1986; Demple and Harrison, 1994) and
AP sites for survival and to prevent mutagenic errors during DNA
replication (Loeb and Preston, 1986).
The repair of AP sites is
initiated by class II AP endonucleases, which make incisions on the
immediate 5` side of abasic sites (Warner et al., 1980; Demple
and Harrison, 1994). Many class II AP endonucleases also possess
3`-repair diesterase activity that selectively removes deoxyribose
fragments such as phosphoglycolate esters from the 3`-termini of
oxidatively damaged DNA (Henner et al., 1983; Johnson and
Demple, 1988ab; Levin et al., 1988; Ramotar et al.,
1991b; Demple and Harrison, 1994). Both enzymatic activities, class II
AP endonuclease and 3`-diesterase, yield free 3`-hydroxyl groups that
support DNA repair synthesis. In Escherichia coli, exonuclease
III and endonuclease IV are the main AP endonucleases that function in
cellular resistance to oxidants and alkylating agents and in limiting
spontaneous mutagenesis (Cunningham et al., 1986; Demple et al., 1986; Levin et al., 1988; Kunz et al. 1994). In Saccharomyces cerevisiae, these functions are
effected by a single enzyme, Apn1 (Ramotar et al., 1991b; Kunz et al., 1994), which is homologous to E. coli endonuclease IV (Popoff et al., 1990). Related genes have
been identified in Mycobacterium leprae (Honore et
al., 1993), Mycoplasm genitalium (Fraser et al.,
1995), and in the fission yeast Schizosaccharomyces pombe, ()but their biological roles remain undetermined. No homolog
of exonuclease III has yet been reported for S. cerevisiae,
but exonuclease III-related enzymes have been found in mammalian cells
(Demple et al., 1991; Robson and Hickson, 1991; Robson et
al., 1991; Seki et al., 1991), Drosophila (Sander et al., 1991), plants (Babiychuk et al.,
1994), and Gram-positive bacteria (Puyet et al., 1989).
The
alkylation and oxidation resistance conferred in yeast by Apn1 is due,
respectively, to efficient repair of alkylation-induced AP sites and
blocked oxidative 3`-termini in DNA (Ramotar et al., 1991b).
The mutator phenotype of Apn1-deficient (apn1-1) strains
seems largely due to the generation of AP sites by endogenous mutagens,
with the extra mutations primarily single base pair substitutions
dominated by a 60-fold increase in the rate of AT
CG
transversions (Kunz et al., 1994).
Apn1 protein and endonuclease IV share 41% amino acid identity over a region encompassing 95% of the bacterial protein and the N-terminal 285 residues of the yeast protein (Popoff et al., 1990). In addition, Apn1 has a C-terminal segment of 82 residues including three clusters of basic amino acids (Popoff et al.(1990), see Fig. 1). The most C-terminal of these basic clusters of Apn1 is required to localize Apn1 efficiently to the yeast nucleus, but not for enzymatic activity (Ramotar et al., 1993).
Figure 1: Schematic structures of Apn1, endonuclease IV, and endonuclease IV derivatives. The numbers above each bar indicate the amino acid residues. The dark shaded areas represent the basic amino acid clusters of the Apn1 protein. The homologous regions are residues 5-272 of endonuclease IV and 18-287 of Apn1 (Popoff et al., 1990).
In an earlier
report, we demonstrated that Apn1 expressed in E. coli can
substitute specifically for endonuclease IV in the repair of damaged
DNA in vivo (Ramotar et al., 1991a). It thus appears
that Apn1 and endonuclease IV share in vivo substrate
specificity in DNA repair consistent with their homology and similar in vitro properties (Johnson and Demple, 1988b; Levin et
al., 1988). We show here that native E. coli endonuclease
IV can functionally substitute for Apn1 in yeast when expressed at high
but not low levels and is not localized to any specific organelle. A
derivative of endonuclease IV equipped with the distal two basic
clusters of Apn1 accumulated in the yeast nucleus and lowered the apn1-1 mutation rate even when expressed at a lower
level.
Indirect immunofluorescence to detect endonuclease IV expressed in
yeast was performed according to Pringle et al.(1991) modified
as follows. Cells were grown overnight in 1 ml of uracil omission
medium (Sherman et al., 1983) containing 2% glucose; the
following day, the cells were washed twice, each time with 1 ml of 20
mM potassium phosphate (pH 7.0), resuspended in 1 ml of uracil
omission medium containing 2% (v/v) glycerol, and allowed to grow for 8
h at 30 °C. Cells were washed as above, resuspended in 5 ml of
uracil omission medium containing 2% galactose, and grown overnight at
30 °C to permit induction of endonuclease IV. The cells were then
fixed in 4.4% formaldehyde for 90 min at room temperature. After
fixation, the cells were permeabilized by a 20-min incubation with
zymolyase, washed twice with 1 M sorbitol, 10 mM Tris-HCl, pH 7.5, and resuspended in 50 µl of the same buffer.
Ten µl of the resulting spheroblasts was placed on a
polylysine-coated glass microscope slide and incubated overnight at
room temperature with 20 µl of anti-endonuclease IV antiserum at a
dilution of 1:1000 in 137 mM NaCl, 2.6 mM KCl, 7.9
mM NaHP0
, 1.4 mM KH
PO
, pH 7.3, 1 mg/ml bovine serum albumin
(PBS-BSA; Pringle et al.(1991)). The cells were then washed
for 30 min with 10 drops of PBS-BSA. RNase was added to 50 µg/ml in
a final volume of 20 µl, and the incubation continued at room
temperature for 30 min. Cells were then washed for 10 min with 5 drops
of PBS-BSA, and the secondary antibody (fluorescein-conjugated
``Affini-pure'' donkey anti-rabbit IgG (Jackson
ImmunoResearch Laboratories)) was added to the cells as 20 µl of a
1:50 dilution, and the incubation continued for 2 h at room
temperature. The cells were then washed for 1 h with 20 drops of
PBS-BSA, and 20 µl of 200 µg/ml propidium iodide was added to
each well for 10 min. Cells were then washed only twice, each time with
2 drops of PBS-BSA (excessive washing removed the propidium iodide and
weakened the DNA staining). The results were documented using a Zeiss
fluorescence microscope equipped with an Axiophot camera.
A plasmid was constructed to express E. coli endonuclease IV in yeast by placing the nfo gene behind
the yeast galactose-inducible GAL1 promoter in the multicopy
vector pYES2.0 (Ramotar et al., 1993). The resulting construct
(pGAL-EndoIV) was transformed into the yeast strain FY86 and its apn-1 derivative DRY377, and the transformants were
examined for expression of AP endonuclease activity. AP endonuclease
activity was not detectable in cell-free extracts of the apn1-
1 mutant harboring the vector pYES2.0, while the
expected activity (
60 units/mg) was seen in the APN1
strain for cells grown in either glucose
or galactose (Table 1). The plasmid pGAL-EndoIV directed the
synthesis of AP endonuclease activity in the apn1-
1 strain at
25% of the level for glucose-grown, wild-type
yeast, with an induction of
50-fold by galactose (Table 1).
Under the same conditions, plasmid pDR6 containing the APN1
gene under pGAL1 control (Ramotar et
al., 1993) gave both basal and induced AP endonuclease levels in
DR377 at
6-fold higher than observed for pGAL-Endo IV (Table 1). We do not know whether this difference reflects
differences in the synthesis or the stability of endonuclease IV and
Apn1 or a failure of the bacterial enzyme to be generated in the fully
active form in yeast. A more remote possibility is that endonuclease IV
is somehow poorly extracted from yeast cells under our conditions. The
lower induced activity of endonuclease IV in yeast extracts was not due
to the presence of an inhibitor, because the addition of purified
endonuclease IV to apn1-
1 extracts did not alter the
activity of the bacterial enzyme (data not shown).
The expression in
yeast of active, full-length endonuclease IV was confirmed by activity
gel analysis. In this approach, enzymatic cleavage of a synthetic
3`-[P]phosphoglycoaldehyde substrate releases
the label, which then diffuses out of the gel to leave a clear band
upon autoradiography (Bernelot-Moens and Demple, 1989; Ramotar et
al., 1991a). Extracts prepared from the apn1-
1 strain failed to form a detectable band, while the presence of
plasmid pGAL-EndoIV gave rise to a clear activity band (Fig. 2, lane 3) at the position expected for endonuclease IV (lane
7). Growth of this strain in galactose allowed the endonuclease IV
activity band to be easily detected with 20-fold less protein (lane
3 versus 4). Samples of wild-type yeast (lane 1) or
strains bearing plasmid pDR6 (lanes 5 and 6) produced
bands at the position expected for Apn1 (lane 8), in
proportion to the amount of AP endonuclease activity detected in the
quantitative enzymatic assay (Table 1).
Figure 2:
Activity gel analysis of yeast extracts. Lane 1, FY86 (APN1), 40 µg; lane 2, DRY377 (apn1-
1), 200 µg; lane
3, DRY377/pGAL-EndoIV grown in glucose, 100 µg; lane
4, DRY377/pGAL-EndoIV grown in galactose, 5 µg; lane
5, DRY377/pDR6 grown in glucose, 20 µg; lane 6,
DRY377/pDR6 grown in galactose, 2 µg; lane 7, purified
endonuclease IV (1.0 unit); lane 8, purified Apn1 (1.0 unit).
The positions of purified Apn1 and endonuclease IV are
indicated.
Figure 3:
Complementation of an S. cerevisiae
apn1- deletion mutant by E. coli endonuclease IV.
Exponential-phase cells grown in medium containing either glucose (A and C) or SD-galactose medium (B and D) were challenged for 60 min with the indicated
concentrations of MMS (A and B) or
H
O
(C and D) and plated on
YPD agar to determine the surviving fraction of cells (scored as
colony-forming units).
Yeast cells that lack Apn1 accumulate mutations at a high rate, as measured using two different ochre alleles, ade2-1 and lys2-1 (Ramotar et al., 1991b). Basal expression of endonuclease IV from pGAL-Endo IV in glucose failed to suppress this mutator phenotype (Table 2). However, pGAL-EndoIV expressed in galactose-containing medium eliminated the excess spontaneous mutagenesis for both the ade2-1 and the lys2-1 alleles in strain DRY373 (Table 2). It is noteworthy that the substantial overproduction of either endonuclease IV (Table 2) or of Apn1 from pDR6 (data not shown) did not significantly lower the spontaneous mutation rate below that of wild-type levels. However, the results clearly indicate that endonuclease IV can functionally replace Apn1 in repairing both the endogenously generated DNA lesions that potentiate spontaneous mutagenesis (Kunz et al., 1994), as well as lethal lesions produced by treatment with exogenous DNA damaging agents.
Previously, we reported that
basic cluster 1 of the Apn1 C terminus (Fig. 1) is essential for
efficient targeting of the protein to the yeast nucleus (Ramotar et
al., 1993). We tested whether basic cluster 1, or a combination of
clusters 1 and 2, could act as nuclear transport signals for
endonuclease IV. A series of plasmids was constructed (see
``Materials and Methods'') to express various endonuclease
IV-Apn1 hybrid proteins (Fig. 1). The plasmid series
plac-EndoIV, plac-EndoIV, and plac-EndoIV
were designed for expression of these hybrid proteins in E.
coli, under control of the lac promoter. When expressed
even under noninducing conditions in the AP endonuclease-deficient, lacI
strain BW528, all three plasmids
directed the synthesis of substantial AP endonuclease activity (Table 3). All three plasmids also conferred resistance in strain
BW528 to both the alkylating agent MMS and the oxidant t-butyl
hydroperoxide (Fig. 4). A slightly truncated (by two residues)
endonuclease IV derivative, Endo IV*, produced during the construction
of the chimeric proteins (see ``Materials and Methods''),
also retained functional activity in E. coli (Table 3, Fig. 4).
Figure 4:
Resistance conferred by plac-EndoIV and
its derivatives in AP endonuclease-deficient E. coli. Gradient
challenge plates were prepared and scored as indicated under
``Materials and Methods.'' 100% growth corresponds to the
full length of the tested gradient. Bars 1-6 in each
panel represent the following strains: 1, AB1157/pKEN2; 2, BW528/pKEN2; 3, BW528/plac-EndoIV; 4,
BW528/plac-EndoIV*; 5, BW528/plac-EndoIV; and 6,
BW528/plac-EndoIV
.
For expression in yeast, EndoIV and
EndoIV
were positioned next to the GAL1 promoter
to produce, respectively, pGAL-EndoIV
and
pGAL-EndoIV
(see ``Materials and Methods'').
These plasmids were transformed into the yeast apn1-
1 mutant DRY377, and the expression of endonuclease IV derivatives
was assessed by assaying AP endonuclease activity (Table 1). Both
pGAL-EndoIV
and pGAL-EndoIV
directed basal
AP endonuclease expression at
15-20% the level detected for
wild-type yeast and galactose-induced levels
10-fold higher than
wild-type (Table 1).
The expression in yeast of endonuclease
IV and the endonuclease IV-Apn1 hybrid proteins was confirmed by
immunoblot analysis using anti-endonuclease IV polyclonal antibodies.
Extracts of strain DRY377/pGAL-EndoIV expressed a single immunoreactive
polypeptide of the same size as purified endonuclease IV (Fig. 5; compare lanes 2 and 6), while
pGAL-EndoIV gave rise to two polypeptides of
38 and
35 kDa in size (Fig. 5, lane 4); the slower
migrating polypeptide of
38 kDa was of the size predicted for
EndoIV
and accounted for most of the cross-reacting
material. The cross-reacting polypeptide of
35 kDa was likely a
proteolytic fragment of EndoIV
that arose during
extractions rather than an endogenous product (see below). Crude
extracts of strain DRY377/pGAL-EndoIV
expressed a single
polypeptide of
31 kDa in nearly the same amount as native
endonuclease IV (Fig. 5, compare lanes 5 and 2). Since cluster 1 contains only 12 residues, this small
segment was not expected to alter the molecular size of EndoIV
significantly relative to native endonuclease IV. By the same
token, immunoblotting could not establish that cluster 1 was retained
in EndoIV
.
Figure 5:
Immunoblot analysis of extracts of yeast
expressing endonuclease IV and its derivatives. Lanes 1-5 contained 120 µg of crude extract protein prepared from
galactose-induced cells. Lane 1, DRY377/pYES2.0; lane
2, DRY377/pGAL-EndoIV; lane 3, DRY377/pGAL-EndoIV*; lane 4, DRY377/pGAL-EndoIV; lane 5,
DRY377/pGAL-EndoIV
; lane 6, 5 units of purified
endonuclease IV.
Figure 6:
Intracellular localization of endonuclease
IV and its derivatives in yeast. All the strains were grown in
galactose-containing SD medium. A and B, strain
DRY377/pGAL-EndoIV; C and D, strain
DRY377/pGAL-EndoIV; E and F, strain
DRY377pGAL-EndoIV
. A, C, and E show staining for DNA with propidium iodide. B, D, and F show staining with endonuclease IV-specific
polyclonal antiserum.
A gradient plate assay was used to compare the
resistance to MMS of the apn1-1 strains expressing native
endonuclease IV and its derivatives. The apn1-
1 strain
bearing the vector pYES2.0 was hypersensitive to MMS compared to the APN1
strain in both glucose and galactose
media (Fig. 7). In glucose medium, none of the endonuclease IV
expression plasmids (pGAL-EndoIV, pGAL-EndoIV
, and
pGAL-EndoIV
) provided detectable MMS resistance to the apn1-
1 strain (Fig. 7A). Thus, even
though EndoIV
enters the yeast nucleus, the basal
expression of this protein (20% of wild-type yeast levels) was unable
to correct the repair defect in the Apn1-deficient strain. In contrast,
plasmid pDR6, with the APN1 gene under the control of the GAL1 promoter, provided apparently full resistance to the apn1-
1 mutant even in glucose medium (Fig. 7A, bar 6), in which Apn1 activity is
present at a level 50% higher than in wild-type yeast (Table 1).
In galactose medium, all the plasmids (except the pYES2.0 vector)
provided full MMS resistance to the apn1-
1 strain (Fig. 7B).
Figure 7:
Resistance to MMS conferred by pGAL-EndoIV
and its derivatives in yeast apn1-1 cells. Gradient
plates were prepared and analyzed as described under ``Materials
and Methods''; ``100% growth'' corresponds to the full
length of the tested gradient. Bars 1-6 in each panel
represent the following yeast strains: 1, FY86 (APN1
)/pYES2.0; 2, DRY377 (apn1-
1)/pYES2.0; 3, DRY377/pGAL-EndoIV; 4, DRY377/pGAL-EndoIV
; 5,
DRY377/pGAL-EndoIV
; 6, DRY377/pDR6 (with the APN1 gene under control of the GAL1 promoter).
Although the basal expression of
pGAL-EndoIV did not affect cell survival in the face of
high-level damage by MMS, an effect on the apn1-
1 mutator
phenotype was observed. The uninduced level of the EndoIV
hybrid protein diminished the spontaneous mutator rate in
Apn1-deficient cells >50%, while induced expression of this protein
provided a wild-type mutation rate (Table 4). The pronounced
antimutator effect of EndoIV
confirms that the hybrid
protein is efficiently transported to the yeast nucleus, although some
proportion of EndoIV
could remain in the cytoplasm.
The results presented here demonstrate that E. coli endonuclease IV is actively expressed in yeast and can functionally substitute for its S. cerevisiae homolog Apn1. Both endonuclease IV and Apn1 were shown previously to repair damaged DNA containing AP sites and blocked 3`-termini (Levin et al., 1988; Johnson et al., 1988ab; Ramotar et al., 1991b). Thus, the observed substitution of yeast Apn1 by E. coli endonuclease IV is likely a direct effect of endonuclease IV acting in vivo to repair damaged chromosomal DNA in yeast cells. The cross-species complementation by both endonuclease IV and Apn1 (Ramotar et al., 1991a) suggests that their dual enzymatic activities, AP endonuclease and 3`-diesterase, were selected and conserved during evolution. Moreover, the need for these enzymes to repair spontaneous DNA lesions strengthens the likelihood that similar functions will be present in more complex eukaryotic cells.
The
ability of EndoIV to diminish the spontaneous mutation
rate in Apn1-deficient cells, even when expressed at only
1000
molecules per cell (Table 1), indicates that this effect occurs
via repair of major substrate(s) of the enzyme. Since the high-level
expression of either native endonuclease IV or EndoIV
restored normal spontaneous mutation rates to apn1-
1 strains, a mutagenic DNA damage that is a substrate for both the
bacterial and the yeast enzyme is formed spontaneously in
vivo. A major contributor is evidently the endogenous production
of AP sites, since about half of the observed apn1-
1 mutator effect depends on a DNA glycosylase encoded by the yeast MAG1 gene (Xiao and Samson, 1993; Kunz et al., 1994).
Oxidative DNA strand breaks could still constitute potentially
mutagenic damages in vivo (Ramotar et al., 1991b),
but such a hypothesis is undermined by the recently observed ability of
the human Ape AP endonuclease to restore normal mutation rates in
Apn1-deficient yeast (Wilson et al., 1995). Ape protein
exhibits powerful AP endonuclease activity, but only weak 3`-repair
diesterase (Chen et al., 1991).
Despite its active
3`-repair diesterase, endonuclease IV did not completely replace the
function of Apn1 in providing resistance to the oxidant hydrogen
peroxide, which produces a variety of DNA lesions including modified
bases, oxidized abasic sites, and single strand breaks with blocked
3`-termini (von Sonntag, 1987). One explanation for the only partial
restoration of HO
resistance in yeast apn1-
mutants expressing endonuclease IV is that this
agent may produce lesions in vivo that are not efficiently
repaired by endonuclease IV, but no such damage has yet been identified
(Johnson and Demple, 1988b; Levin et al., 1988; Demple and
Harrison, 1994). Recently, it has been shown that endonuclease IV
cleaves the oxidative DNA lesion
-deoxyadenosine (Ide et
al., 1994), which arises by abstraction at C1` of deoxyribose and
subsequent hydrogen donation by compounds such as glutathione (von
Sonntag, 1987). A number of other DNA repair enzymes, including E.
coli exonuclease III, endonuclease III, endonuclease VIII, Fpg
glycosylase, and T4 endonuclease V do not cleave at
-deoxyadenosine, but Apn1 was not tested (Ide et al.,
1994). Other candidate oxidative lesions include deoxyribonolactone
(1`-oxidized) and deoxypentos-4-ulose (4`-oxidized) residues, the
latter actually cleaved preferentially by endonuclease IV
(Häring et al., 1994). The activity of
Apn1 against these oxidative abasic lesions is unknown. It is also
possible that partial complementation of H
O
resistance in apn1-
1 yeast is due to an inability
of the bacterial protein to gain access to all of the oxidative damages
present in yeast chromatin or by a failure of endonuclease IV to
mediate productive interactions with (unknown) accessory repair
proteins in yeast.
E. coli endonuclease IV lacks a eukaryotic nuclear localization signal and hence was not concentrated in the yeast nucleus. In this study, we achieved nuclear targeting of endonuclease IV in yeast by attaching both basic clusters 2 and 1 of Apn1 to the bacterial protein; cluster 1 by itself was insufficient to mediate such localization. Together with the delocalizing effect of deleting only cluster 1 from Apn1 (Ramotar et al., 1993), this result indicates that nuclear transport of Apn1 is achieved by a bipartite nuclear localization signal, analogous to the native signals reported for a variety of nuclear proteins (Robbins et al., 1991).