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J. Biol. Chem., Vol. 278, Issue 33, 31434-31443, August 15, 2003
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From the
Department of Pathology, University of
Michigan Medical School, Ann Arbor, Michigan 48109-0602, the
Hôpital Maisonneuve-Rosemont, Centre de
Recherche, Université de Montréal, 5415 Boul de l'Assomption,
Montreal, Québec H1T 2M4, Canada, and
¶Plantaceutica, Inc., Research Triangle Park,
North Carolina 27709-2060
Received for publication, May 2, 2003 , and in revised form, May 28, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-lyase activity in S. cerevisiae, since
expression of bacterial Fpg conferred Tpp1-dependent resistance to
methylmethane sulfonate in yeast lacking the abasic endonucleases Apn1 and
Apn2. In contrast, 
-only lyases increased methylmethane sulfonate
sensitivity independently of Tpp1, which was explained by the inability of
Tpp1 to cleave 3' 
,-unsaturated aldehydes. In parallel
experiments, mutations of TPP1 and RAD1, encoding part of
the Rad1/Rad10 3'-flap endonuclease, caused synthetic growth defects in
yeast strains lacking Apn1. In contrast, Fpg expression led to a partial
rescue of apn1 apn2 rad1 synthetic lethality by converting lesions
into Tpp1-cleavable 3'-phosphates. The collected experiments reveal a
profound toxicity of strand breaks with irreparable 3' blocking lesions,
and extend the function of the Rad1/Rad10 salvage pathway to
3'-phosphates. They further demonstrate a role for Tpp1 in repairing
endogenously created 3'-phosphates. The source of these phosphates
remains enigmatic, however, because apn1 tpp1 rad1 slow growth could
be correlated with neither the presence of a yeast -lyase, the activity
of the 3'-phosphate-generating enzyme Tdp1, nor levels of endogenous
oxidation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Several enzyme families act at base lesions and single-strand breaks,
collectively forming the base excision repair (BER) pathways depicted in
Fig. 1 (reviewed in Refs.
1,
3–6).
Individual enzymes frequently only interconvert one form of DNA damage to
another. It is through their concerted action that repair is achieved, and
there is often more than one sequence leading to repair. Glycosylases remove
modified bases to leave abasic sites
(9,
10), such as the Mag1 enzyme
of Saccharomyces cerevisiae that cleaves alkylated bases
(11). A subset of
glycosylases, referred to as glycosylase/lyases, additionally cleave the
resulting abasic site by a non-hydrolytic -elimination, or lyase,
reaction to leave a 3' ,-unsaturated aldehyde.
-Lyases include bacterial endonuclease III (Nth), and S.
cerevisiae Ntg1, Ntg2, and Ogg1
(9,
10,
12). Yet a further subset of
glycosylase/lyases catalyze a second sequential -elimination reaction,
more commonly known as -elimination, to remove the aldehyde and leave a
3'-phosphate. -Lyases include bacterial Fpg and endonuclease VIII
(Nei), and recently identified human homologues
(9,
10,
13,
14). The more common form of
BER entails hydrolytic cleavage 5' to an abasic site by two distinct but
functionally similar classes of apurinic/apyrimidinic (i.e. abasic)
endonucleases that leave a 3'-hydroxyl and 5'-dRP. These include
bacterial endonuclease IV (Nfo) and its yeast homologue Apn1
(15), and bacterial
exonuclease III (Xth) and its yeast homologue Apn2
(16). Each of the abasic
endonuclease families also cleave essentially all 3' blocking lesions
through the same active site, an activity named 3'-phosphodiesterase
(17,
18). Finally, although the
3'-flap endonuclease Rad1/Rad10 is best known for its functions in
nucleotide excision repair (NER)
(19) and trimming
nonhomologous ends during recombination
(20), recent studies have
suggested a role for Rad1/Rad10 in repair of strand breaks with blocked
3'-termini
(21–23).
DNA 3'-phosphatase is a more recently discovered BER enzyme. We have identified and begun to characterize Tpp1, the S. cerevisiae phosphatase (24, 25). Tpp1 is homologous to one portion of the bifunctional enzyme polynucleotide kinase/3' phosphatase (PNKP) found in Schizosaccharomyces pombe and metazoans (26–29). It is also homologous to a family of plant enzymes, including characterized Zea mays and Arabidopsis thaliana counterparts (30, 31). Bacteria, in contrast, lack a related DNA 3'-phosphatase. Like the plant enzymes, Tpp1 is devoid of a polynucleotide kinase domain, and we have provided functional evidence that budding yeast in fact do not possess this activity (24). Thus, only the 3'-phosphatase function of this enzyme family is consistently conserved, and only in eukaryotes. Tpp1 is functionally redundant with yeast Apn1 and Apn2, which are themselves capable of removing 3'-phosphates (17, 18). A deficiency of 3'-phosphate repair is only detected when APN1 and TPP1 both are mutated, with the greatest deficiency caused by simultaneous apn1 apn2 tpp1 mutation (25). Unlike Apn1 and Apn2, however, Tpp1 activity appears restricted to 3'-phosphates (25).
Here, we have used a variety of methods to explore the cellular function of
Tpp1. We find that Tpp1 can function in bacteria to cleave 3'-phosphates
left by -elimination, but that budding yeast lack a -lyase to
account for the conservation of Tpp1. Tpp1 functions in the repair of
endogenous DNA damage, but this effect is not realized until a redundant
Rad1/Rad10 salvage pathway is also disabled. This and other results
demonstrate that strand breaks with persistent irreparable blocking lesions
are highly cytotoxic. The source of the requisite endogenously created
3'-phosphates remains enigmatic, but does not appear related to ROS.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Bacterial and Yeast Strains—Bacterial strains were BW528
(
(xthpnc), nfo1::kan) and its parent AB1157, kindly
provided by Dr. B. Weiss (Emory, Atlanta, GA). Yeast strains YW636
(SOD1) and YW1094 (sod1) were created by the Yeast Genome
Deletion Project (32) and were
obtained from Research Genetics. All other yeast strains were isogenic
derivatives of YW465 (21);
their genotypes are listed in Table
I. Gene disruptions were created by one-step PCR-mediated gene
replacement (33). The
can1::Myc-Fpg and can1::Fpg chromosomal
alleles, expressing Myc-Fpg and Fpg from the ADH1 promoter,
respectively, were created by replacement of CAN1 with PCR products
made from pMyc-Fpg and pFpg (see below). Transformants were selected by
replica plating 1-day YPD plates to plates containing 50 µg/ml canavanine.
All gene replacements were verified by PCR; can1::Fpg was also
sequenced. Standard techniques of mating and tetrad dissection were used to
create the further strain derivatives used in these studies.
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Media and Growth Conditions—Bacteria were grown in Luria broth at 37 °C unless otherwise indicated. Yeast were grown at 30 °C, with shaking at 280 rpm for liquid cultures. Routine yeast media were either YPD (1% yeast extract, 2% peptone, 2% dextrose, 40 µg/ml adenine) or synthetic defined medium (1.7 g/liter yeast nitrogen base (Difco), 5 g/liter ammonium sulfate, 2% dextrose) supplemented with the appropriate complete synthetic medium dropout mix (QBiogene; adenine concentration was 10 µg/ml for plates and 40 µg/ml for liquid cultures). When indicated, 2% ethanol plus 3% glycerol was substituted for dextrose (YPEG). Anaerobic growth was in sealed jars containing an AnaeroGen anaerobic pack (Camp Micro Inc.) on YPD supplemented with 0.5% Tween 80 and 20 µg/ml ergosterol (34). When indicated, 25 mM ascorbic acid or 25 mM N-acetylcysteine were added as free radical scavengers.
Tpp1 Expression Plasmids and Mutagenesis—pTW300 and pTW367 are related LEU2/CEN/ARS vectors that allow for expression of genes in yeast from the strong constitutive ADH1 promoter (35, 36). pTW375 is a pTW300 derivative that expresses His9-Myc3-Tpp1. It was created by making a PCR product corresponding to the TPP1 coding sequence with 5'-BamHI and 3'-SalI tails and ligating it into pTW300 digested with these enzymes. For bacterial expression, the Tpp1 open reading frame was excised from pTW375 by BamHI/SalI digestion and ligated into similarly digested pGEX-5X-1 to form pGST-Tpp1. Tpp1 point mutations D35A and D37A were introduced into pGST-Tpp1 using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Mutamer oligonucleotides were (mutated bases are underlined): TppD35Aup (5'-GGTCTAAACGTTTATGCCTTTGCTCTGGACCATACAATTATC; TppR-D35Adwn (5'-GATAATTGTATGGTCCAGAGCAAAGGCATAAACGTTTAGACC); TppD37Aup (5'-CGTTTATGCCTTTGATCTGGCCCATACAATTATCAAACC); and TppR-D37Adwn (5'-GGTTTGATAATTGTATGGGCCAGATCAAAGGCATAAACG). Mutants were subsequently transferred from pGST-Tpp1 to pTW375 by BamHI/SalI digestion and ligation. Mutant pTW375 derivatives were sequenced to confirm the expected mutation and rule out non-directed Tpp1 mutations.
Fpg and Nth Expression Plasmids—pMyc-Fpg and pMyc-Nth, which express the bacterial fpg and nth genes in yeast with single amino-terminal Myc tags, respectively, were made by gap repair cloning into pTW367 as previously described (forward primer, 5'-ACCATGGCGTCCGAGCAAAAGCTCATTTCTGAAGAGGACTTGCGC; reverse primer, 5'-TTTATGTAACGTTATAGATATGAAGGATTTCATTCGTCTGTCGAC) (36). pFpg, the pTW367 derivative that expresses untagged Fpg, was cloned similarly except that a different forward primer was used whose 5'-tail caused deletion of the Myc tag contained in the vector (5'-CAATATTTCAAGCTATACCAAGCATACAATAAGCTTCTCACC). Yeast strains bearing successful gap repair constructs were verified by PCR. In all cases, multiple PCR-positive transformants were tested for MMS sensitivity.
H2O2 and MMS Sensitivity—Bacteria were transformed with the appropriate plasmid by the CaCl2 method. Cultures of BW528 strains bearing the respective plasmids were grown in LB medium supplemented with 100 µg/ml ampicillin. Overnight cultures were diluted 1:10 in the same medium, grown for 2.5 h at 30 °C, and then the absorbance at 600 nm (OD600) was adjusted to 0.9. The cultures were grown for a further 10 min before adding drugs. H2O2 was added to a final concentration of 0.1%, samples were drawn at 0, 5, 10, and 15 min, serially diluted to 1 x 10–5 or 1 x 10–6, and plated onto LB agar plates containing 100 µg/ml ampicillin. Colonies were scored after overnight incubation at 30 °C. MMS was added to cultures at a final concentration of 0.17%, samples were withdrawn at 0, 15, 30, and 45 min and processed as with H2O2 treatment.
For determination of yeast MMS sensitivity, exponentially growing cells were exposed to varying concentrations of MMS in 50 mM potassium phosphate, pH 7.5, at 30 °C for 30 min as previously described (25). Survival was determined by plating serial dilutions to YPD (or appropriate dropout plates in the case of yeast strains with plasmids), with colonies counted after 3 days growth at 30 °C.
Primer Extension Assay—Exponentially growing cells in 5 ml of LB were either untreated or treated with 25 mM H2O2 for1hat37 °C. Cells were harvested, washed twice with M9 buffer, and the cell pellet stored frozen for 1 h at –80 °C. Extraction of the chromosomal DNA was performed as previously described (37). To measure the incorporation of [methyl-3H]dTMP, 150 µM of chromosomal DNA in 25 µl of HE buffer (10 mM Hepes-KOH, pH 7.0; 1 mM EDTA) was added to 225 µl of an ice-cold reaction mixture. This mixture consisted of 25 mM Hepes-KOH pH 7.6; 25 mM KCl; 10 mM MgCl2; 50 µg/ml bovine serum albumin; 100 µM dATP; 100 µM dCTP; 100 µM dGTP; 30 µM dTTP, and 3 units of Escherichia coli DNA polymerase per ml. Labeled [methyl-3H]dTTP (NET221X from PerkinElmer Life Sciences; 37.0 MBq) was next added to a specific activity of 1260 cpm/pmol. The reaction was started when the samples were immersed into a 37 °C water bath. At the indicated times, 40-µl samples were withdrawn and added to tubes containing 200 µl of 0.1 M sodium pyrophosphate and 1 mg/ml of bovine serum albumin, followed by the addition of 200 µl of 0.8 M trichloroacetic acid. Tubes were mixed and placed on ice for 10 min. The samples were processed on a 12-hole filtration apparatus (Millipore, Bedford, MA) using GF/C circle filters (Whatman). The trapped DNA was washed three times with 3 ml of 0.1 M sodium pyrophosphate, briefly rinsed with ethanol, air-dried, and counted with 5 ml of scintillation fluid (BCS, Amersham Biosciences). In the case of chromosomal DNA pretreated with either Nfo or hPNKP, 20 ng of the purified enzyme was incubated with the DNA for 20 min at 37 °C in 10 µl of reaction buffer (25 mM Hepes-KOH, pH 7.6; 50 mM KCL, 5 mM MgCl2, 1 mg/ml bovine serum albumin). The enzyme was heat-inactivated at 70 °C for 3 min.
Enzyme Activity Assays—GST-Tpp1 and GST-Apn1 were purified from S. cerevisiae as previously described (24). Duplex oligonucleotides bearing a uracil, or a 3'-phosphate at a nick, were labeled and annealed as previously described (24, 25). The uracil was converted to a singlebase gap with a 3'-aldehyde by treatment with uracil-DNA glycosylase (Udg, 0.01 units/5 pmol oligonucleotide) and Nth (28 ng/pmol oligonucleotide) in Udg buffer (NEB) for 1 h at 37 °C. Activity assays contained 50 fmol of DNA substrate and appropriate amounts of enzyme in a reaction volume of 10 µl such that the final buffer was 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 50 µg/ml bovine serum albumin. Following incubation at 30 °C for 10 min, the reaction was stopped with formamide/EDTA loading buffer, and samples were electrophoresed on 7 M urea, 12% polyacrylamide gels followed by autoradiography.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Examining the survival of bacteria treated with H2O2 gave similar results, in that GST-Tpp1 could largely, but not completely, rescue survival of BW528 as compared with wild type (Fig. 3A). This result parallels previous experiments in yeast showing that Tpp1 confers H2O2 resistance (25). We were initially surprised to see that GST-Tpp1 could also confer partial BW528 resistance to MMS, however (Fig. 3B). This was unexpected since MMS leads principally to the formation of abasic sites through the action of DNA glycosylases (see Fig. 1), not 3'-phosphates. Indeed, apn1 apn2 tpp1 triple mutant yeast are no more sensitive to MMS than apn1 apn2 double mutant yeast (25). In the following experiments we sought to determine the basis of the differing effects of Tpp1 expression on MMS sensitivity in yeast and bacteria.
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Tpp1 Cannot Remove 3'-Aldehydes—One
explanation for the bacterial result would be that Tpp1 in fact has a
3'-phosphodiesterase activity, beyond 3'-phosphatase, that is able
to cleave a subset of blocking lesions created by MMS treatment. We have
previously shown that Tpp1 can not cleave abasic sites or
3'-phosphoglycolates, nor is it an exonuclease
(25). An untested high
frequency 3'-blocking lesion was the ,-unsaturated aldehyde
resulting from -elimination at a deoxyribose. As shown in
Fig. 4, GST-Tpp1, unlike
GST-Apn1, showed no activity at this lesion, even at enzyme concentrations
40-fold higher than those required to completely cleave a similar amount of
3'-phosphates. It is therefore highly likely that the beneficial effect
of Tpp1 in bacteria is explainable purely by its 3'-phosphatase
activity.
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Fpg, but Not Nth, Confers Tpp1-dependent Phenotypes in apn1 apn2
Yeast—Our preferred hypothesis for the unexpected rescue of BW528
MMS resistance by GST-Tpp1 relates to the secondary metabolism of abasic sites
(see Fig. 1). Specifically,
action of the bacterial -lyases Fpg and Nei at MMS-induced abasic sites
would produce 3'-phosphates that are a suitable substrate for GST-Tpp1.
In contrast, yeast are not known to express a -lyase, although the
recent unanticipated demonstration of Nei-related enzymes in humans makes this
claim uncertain (13,
14). We reasoned that if the
difference between the bacterial and yeast MMS experiments was indeed a result
of -lyase expression, then yeast should become more like bacteria if a
-lyase was expressed ectopically.
Our goal was to express the E. coli fpg gene in S.
cerevisiae. This experiment was complicated by the fact that the
amino-terminal proline of Fpg acts as the Schiff base nucleophile during the
sequential - and -elimination reactions
(40). Consequently, even a two
residue (Gly-Met) N-terminal extension impairs Fpg activity by more than
100-fold (41). The extent to
which the obligatory initiator methionine would be removed in yeast to leave
an N-terminal proline could not be predicted, but fpg is known to
complement ogg1 mutation in S. cerevisiae, indicating that
some function is retained
(42). We attempted to express
Fpg both with and without an amino-terminal Myc tag.
Fig. 5A and
Table II show the interesting
result that only very poorly growing colonies were obtained when untagged Fpg
was expressed in apn1 apn2 tpp1 yeast. In contrast, Fpg was entirely
tolerated by apn1 apn2 yeast. Although these results largely
precluded meaningful drug sensitivity testing with untagged Fpg, they already
demonstrated that Fpg could confer a Tpp1-dependent phenotype in yeast,
presumably by cleaving spontaneously arising DNA lesions (see more below).
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-elimination.
In fact, Myc-Fpg dramatically increased the MMS sensitivity of apn1 apn2
tpp1 yeast, such that cultures were effectively sterilized by a 30 min
treatment with the extremely low MMS concentration of 0.025%. Tpp1-dependent
MMS resistance was also seen when Myc-Nei was heterologously
expressed.2
The implication of the above results is that Myc-Fpg was able to convert
less toxic abasic lesions to strand breaks that became highly toxic in the
absence of enzymes able to remove 3'-phosphate blocking lesions. This
would predict that (over)expression of a -only lyase would lead to an
increase in MMS killing regardless of the presence of Tpp1, since Tpp1 cannot
remove 3'-aldehydes (see above). Fig.
5C shows that Myc-Nth did indeed lead to an equivalent
increase in MMS sensitivity in both apn1 apn2 and apn1 apn2
tpp1 yeast. A similar pattern was observed when the yeast glycosylases
Ogg1, Ntg1, or Ntg2 were overexpressed as Myc fusion proteins from the strong
constitutive ADH1 promoter.2 These results provide clear
in vivo corroboration of the lesion specificity of Tpp1.
Tpp1 Amino Acids Asp-35 and Asp-37 Are Essential for Its in Vivo Function—In addition to conferring H2O2 and MMS resistance in bacteria (Fig. 3), plasmid-expressed Tpp1 reduced MMS sensitivity of apn1 apn2 tpp1 Myc-Fpg yeast more than 1500-fold (Fig. 5D). To validate dependence of these assays on Tpp1 catalytic function, we examined mutations of two universally conserved Tpp1 aspartate residues, Asp-35 and Asp-37. Based on homology with the L-haloacid dehalogenase super-family of hydrolases, these residues are believed to participate in catalysis by forming a covalent bond between the phosphate-leaving group and the first aspartate (24). Mutation of either aspartate to alanine (D35A and D37A) completely abolished Tpp1-dependent repair of H2O2-induced lesions (Fig. 2 and data not shown) and resistance to both H2O2 and MMS in bacteria (Fig. 3), as well as resistance to MMS in Myc-Fpg-expressing yeast (Fig. 5D). GST-Tpp1-D35A and GST-Tpp1-D37A were also completely inactive in vitro.2
Synthetic Growth Defects Caused by apn1, apn2, tpp1, and rad1 Mutant
Combinations—We have previously seen that apn1 apn2 tpp1
yeast grow similarly to wild-type but die when the recombination gene
RAD52 is deleted, and that apn1 tpp1 rad52 and apn1 apn2
rad52 yeast grow extremely poorly
(25). This suggested a
recombination-dependent mechanism for removal of 3' blocking lesions. To
explore this hypothesis further, we performed analogous tetrad dissection
experiments with the RAD1 gene, which encodes part of the Rad1/Rad10
endonuclease that acts to trim 3'-ends during recombination
(20). As shown in
Fig. 6, apn1 tpp1 rad1
strains grew very poorly, similar to apn1 tpp1 rad52. This effect was
not due to the NER deficiency of rad1 mutants
(19) since rad2 and
rad14 NER mutants did not share the tpp1 synthetic
phenotype.2 Strikingly, apn1 apn2 rad1 strains showed an
even more severe phenotype of synthetic lethality, similar to a recent report
(23). Microscopic examination
of inferred apn1 apn2 rad1 microcolonies showed them to be typically
comprised of 10 to 
50 large, budded, and misshapen cells
(Table II and data not shown),
characteristic of persistent irreparable DNA damage. When tpp1
mutation was added this phenotype became even more severe, such that apn1
apn2 tpp1 rad1 cells rarely formed microcolonies of more than 10 cells.
The collected results demonstrate that while the synthetic interaction with
rad1 mutation is most severe for apn1 apn2 mutation, the
phenotype extends to tpp1 mutation, which is informative because Tpp1
is a pure DNA 3'-phosphatase.
Partial Suppression of the apn1 apn2 rad1 Growth Defect by
Fpg—The most likely explanation for apn1 apn2 rad1
synthetic lethality is that spontaneously arising abasic sites either persist
or are converted to strand breaks with 3'-aldehyde blocking lesions by
-lyases (see "Discussion"). In the absence of a
-lyase or abasic endonuclease/3'-phosphodiesterase, these lesions
would persist and require Rad1/Rad10 for removal. A prediction of this model
is that ectopic expression of a -lyase should rescue apn1 apn2
rad1 lethality in a Tpp1-dependent fashion. Indeed, apn1 apn2
rad1 yeast expressing untagged Fpg did form visible colonies, but not
when TPP1 was also mutated (Table
II). The rescue was partial, however, since these colonies were
very small and failed to continue growing. This may be because only a small
portion of the Fpg expressed in yeast is made competent for
-elimination by removal of the amino-terminal methionine.
Alternatively, apn1 apn2 rad1 lethality may depend on functions in
addition to processing of abasic sites and 3'-aldehydes.
Suppression of apn1 tpp1 rad1 Synthetic Slow Growth—
Extending the above logic, endogenous DNA damage must also explain the
tpp1 rad1 synthetic interaction, and this damage must include
3'-phosphates. There are three known mechanisms by which
3'-phosphates are generated in growing cells. First is through the
action of -lyases as discussed, but the above experiments functionally
demonstrate that S. cerevisiae does not possess a -lyase (see
"Discussion").
Second, we and others have described a pathway in which the enzyme Tdp1 removes the DNA topoisomerase Top1 when the latter is irreversibly and covalently bound to a DNA 3'-terminus (21, 25, 43, 44). Tdp1 leaves a 3'-phosphate (43) that is removed by the combined action of Tpp1, Apn1, and Apn2 (see Fig. 1) (25). The slow growth phenotype of tdp1 rad1 yeast demonstrates that the Tdp1 pathway is active to some extent even in the absence of Top1 inhibitors (21). This tdp1 rad1 slow growth is much less severe than even the apn1 tpp1 rad1 phenotype, however, making it unlikely that the Top1 pathway contributes substantially to the tpp1 rad1 synthetic interaction. Indeed, mutation of neither TDP1 nor TOP1 led to a measurable suppression of the apn1 tpp1 rad1 growth defect (Table II).
Third, endogenous reactive oxygen species can lead directly to the formation of strand breaks with 3'-phosphates in the same manner as exogenous H2O2. We therefore compared the growth of apn1 tpp1 rad1 and apn1 tpp1 yeast in aerobically and anaerobically grown streaks (Fig. 7). The profound growth difference of these strains was preserved on aerobic re-streaking, i.e. small spore colonies were not an artifact of slow germination. In contrast, anaerobic growth tended to normalize the appearance of the two strains. This initially suggested that endogenous oxidation is a significant contributor to apn1 tpp1 rad1 slow growth. However, when we attempted to verify this phenotype by additional rounds of restreaking, we found that cells in anaerobically grown colonies were nearly all dead, regardless of genotype and despite the inclusion of lipid nutrients essential for anaerobically grown S. cerevisiae (34). We therefore sought to corroborate the anaerobic findings by independent means, but without success. Growth in a reduced O2 environment in CampyPaks (BBL) relieved the cell death observed in anaerobic chambers, but did nothing to suppress the apn1 tpp1 rad1 growth defect.2 Furthermore, apn1 tpp1 rad1 growth was not improved by growth in the presence of the ROS scavengers ascorbic acid (a superoxide scavenger, Ref. 45) and N-acetylcysteine (a hydroxyl radical scavenger, Ref. 46) (Fig. 7). The slow growth of an ROS-sensitive superoxide dismutase mutant strain (sod1) was normalized on these same plates, demonstrating the efficacy of the scavenger treatments.
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The majority of endogenous ROS are believed to arise through mitochondrial generation of superoxide during respiration, which is ultimately converted to the DNA-damaging hydroxyl radical (1, 7, 47). We therefore reasoned that increasing or decreasing mitochondrial respiration should exacerbate or alleviate apn1 tpp1 rad1 slow growth, respectively. This again did not prove to be the case. Disruption of the mitochondrial electron transport chain at an early step by mutation of the nuclear COQ3 gene blocks respiration, and so suppresses stationary-phase death of sod1 mutants (7). No such suppression of apn1 tpp1 rad1 slow growth was observed; all coq3 apn1 tpp1 rad1 spore colonies remained slow growing (colony size 3, Table II). In the complementary approach, growth on plates containing the non-fermentable carbon sources ethanol and glycerol forces respiratory metabolism. Remarkably, ethanol-glycerol medium did not exacerbate apn1 tpp1 rad1 slow growth, but in fact completely suppressed it (Fig. 7). This phenotype was reversible because apn1 tpp1 rad1 colonies streaked from ethanol-glycerol back to dextrose once again showed severely impaired growth.2 Finally, we observed spontaneously arising slow-growth suppressors in aerobic streaks of apn1 tpp1 rad1 yeast, but none of 18 tested proved to be respiratory (i.e. petite) mutants. The implications of these findings are addressed in "Discussion."
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-Elimination Is Not Required for Genome Maintenance in S.
cerevisiae—It was initially surprising that Tpp1 conferred
bacterial resistance to MMS as well H2O2. This finding
was best explained by the presence of lyases in bacteria that can create
3'-phosphates from MMS-induced abasic sites by consecutive
/ elimination (Fig.
1). We validated this hypothesis by demonstrating that even the
crippled Myc-Fpg led to a clear and predictable Tpp1-dependent MMS resistance
when expressed in yeast (Fig.
5). The complete lack of Tpp1-dependent MMS resistance in the
parent strains thus demonstrates functionally that S. cerevisiae does
not possess a detectable lyase. This conclusion is supported by the
fact that the characteristic structural features of the Fpg/Nei
glycosylase/lyase family are not shared by any hypothetical protein in S.
cerevisiae, as determined by BLAST searches or by considering all open
reading frames with a proline as the second residue
(14).2 It is thus
apparent that yeast ordinarily maintain their genomes without the benefit of
-elimination, and must instead rely on the abasic endonucleases to
remove 3'-aldehydes left by chemical or enzymatic -elimination.
This is important in light of the recent unexpected discovery of functional
homologues of Fpg/Nei in many eukaryotes, including humans and the fungus
Candida albicans (13,
14).
The paradox is this: why do bacteria possess -lyases but no pure
3'-phosphatase, while yeast possess a 3'-phosphatase but no
-lyase? At a minimum, this paradox makes clear that Tpp1 does not exist
to cleave 3'-phosphate lesions left by -elimination. Indeed, in
bacteria this need is satisfied by Nfo and Xth. The question remains why Tpp1
is ever necessary, or preferred, in yeast cells that already possess Apn1 and
Apn2.
A General Rad1/Rad10-dependent Salvage Pathway for Repair of 3'-Blocked Strand Breaks—Our results support the conclusion that Tpp1 participates in the repair of endogenous DNA damage, but in a fashion redundant with other 3' repair mechanisms. This was not initially obvious because even apn1 apn2 tpp1 cells grow at a normal rate. However, the addition of either rad52 (25) or rad1 (Fig. 6, Table II) mutation leads to lethality in this background, suggesting that Rad52 and Rad1/Rad10 act in a redundant pathway that repairs persistent strand breaks. These results parallel similar recent findings. For example, Rad1/Rad10 participates in repair of strand breaks when Top1 peptide fragments are covalently bound to the 3' terminus, but only in the absence of Tdp1, the enzyme that normally removes these peptides (21, 22). In addition, the apn1 apn2 rad1 mutation combination is lethal due to absence of Rad1/Rad10-dependent processing of 3'-aldehyde lesions that persist in apn1 apn2 cells (23). Analysis of tpp1 phenotypes is necessarily more complex, because depriving cells of 3'-phosphatase function requires apn1 and apn2 mutation and thus concomitant loss of abasic endonuclease/phosphodiesterase activity. Nonetheless, tpp1 mutation causes an incremental growth defect in both apn1 rad1 and apn1 apn2 rad1 backgrounds (Fig. 6 and Table II). The lesion specificity of Tpp1 dictates that this effect must be due to failed repair of endogenously generated 3'-phosphates, and in turn that 3'-phosphates can be repaired by the Rad1/Rad10 pathway. The capacity of this pathway is clearly limited, however, since it cannot protect apn1 apn2 tpp1 cells from higher levels of H2O2-induced damage (25). Taken together, we argue that Rad1/Rad10-dependent repair of 3'-phosphates is truly a salvage pathway not normally active in cells expressing Tpp1. Whether there is any normal function of this Rad1/Rad10 pathway of strand break repair remains to be determined.
Sources of Endogenously Generated 3'-Phosphates—Surprisingly, there appears to be little correlation between the endogenous mitochondrial ROS burden and the apn1 tpp1 rad1 growth defect. Anaerobic growth did normalize the growth of apn1 tpp1 rad1 strains (Fig. 7), but reducing mitochondrial ROS production by coq3 mutation did not (Table II). Instead, attempting to increase mitochondrial ROS production by growth on non-fermentable carbon sources antithetically suppressed apn1 tpp1 rad1 slow growth (Fig. 7). It is possible that the induction of the oxidative stress response that occurs during respiratory growth (48) more than compensates for the increased oxidative load. Exogenous ROS scavengers were ineffective in suppressing apn1 tpp1 rad1 slow growth, however, arguing against this hypothesis (Fig. 7).
A different explanation would be that the anaerobic environment and
non-fermentable carbon sources each suppress the apn1 tpp1 rad1
growth defect independently of oxidation. Slow growth might simply allow more
time for repair. Alternatively, faster replication and/or growth on glucose
might itself result in increased production of 3'-phosphates. Given that
S. cerevisiae does not possess a -lyase, the enzyme Tdp1 is
the only remaining known non-oxidative source of endogenous
3'-phosphates in this organism
(43). However, while Top1
damage might be exacerbated by rapid replication, the Top1-Tdp1 pathway
apparently does not lead to enough basal damage to account for the apn1
tpp1 rad1 slow growth phenotype (Table
II). We must therefore consider the possibility that there is an
as yet unidentified intracellular source of 3'-phosphates. This would
most likely be an enzyme, perhaps one under carbon source regulation.
-Lyases and Tdp1 give clear precedent for the notion that a DNA
metabolic enzyme might leave a 3'-phosphate in need of further
repair.
Persistent Strand Breaks Are Especially Cytotoxic—Xiao
et al. (11) have
demonstrated that metabolism of modified bases to abasic sites is necessary to
achieve cell killing and mutagenesis by agents such as MMS. A consistent
implication of our studies is that it is secondary strand breaks, rather than
abasic sites themselves, that are most cytotoxic. Guillet et al.
(23) recently came to a
similar conclusion. They demonstrated that the lethality of apn1 apn2
rad1 yeast was suppressed by the combined mutation of the -only
lyases expressed in S. cerevisiae, OGG1, NTG1, and NTG2,
which suggested that it is 3'-aldehydes that are least well tolerated.
We have extensively employed the complementary approach of expressing
glycosylase/lyases to interconvert lesions to predictable endpoints.
Overexpression of -lyases, including Nth, Ogg1, Ntg1, and Ntg2, enhanced
the already substantial MMS sensitivity of apn1 apn2 yeast
(Fig. 5).2 In
contrast, even Fpg weakened by an amino-terminal fusion was able to reduce
this MMS sensitivity, presumably by converting toxic 3'-aldehydes into
Tpp1-reparable 3'-phosphates (Fig.
5). Indeed, Myc-Fpg-expressing apn1 apn2 tpp1 yeast were
exquisitely MMS hypersensitive. Similarly, untagged Fpg was able to partially
suppress apn1 apn2 rad1 lethality
(Table II), but conversion of
even endogenous levels of base lesions to 3'-phosphates by Fpg
over-whelmed the capacity of Rad1/Rad10 in the absence of
3'-phosphatases (Fig. 5
and Table II).
We conclude that it is not strand breaks themselves that are toxic, but
rather their persistence due to irreparable 3' blocking lesions.
Replication would thus lead to the formation of double-strand breaks still
bearing the blocking lesion, as we have modeled in detail for
camptothecin-induced damage
(21). Interestingly, this
overall pattern is apparently not restricted to 3' blocking lesions. It
is the lyase activity of mammalian DNA polymerase , which removes
5'-dRP blocking lesions, that is most critical during BER
(49). In lyase-deficient
mutants, 5'-blocked strand breaks persist and are thought to lead to a
similar catastrophe during replication.
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FOOTNOTES |
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|| Supported by a career scientist award from the National Cancer Institute of
Canada and currently by a senior award from the Fonds de la recherche en sante
du Quebec.
** To whom correspondence should be addressed. Tel.: 734-936-1887; Fax: 734-763-6476; E-mail: wilsonte{at}umich.edu.
1 The abbreviations used are: ROS, reactive oxygen species; dRP, deoxyribose
phosphate; MMS, methylmethane sulfonate; H2O2, hydrogen
peroxide; BER, base excision repair; NER, nucleotide excision repair; PNKP,
polynucleotide kinase/3' phosphatase; GST, glutathione
S-transferase.
2 A. S. Karumbati, R. A. Deshpande, A. Jilani, J. R. Vance, D. Ramotar, and
T. E. Wilson, unpublished observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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