JOURNAL OF BACTERIOLOGY, Sept. 2003, p. 5380–5390 Vol. 185, No.

18
0021-9193/03/$08.00⫹0 DOI: 10.1128/JB.185.18.5380–5390.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

YqfS from Bacillus subtilis Is a Spore Protein and a New Functional

Member of the Type IV Apurinic/Apyrimidinic-Endonuclease Family
José M. Salas-Pacheco,1 Norma Urtiz-Estrada,1 Guadalupe Martínez-Cadena,1
Ronald E. Yasbin,2 and Mario Pedraza-Reyes1*
Institute of Investigation in Experimental Biology, Faculty of Chemistry, University of Guanajuato, Guanajuato 36060, Mexico,1 and
Department of Molecular and Cell Biology, The University of Texas at Dallas, Richardson, Texas 750832
Received 23 April 2003/Accepted 25 June 2003

Downloaded from http://jb.asm.org/ on April 2, 2015 by UNIV OF MASS MED SCH

The enzymatic properties and the physiological function of the type IV apurinic/apyrimidinic (AP)-endonu-
clease homolog of Bacillus subtilis, encoded by yqfS, a gene specifically expressed in spores, were studied here.
To this end, a recombinant YqfS protein containing an N-terminal His6 tag was synthesized in Escherichia coli
and purified to homogeneity. An anti-His6-YqfS polyclonal antibody exclusively localized YqfS in cell extracts
prepared from B. subtilis spores. The His6-YqfS protein demonstrated enzymatic properties characteristic of
the type IV family of DNA repair enzymes, such as AP-endonucleases and 3ⴕ-phosphatases. However, the
purified protein lacked both 5ⴕ-phosphatase and exonuclease III activities. YqfS showed not only a high level
of amino acid identity with E. coli Nfo but also a high resistance to inactivation by EDTA, in the presence of
DNA containing AP sites (AP-DNA). These results suggest that YqfS possesses a trinuclear Zn center in which
the three metal atoms are intimately coordinated by nine conserved basic residues and two water molecules.
Electrophoretic mobility shift assays demonstrated that YqfS possesses structural properties that permit it to
bind and scan undamaged DNA as well as to strongly interact with AP-DNA. The ability of yqfS to genetically
complement the DNA repair deficiency of an E. coli mutant lacking the major AP-endonucleases Nfo and
exonuclease III strongly suggests that its product confers protection to cells against the deleterious effects of
oxidative promoters and alkylating agents. Thus, we conclude that YqfS of B. subtilis is a spore-specific protein
that has structural and enzymatic properties required to participate in the repair of AP sites and 3ⴕ blocking
groups of DNA generated during both spore dormancy and germination.

During unpredicted periods of dormancy Bacillus subtilis cleotide excision repair system (UVR) and Rec proteins (re-
spores are constantly exposed to environmental conditions that viewed in reference 16).
have the potential to cause several types of DNA damage. AP sites can be potentially generated during spore germina-
Therefore, the existence of spore-specific protecting mecha- tion not only by the action of DNA glycosylases but also by the
nisms would seem to be fundamental for spore survival. One of spontaneous depurination and depyrimidination of DNA. AP
the factors intricately involved in protecting spore DNA from sites are inherently toxic and highly mutagenic; therefore, they
several types of damage, such as oxidative stress, UV-C irra- should be rapidly processed and eliminated during spore ger-
diation, and desiccation, is the presence of ␣/␤ type small mination. Moreover, 3⬘-blocking groups such as phosphates,
acid-soluble proteins (reviewed in references 16, 28, and 27). phosphoglycolates, and 3⬘␣,␤-unsaturated aldehydes existing
Although ␣/␤ type small acid-soluble proteins protect spore in DNA as products of reactive oxygen species attack or gen-
DNA from several stresses, they confer protection neither to erated by the combined action of glycosylase/lyase activities
base alkylation (29) nor to UV-induced DNA strand break must be also eliminated by AP-endonucleases as they inhibit
formation (30). Thus, while the physiological state of the B. DNA replication (4).
subtilis spores prevents or dramatically slows DNA damage The first catalytic event during repair of AP sites is carried
during the long periods of dormancy, it is clear that spores do out by AP-endonucleases which cleave the DNA backbone
accumulate potentially lethal and mutagenic DNA lesions such immediately 5⬘ of an AP site, generating a 5⬘ deoxyribose-
as the spore photoproduct, strand breaks, cyclobutane pyrim- phosphate group and a 3⬘ deoxyribose-hydroxyl group (6). On
idine dimers, chemically altered bases and apurinic/apyridi- the other hand, 3⬘ blocking groups on DNA strand breaks are
minic (AP) sites which could affect transcription and replica- also processed by AP-endonucleases to generate a 3⬘-OH
tion processes during germination (16, 26, 29). To remove group (4).
these potentially deleterious DNA damages and alterations, B. Analysis of the genome of B. subtilis (10) revealed the exis-
subtilis spores utilize spore-specific and general DNA repair tence of two open reading frames (ORFs), named exoA and
systems such as the spore photoproduct lyase (SplB), the nu- yqfS, whose predicted products share amino acid sequence
homology with Escherichia coli exonuclease III (ExoIII) and
Nfo, respectively. Except for a lower 3⬘-5⬘exonuclease activity
the biochemical properties of a B. subtilis ExoA purified pro-
* Corresponding author. Mailing address: Institute of Investigation tein were very similar to those reported for E. coli ExoIII (30).
in Experimental Biology, Building L, Faculty of Chemistry, University
of Guanajuato, Noria Alta S/N, P.O. Box 187, Guanajuato 36050, Gto,
Interestingly a B. subtilis mutant lacking the ExoA function was
Mexico. Phone: (473) 73 2 00 06, ext. 8161. Fax: (473) 73 2 00 06, ext. as tolerant to hydrogen peroxide and alkylating agents as was
8153. E-mail: pedrama@quijote.ugto.mx. the repair proficient isogenic parental strain (30), suggesting

5380
VOL. 185, 2003 B. SUBTILIS TYPE IV AP-ENDONUCLEASE 5381

TABLE 1. Strains and plasmids used in this study

Strain or plasmid Genotype or description Source or reference

E. coli strains
SURE e14⫺ McrA⫺ (mcrCB-hsd SMR-mrr)171 endA1 supE44 thl-1 gyrA96 relA1 lac recB recJ Stratagene
sbcC umuC::Tn5(Kmr) uvrC [F⬘ proAB lacqZ M15 Tn10(Tcr)]
XL10-Gold Tetr ⌬(mcrA)183 ⌬(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Stratagene
Kan Hte [F⬘ proAB lac1qZ⌬M15 Tn10(Tetr) Tn5(Kanr) Amy]
PERM282 Strain E. coli SURE carrying plasmid pPERM 282 This study
PERM337 Strain E. coli XL10-Gold Kanr carrying plasmid pPERM 337 This study
PERM348 Strain E. coli XL10-Gold Kanr carrying plasmid pPERM348 This study
RPC501 nfo-1::Kan ⌬(xth-pncA) R. P. Cunningham
PERM398 RPC 501 carrying pPERM 348 This study
PERM399 RPC 501 carrying pQE30 This study

Downloaded from http://jb.asm.org/ on April 2, 2015 by UNIV OF MASS MED SCH

Plasmids
pUC18 Multisite E. coli cloning vector; Apr Laboratory stock
pQE30 Vector that contains a T5 promoter that enables six-His-tagged protein expression; Apr Qiagen
pPERM282 pUC18 with 1.07-kb XbaI-BamHI PCR product containing yqfS; Apr This study
pPERM348 pQE30 with 1.07-kb BamHI-BamHI yqfS fragment from pPERM282; Apr This study

that YqfS or other noncharacterized AP-endonucleases might into E. coli XL-1 Blue (Stratagene, La Jolla, Calif.). pPERM282 was digested
compensate the functions of ExoA in B. subtilis. with BamHI, and the 1,070-bp yqfS fragment was inserted in-frame into the
BamHI site of pQE30 (QIAGEN Inc., Valencia, Calif.); the resulting construc-
In E. coli the expression of nfo is linked to the oxidative tion pPERM348 was replicated in E. coli XL10-Gold Kan (Stratagene). The
stress generated by superoxide radicals (2). However, in B. proper in-frame insertion of the yqfS fragment was assessed by both restriction
subtilis the regulation of yqfS expression occurs in a temporal analysis and DNA sequencing as previously described (22, 23).
manner and the mRNA for this gene is apparently localized Purification of His6-YqfS. E. coli PERM348 was grown in 50 ml of LB medium
supplemented with AMP to an optical density of 0.5. Expression of the yqfS gene
within the forespore (32). Furthermore, the promoter respon-
was induced during 4 h at 37°C by addition of isopropyl-␤-D-thiogalactopyrano-
sible for the regulation of yqfS expression appears to be part of side (IPTG) to 0.5 mM. Cells were collected by centrifugation and washed two
the ␴G regulon (32). In addition the lack of induction of a times with 10 ml of 50 mM Tris-HCl (pH 7.5)–300 mM NaCl (buffer A). The cells
yqfS-lacZ fusion inserted at the yqfS locus of the B. subtilis were disrupted in 10 ml of buffer A containing lysozyme (10 mg/ml) for 30 min
chromosome following treatment by either hydrogen peroxide at 37°C. The cell homogenate was subjected to centrifugation (29,200 ⫻ g) to
eliminate undisrupted cells and cell debris and the supernatant was applied to a
or the DNA damaging agent mitomycin C revealed that this
5 ml Ni-nitrilotriacetic acid (NTA)-agarose (QIAGEN Inc.) column, previously
gene is not under the control of the PerR or SOS regulons equilibrated with buffer A. The column was washed with 50 ml of buffer A
(32). containing 10 mM imidazole plus 50 ml of buffer A containing 20 mM imidazole,
The research reported in this manuscript demonstrates that and the protein bound to the resin was eluted with 15 ml of buffer A containing
the AP-endonuclease YqfS exists in mature spores and that its 100 mM imidazole, 2-ml fractions were collected during this last step. Aliquots
(15 ␮l) of the cell homogenate and the flowthrough as well as the bound fractions
DNA coding sequence possesses the ability to genetically com- were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
plement the DNA repair deficiency of an E. coli mutant lacking (SDS-PAGE) as previously described (11).
the major AP-endonucleases Nfo and ExoIII. Furthermore, a Complementation of the DNA repair-deficient strain E. coli RPC501
His6-YqfS protein synthesized in E. coli and purified to homo- [nfo-1::Kan ⌬(xth-pncA)]. The plasmid pPERM348 was introduced by transfor-
geneity has biochemical properties similar to those exhibited mation into competent cells of the strain E. coli RPC501 (kindly provided by
Richard P. Cunningham), following the calcium chloride protocol (22). Trans-
by the type IV family of endonucleases. Therefore, we con- formant colonies were selected in LB medium supplemented with AMP. The
clude that the spore protein YqfS of B. subtilis is a new func- presence of the plasmid pPERM348 in the transformant strain (E. coli
tional member of the type IV family of AP-endonucleases. PERM398) was determined by restriction analysis of plasmid DNA mini-prep-
arations (22). The overproduction of His6-YqfS in the strain E. coli RPC501
harboring pPERM348 was corroborated by taking a culture to an OD at 600 nm
MATERIALS AND METHODS (OD600) of 0.5 and inducing with 0.1 mM IPTG for 1 h. After disrupting the cells
Bacterial strains and plasmids. Strains and plasmids used in this work are with lysozyme, the cell extracts were analyzed by SDS-PAGE using as a marker
shown in Table 1. The medium used was Luria-Bertani (LB) medium (15). When the His6-YqfS protein purified as described above. The sensitivity of E. coli
appropriate, ampicillin (AMP) (100 ␮g/ml) or kanamycin (25 ␮g/ml) was added RPC501 and its derivative E. coli PERM398 to the DNA damaging agents H2O2
to the medium. Liquid cultures were incubated with aeration (shaking at 250 and methyl methanosulfonate (MMS) was determined. Essentially, strains were
rpm) at 37°C. Cultures on solid media were grown at 37°C. The optical density grown overnight in LB containing the appropriate antibiotics and then diluted
(OD) of liquid cultures was monitored with a Pharmacia Ultrospec 2000 spec- (1:50) into fresh medium. The cultures were shaken at 37°C to an OD600 of 0.5
trophotometer set at 600 nm. then IPTG (0.1 mM) was exclusively added to the strain RPC501 (a subculture
Design of a plasmid to overexpress yqfS and purify a His6-YqfS protein. The of this strain was left with no IPTG) and incubation was continued for 15 min.
ORF of yqfS lacking the first codon and extending through 157 bp downstream The cells were collected by centrifugation, washed once and suspended in 10 mM
of the yqfS stop codon was amplified by PCR, using 0.1 ␮g of chromosomal DNA sodium phosphate (pH 7.5)–150 mM NaCl (buffer B). The cultures of each strain
from B. subtilis 168 and the oligonucleotide primers 5⬘-GCGGATCCCTG AGA were treated with different concentrations of either MMS or H2O2 and incuba-
ATA GGC TCA CAC G-3⬘ (forward) and 5⬘-CGGGATCCGGC CGT TGA tion was continued for 1 h at 37°C with shaking. The cell suspensions were
AGT AGC GAA CC-3⬘ (reverse). The primers were designed to insert BamHI diluted serially 10-fold in buffer B and plated on solid LB containing the appro-
restriction sites into the cloned DNA (underlined). Amplification was performed priate selective antibiotics. The viable colonies were counted after 1 to 2 days of
on a MJ Research (Watertown, Mass.) Minicycler using Vent DNA polymerase incubation at 37°C to estimate survival.
(New England Biolabs, Beverly, Mass.). The PCR fragment (1,070 bp) was first Substrates and enzyme assays for AP-endonuclease activity. AP-endonuclease
ligated into HincII-treated pUC18 to generate pPERM282 which was replicated activity of His6-YqfS was assayed against pBluescript (pBS) (Stratagene) which
5382 SALAS-PACHECO ET AL. J. BACTERIOL.

was partially depurinated following a previously described protocol (7). A typical conserves important amino acid residues present in several
mixture reaction in a volume of 25 ␮l contained 600 ng of purified His6-YqfS, 100 members of the AP-endonuclease family, including the nine
ng of substrate in 50 mM Tris-HCl (pH 7.5) containing 1 mM dithiothreitol
(DTT). The reactions were incubated at 37°C during 30 min, and analyzed by
residues, His-69, His-110, His145, Asp-179, His-182, His-214,
electrophoresis on a 1% agarose gel which was stained with ethidium bromide. Asp-227, His-229, and Glu-25, putatively involved in the coor-
AP-endonuclease activity of His6-YqfS was also determined utilizing a radioac- dination of three Zn atoms in the active site of E. coli Nfo (6).
tive double-stranded 19-mer nucleotide containing a single AP site which was Moreover, equivalent residues, His-7, Phe-31, Tyr-72, Trp-266,
synthesized as previously described (5). Essentially, the nucleotide 5⬘-GCAGC
and the Zn2⫹-ligand Glu-259, involved in forming a deep AP
GCAGUCAGCCGACG-3⬘ was treated with uracil-DNA glycosylase following
the instructions of the provider (Roche, Mannheim Germany). The AP site site pocket for cleaving the phosphodiester bond of the AP-site
containing 19-mer nucleotide was labeled on its 5⬘end with [␥-32P]ATP and T4 in E. coli Nfo (6), are also conserved in YqfS (Fig. 1).
polynucleotide kinase (Promega, Madison, Wis.) as previously reported (5). Among the type IV AP-endonuclease homologs described in
Finally, the AP radioactive oligonucleotide was annealed to a threefold excess of the literature, only the functions of those from E. coli (12), S.
the complementary oligonucleotide 5⬘-CGTCGGCTGACTGCGCTGC-3⬘ on
cerevisiae (9, 20), and T. maritima (5) have been assessed. As

Downloaded from http://jb.asm.org/ on April 2, 2015 by UNIV OF MASS MED SCH

ice for 3 h (5).
The reactions were performed in a total volume of 15 ␮l containing 50 mM shown in Fig. 1, the predicted product of B. subtilis yqfS con-
Tris-HCl (pH 7.5), 1 mM DTT, and 500 nM unlabeled and 10 nM double- serves a high level of sequence homology with E. coli Nfo,
stranded radioactive 19-mer containing a single AP-site. His6-YqfS (300 ng) was therefore we investigated whether yqfS encodes a functional
added to the mixture reaction and incubated for 30 min at 37°C. The reactions type IV AP-endonuclease homolog of B. subtilis. As a neces-
were separated on a 20% denaturing polyacrylamide gel which was dried and
then exposed to X-Omat films (Kodak) during 12 h.
sary step to accomplish this goal a protocol was devised to
To assay 5⬘-phosphatase activity the 19-mer nucleotide 5⬘-GCAGCGCAGU purify a recombinant YqfS protein. Thus, the yqfS gene was
CAGCCGACG-3⬘ was labeled on its 5⬘end with [␥-32P]ATP and T4 polynucle- amplified by PCR and expressed from the IPTG inducible T5
otide kinase. 5⬘-Phosphatase reactions were performed in 25-␮l reactions which promoter of the plasmid pQE-30 to generate a protein tagged
contained 500 ng of His6-YqfS, 20 ng of the 5⬘-end radiolabeled 19-mer (22,000
with 6 histidines on its N terminus. Initially, induction of yqfS
cpm/ng of DNA) in 50 mM Tris-HCl (pH 7.5)–1 mM DTT. As a positive control
1 U of alkaline phosphatase (New England Bio Labs) was added to the mixture was performed for 2 h with 5 mM IPTG; however, these con-
reaction instead of His6-YqfS. Mixture reactions were incubated at 37°C for up ditions caused a very high level of His6-YqfS synthesis, result-
to 30 min. The amount of radioactive phosphate released was determined from ing in the formation of inclusion bodies in the E. coli host cells
the norit-nonadsorbed fraction by liquid scintillation, as previously described (results not shown). To improve the amount of soluble His6-
(12).
The 3⬘-exonuclease activity of His6-YqfS was determined against a substrate
YqfS protein, the strain E. coli PERM 348 was grown at 37°C
containing 3⬘-terminal [␣-32P]dCMP, which was synthesized by treating pUC19 to an OD600 of 0.5 and then induction was carried out at 37°C
with EcoRI followed by end-filling with [␣-32P]dCTP and the Klenow fragment with 0.5 mM IPTG, for 2 h. SDS-PAGE analysis confirmed the
of DNA polymerase (Promega) according to the manufacturer’s procedures. To existence of a highly abundant soluble 36-kDa protein in cell
this end, mixture reactions of 25 ␮l were mounted, which contained, 500 ng of
homogenates obtained from E. coli cells subjected to these
His6-YqfS, 13 ng of the [␣-32P]dCTP radioactively labeled pUC19 substrate
(11,500 cpm/ng of DNA) in 50 mM Tris-HCl (pH 7.5) containing 1 mM DTT. A induction conditions (Fig. 2A, lane 2). When the His6-YqfS
mixture reaction containing 100 U of ExoIII (New England Bio Labs) instead of protein present in the cell extracts was subjected to purification
His6-YqfS was used as a positive control. The 3⬘-exonuclease activity was deter- by metal chelate affinity chromatography, on a Ni-NTA-aga-
mined by acid precipitation as previously published (12). The 3⬘-phosphatase rose column, a major protein band with a molecular mass of
activity of His6-YqfS was determined by measuring the ability of His6-YqfS to
around 36 kDa was eluded from the column with 100 mM
stimulate nick translation of DNA containing 3⬘-phosphate termini, according to
a previously described protocol (7). Briefly, micrococcal nuclease-treated pBS imidazole, as revealed by SDS-PAGE (Fig. 2A, lanes 4 and 5).
(50 nmol) was incubated with either 1 U of E. coli Nfo, 500 ng of His6-YqfS, or YqfS is located in mature spores. Our recent results dem-
1 U of alkaline phosphatase for 30 min at 37°C. Each sample of enzyme-treated onstrated that in B. subtilis the expression of yqfS is neither
plasmid was incubated with the Klenow fragment of DNA polymerase at 37°C in constitutive nor induced by oxidative or DNA damaging agents
the presence of deoxynucleoside triphosphates (10 ␮M) and [␣-32P]dCTP in 50
mM Tris-HCl (pH 7.5) containing 10 mM MgCl2 and 1 mM DTT. Reactions
but regulated in a spatial and temporal manner (32). The
were terminated by adding bovine serum albumin (2 mg/ml) and ice-cold 5% expression of yqfS was shown to occur during the last steps of
trichloroacetic acid. The radioactivity incorporated into the material precipitated sporulation in the developing spores from a sigG type pro-
by trichloroacetic acid was quantified by liquid scintillation. moter (32). Therefore, we wanted to determine if the site and
Electrophoretic mobility shift assays (EMSA). Protein-DNA interactions were
timing of yqfS expression was consistent with the location of
carried out in 20-␮l reaction mixtures that typically contained Tris-HCl 50 mM
(pH 7.5), 1 mM DTT, 300 mM NaCl, ⬃20 pmol of the [32P] labeled 19-mer the YqfS protein. Specifically, is YqfS found in the spore
containing a single AP site, and the indicated amounts of purified His6-YqfS and/or in growing cells? To this end, cellular extracts prepared
protein. Reactions were incubated at 4°C for 10 min and then loaded onto 3% from vegetatively growing cells and mature spores of B. subtilis
agarose gels. Gels were first subjected to electrophoresis at 70 V in 1⫻ Tris- 168 were separated by SDS-PAGE, transferred to a nitrocel-
acetate buffer and after drying subjected to autoradiography.
lulose filter and then probed with a polyclonal chicken anti-
serum against His6-YqfS. As expected, the antibodies recog-
nized the His6-YqfS protein purified from E. coli cells (Fig. 2B,
RESULTS
lane 1); on the other hand, the anti-His6-YqfS antibodies ex-
Expression and purification of YqfS from E. coli. A gene clusively recognized a band of about 33 kDa in cell extracts
designated yqfS was retrieved from the genome of B. subtilis prepared from B. subtilis spores (Fig. 2B, lane 2) but not in
(10); whose primary structure revealed an ORF of 891 bp with those prepared from vegetative cells (Fig. 2B, lane 3). These
enough information for the synthesis of a predicted protein of results, revealed for the first time that the type IV AP-endo-
32, 915 Da. Amino acid alignments (1) showed that YqfS nuclease enzyme encoded by yqfS exists in B. subtilis dormant
posses homologies of 53, 52, and 32% with E. coli Nfo (24), spores.
Saccharomyces cerevisiae Apn1 (18), and Thermotoga maritima Enzyme activities of His6-YqfS. To investigate whether the
endonuclease IV (5), respectively. As shown in Fig. 1, YqfS purified His6-YqfS protein possessed AP-endonuclease activity
VOL. 185, 2003 B. SUBTILIS TYPE IV AP-ENDONUCLEASE 5383

Downloaded from http://jb.asm.org/ on April 2, 2015 by UNIV OF MASS MED SCH

FIG. 1. Amino acid sequence alignment of B. subtilis YqfS with homologs from E. coli (12), S. cerevisiae (18), and T. maritima (5). Symbols:
*, residues involved in the coordination of three Zn atoms in the active site of E. coli Nfo; ⽧, residues involved in forming a deep AP site pocket
for cleaving the phosphodiester bond of the AP-site in E. coli Nfo.

the recombinant protein was first incubated with partially de- also possessed 3⬘-phosphatase activity. Therefore, plasmid
purinated plasmid DNA (AP-pB). The results analyzed by pBluescript was first treated with micrococcal nuclease to gen-
electrophoresis in agarose gels, revealed that YqfS was able to erate 3⬘-phosphates and then used as a substrate to determine
convert the AP-pB plasmid from the closed covalently circular whether His6-YqfS stimulates the nick translation activity of
form to the open circular form (Fig. 3A; lane 4). The nuclease the Klenow fragment of DNA polymerase (7). The results
reaction performed by His6-YqfS was specific for the AP sub- shown in Fig. 4A revealed that YqfS possessed 3⬘-phosphatase
strate as the enzyme was unable to attack the DNA of the activity since it was able to stimulate the incorporation of
nondepurinated plasmid (U-pB) (Fig. 3A, lane 3). The AP- radioactivity into DNA during the nick translation assay. In
endonuclease activity encoded by the His6-YqfS protein was this experiment, a positive control showed that as expected,
also tested against a 5⬘-end radioactively labeled double- Nfo from E. coli also was able to stimulate the nick translation
stranded 19-mer nucleotide containing a single AP site. The activity of the polymerase. Although His6-YqfS was able to
products of the reaction analyzed on a denaturing acrylamide function as an AP-endonuclease and possessed 3⬘-phosphatase
gel revealed that the endonucleolytic activity of YqfS specifi- activity it showed no activities of either 5⬘-phosphatase (Fig.
cally processes the cleavage of the substrate containing an 4B) or 3⬘-exonuclease (Fig. 4C). These results demonstrated
apurinic site (Fig. 3B, lane 6) since it showed no endonuclease that the product encoded by the yqfS gene shares enzyme
activity against the 32P-labeled 19-mer substrate lacking an AP properties similar to those reported for members of the type
site (Fig. 3B, lane 5). As shown in Fig. 3B (lane 4), the AP IV AP-endonuclease family (5, 8, 12).
32
P-labeled 19-mer was also cleaved by E. coli Nfo, which on Biochemical properties of recombinant His6-YqfS. Elucida-
the other hand was not able to process the cleavage of the tion of the crystal structure of E. coli Nfo revealed that its
intact 19-mer substrate (Fig. 3B, lane 3). Utilizing the 32P- active site consists of a trinuclear Zn center which is putatively
labeled 19-mer substrate containing a single AP site as a sub- involved in catalyzing the phosphodiester cleavage in the AP
strate the apparent Km of YqfS for AP cleavage site was 86 nM. sites of DNA (6). Alignments between the primary structures
Furthermore, we investigated whether, in addition to possess- of YqfS and functional homologs of E. coli Nfo, revealed that
ing AP-endonuclease activity, the purified His6-YqfS enzyme the former contains nine invariably conserved residues puta-
5384 SALAS-PACHECO ET AL. J. BACTERIOL.

Downloaded from http://jb.asm.org/ on April 2, 2015 by UNIV OF MASS MED SCH

FIG. 2. (A) SDS-PAGE analysis of His6-YqfS purification through a Ni-NTA-agarose column. Aliquots (15 ␮l) of each sample were electro-
phoresed on a 10% polyacrylamide gel which was stained with Coomassie blue. Lane 1, molecular weight standards; lane 2, E. coli PERM348 lysate;
lane 3, flowthrough; lanes 4 and 5, fractions eluted from the column with 100 mM imidazole. (B) Immunoblot analysis of protein samples prepared
with 5 ␮g of pure His6-YqfS (lane 1) or 100 ␮g of cell extracts prepared from either mature spores (17) (lane 2) or vegetative cells (lane 3) of B.
subtilis 168, which were separated on an SDS–12% polyacrylamide gel and transferred to a nitrocellulose membrane. The blot was probed with
a polyclonal anti-His6-YqfS chicken antibody which was diluted 5,000-fold and then processed with an ECL Western blotting analysis system
(Amersham Pharmacia, Buckinghamshire, United Kingdom).

tively involved in coordinating three Zn atoms (Fig. 1). There- DNA (6). Due to the evident structural similarities between
fore, we investigated whether YqfS is also a metal dependent YqfS and Nfo (Fig. 1) we analyzed the DNA binding proper-
enzyme. Increasing concentrations of EDTA were added to ties of YqfS by utilizing DNA shift mobility analysis by probing
reactions containing His6-YqfS and the substrate AP-pB. Re- with a 32P 5⬘-end labeled double-stranded 19-mer nucleotide.
sults analyzed on agarose gels, revealed that concentrations as As shown in Fig. 6, the His6-YqfS enzyme was able to recog-
high as 100 mM of EDTA were unable to inhibit the AP- nize and bind to the AP site containing 32P-labeled 19-mer
endonuclease activity of YqfS (Fig. 5A, lanes 2 to 5). In fact, a substrate causing a shift on its electrophoretic mobility. For-
concentration of 250 mM of EDTA was required to inactivate mation of the His6-YqfS:AP-19-mer complex was dependent
the AP-endonuclease activity of this protein (results not on the concentration of the enzyme used in the reaction (Fig.
shown). However, as shown in Fig. 5B, when His6-YqfS was 6, lanes 2 to 4). Moreover, addition of antibody for the His6-
preincubated at room temperature with different concentra- YqfS to the binding reaction between His6-YqfS and the ra-
tions of EDTA and then assayed for endonuclease activity dioactive AP double-stranded 19-mer caused the formation of
against the AP-pB substrate, a concentration of as low as 10 a highly retarded His6-YqfS:AP-DNA:Ab complex (Fig. 6, lane
mM of EDTA was enough to inactivate the enzyme. Taken 5). A control experiment showed that the His6-YqfS antibody
together these results we conclude that B. subtilis YqfS is a was not able to interact with the AP radioactive 19-mer sub-
metal dependent enzyme which shows similar properties of strate by itself (Fig. 6, lane 6).
inactivation by EDTA as those observed for E. coli Nfo (12) Crystallographic analysis of Nfo as well as of a complex
and S. cerevisiae ApnI (8). between Nfo and a 15-bp DNA containing tetrahydrofuran as
DNA binding properties of YqfS. Analysis of the crystal a synthetic abasic site suggested that the TIM barrel fold struc-
structure of Nfo revealed for the first time that an ␣8␤8 TIM ture has molecular properties not only for binding and scan-
barrel possesses structural properties that enable it to bind ning normal DNA but also to specifically recognize AP sites on
VOL. 185, 2003 B. SUBTILIS TYPE IV AP-ENDONUCLEASE 5385

Downloaded from http://jb.asm.org/ on April 2, 2015 by UNIV OF MASS MED SCH

FIG. 3. Endonuclease activity of His6-YqfS against a plasmid con-
taining AP sites (A) and against a double-stranded 19-mer containing
a single AP site (B). (A) Aliquots (0.6 ␮g) of His6-YqfS were incubated
with 0.5 ␮g of either nontreated (pBS) (lane 3) or AP-site-containing
(AP-pB) (lane 4) pBluescript. Lane 1, untreated plasmid incubated
with 50 mM Tris-HCl, (pH 7.5), 300 mM NaCl; lane 2, AP sites-
containing plasmid incubated with 50 mM Tris-HCl (pH 7.5)–300 mM
NaCl. The reactions were incubated at 37°C during 30 min and ana-
lyzed by electrophoresis on a 1% agarose gel which was stained with
ethidium bromide. Abbreviations: CCC, covalent closed circular plas-
mid; OC, open circular plasmid. (B) A 510 nM concentration of 32P-,
5⬘-end-labeled double-stranded 19-mer nucleotide containing a single
AP site was incubated in the absence (lane 2) or presence of 300 ng of
His6-YqfS (lane 6) and 1 U of E. coli Nfo (lane 4). A 32P-labeled
19-mer substrate lacking an AP site (510 nM) was incubated under the
same conditions in the absence (lane 1) or presence of 300 ng of
His6YqfS (lane 5) and 1 U of E. coli Nfo (lane 3). The reactions were FIG. 4. Determination of 3⬘-phosphatase (A), 5⬘-phosphatase (B),
separated on a 20% denaturing acrylamide gel and then subjected to and 3⬘-exonuclease (C) activities for YqfS. (A) The ability of either E.
autoradiography. Abbreviations: U, uncleaved substrate; C, cleaved coli Nfo (1 U), His6-YqfS (YqfS) (500 ng), or alkaline phosphatase
substrate. (A.P.) (1 U) to stimulate the nick translation activity of DNA contain-
ing 3⬘-phosphate termini was determined as described in Materials and
Methods. Error bars show standard deviation. (B) 5⬘-Phosphatase ac-
tivity was determined as described in Materials and Methods to either
DNA (6). To test this hypothesis, the purified His6-YqfS en- E. coli Nfo (1 U), His6-YqfS (500 ng) or alkaline phosphatase (1 U).
zyme was incubated with a radioactive double-stranded 19-mer (C) 3⬘-Exonuclease activity was determined as described in Materials
and Methods to either, E. coli Nfo (1 U), His6-YqfS (500 ng), or ExoIII
substrate containing (Fig. 7A) or not containing (Fig. 7B) a (100 U). C, no added enzyme. For all three panels, the y axis shows
single AP site. In this protocol different amounts of sodium counts per minute incorporated (A, 105; B, 105; C, 104). The data are
chloride were added to the reactions, and the results were then expressed as averages of two independent duplicate determinations.
analyzed by EMSA. The data revealed that in the absence or in
the presence of low concentrations of salt (50 and 100 mM) the
enzyme was able to recognize both types of DNA independent the nondamaged DNA substrate (Fig. 7B, lanes 3 to 4). The
of the presence or absence of an AP site (Fig. 7, lanes 2 to 4). bonding complex formed between YqfS and the double-
However, when the concentrations of the salt were increased stranded 19-mer substrate containing an AP site was strong
to 100 to 250 mM, the enzyme was exclusively detached from since the protein remained attached to the damaged substrate
5386 SALAS-PACHECO ET AL. J. BACTERIOL.

Downloaded from http://jb.asm.org/ on April 2, 2015 by UNIV OF MASS MED SCH

FIG. 5. Effect of EDTA on the AP-endonuclease activity of His6-YqfS. (A). Aliquots of His6-YqfS (0.1 ␮g) were incubated with 0.5 ␮g of AP
containing sites pBluescript (APpB), either in the absence (lane 1) or presence (lanes 2 to 5) of different concentrations of EDTA. The reactions
were incubated at 37°C during 30 min and analyzed by electrophoresis on a 1% agarose gel which was stained with ethidium bromide. (B). Aliquots
of His6-YqfS (0.5 ␮g) were incubated either in the absence (lane 1) or presence (lanes 2 to 6) of different amounts of EDTA for 15 min at 37°C,
and then 0.5 ␮g of APpB was added to each reaction mixture and the mixtures were incubated at 37°C during 30 min and analyzed by
electrophoresis on a 1% agarose gel which was stained with ethidium bromide.

even at salt concentrations as high as 500 mM (Fig. 7A, lanes was investigated by growing the cells to an OD600 of 0.5 and
4 to 6). In fact concentrations of 1 M of NaCl were required to then inducing with IPTG to a final concentration of 0.1 mM.
disrupt the His6-YqfS:AP-DNAcomplex (Fig. 7A, lanes 7). The results of Fig. 8A show that even in the absence of IPTG
yqfS genetically complements an E. coli mutant deficient in the cells produce a soluble protein (Fig. 8A, lanes 2 and 3) with
ExoIII and Nfo activities. To investigate the physiological role a molecular mass identical to the pure His6-YqfS protein (Fig.
played by YqfS, it was determined whether its encoding DNA 8, lane 1). On the other hand, isogenic strains lacking the yqfS
sequence complemented the DNA repair-deficient phenotype ORF were unable to induce the synthesis of a protein band
of the mutant E. coli strain RPC501 which lacks both the exoIII with the molecular mass of His6-YqfS, in the absence (Fig. 8,
and nfo genes (33). Accordingly, the plasmid pPERM348, lane 4) or presence (Fig. 8, lane 5) of IPTG. Once it was
which contained the yqfS ORF cloned in frame into the mul- determined that YqfS was being produced in the appropriate
tiple cloning site of the expression plasmid pQE30, was intro- genetic background, E. coli RP501, the ability of this strain to
duced into the mutant strain E. coli RPC501. The production survive treatment with different concentrations of either H2O2
of a His6-YqfS protein in the resulting strain E. coli PERM398 or MMS was determined. The results revealed that the high
VOL. 185, 2003 B. SUBTILIS TYPE IV AP-ENDONUCLEASE 5387

Downloaded from http://jb.asm.org/ on April 2, 2015 by UNIV OF MASS MED SCH

FIG. 6. EMSA of His6-YqfS binding to an AP site containing 32P-labeled double-stranded 19-mer nucleotide at different protein concentra-
tions. Reaction mixtures (20 ␮l) containing ⬃20 pmol of 32P-AP-19 bp, Tris-HCl (50 mM; pH 7.5), 1 mM DTT, and 300 mM NaCl were incubated
for 10 min at 4°C, either in the absence (lanes 1 and 6) or presence (lanes 2 to 5) of the indicated amounts of His6-YqfS. A polyclonal
anti-His6-YqfS antibody was added to the mixture reactions shown in lane 5. The reaction mixtures were loaded onto an agarose gel which was
developed at 70 V in 1⫻ Tris-acetate buffer and after drying subjected to autoradiography.

sensitivity of E. coli RPC501 to both MMS (Fig. 8B) and H2O2 The His6-YqfS protein was utilized to produce polyclonal
(Fig. 8C) can be complemented by the expression of yqfS in the antibodies which when utilized in Western blot experiments
strain E. coli RPC501. However, this complementation was not recognized in cell extracts a 33-kDa protein from B. subtilis
observed for the strain E. coli PERM399, which harbors only spores. This protein had the expected molecular mass for the
the plasmid pQE30, which lacks the ORF for yqfS. Interest- predicted product of yqfS. No similar protein was recognized
ingly, we observed that the resistance of E. coli PERM398 to from extracts of growing cells. These results are in agreement
H2O2 and MMS treatment was better in the absence of IPTG with data that demonstrated that in B. subtilis the expression of
compared to when the yqfS gene was induced to overproduce yqfS occurs in the developing spores during the last steps of
the recombinant protein (results not shown). These data sug- sporulation from a sigG type promoter (32). It has been re-
gest that high levels of expression of His6-YqfS from the T5 ported that other sigG-dependent genes such as ssp genes and
promoter might have a negative effect on the growth and splAB are transcribed during sporulation and their products
survival of E. coli PERM398.
are packed into the spores to confer protection to DNA against
the mutagenic and deleterious effects of chemical agents and
DISCUSSION environmental stresses (16, 29). The specific location of YqfS
in B. subtilis mature spores strongly suggests that a base exci-
Previous results revealed that in B. subtilis the regulation of
yqfS expression occurs in a temporal and forespore-specific sion repair pathway is involved in protecting spores from the
manner (32), suggesting that its product is involved in protect- environmental damage, which results in the generation of AP
ing spores from the environmental conditions which result in sites and strand breaks during either dormancy or germination.
the generation of AP sites and strand breaks. Therefore, it was It remains to be investigated whether other components of the
of relevance to investigate whether YqfS posses enzymatic and BER pathway follow a spore-specific expression pattern.
biochemical properties characteristic of the type IV AP-endo- The functionality of the pure His6-YqfS protein was dem-
nuclease family (18). Accordingly, the yqfS gene was expressed onstrated by its ability to catalyze the cleavage of a plasmid
in E. coli with a N-terminal hexahistidine tag and purified to containing AP sites as well as of a 32P-labeled double-stranded
apparent homogeneity by Ni-NTA-agarose chromatography. 19-mer nucleotide containing a single AP site. The apparent
As has been described for other His-tagged proteins (21, 25), Km of YqfS for AP cleavage site of the last substrate was 86
His6-YqfS exhibited a slow migration on SDS-PAGE, showing nM. A Km value around three times larger, i.e., 270 nM, was
a molecular mass of ⬃36 kDa, 3 kDa above its predicted mass. found for T. maritima endonuclease IV, during the degrada-
5388 SALAS-PACHECO ET AL. J. BACTERIOL.

Downloaded from http://jb.asm.org/ on April 2, 2015 by UNIV OF MASS MED SCH

FIG. 7. EMSA of His6-YqfS binding to a 32P-labeled double-stranded 19-mer nucleotide containing (A) or not containing (B) a single AP site
under different ionic strengths. Reaction mixtures (20 ␮l) containing ⬃20 pmol of either 32P-AP-19bp (A) or 32P-19bp (B), Tris-HCl 50 mM (pH
7.5), and 1 mM DTT were incubated for 10 min at 4°C, either in the absence (lane 1) or presence (lanes 2 to 8) of 8 ␮g of His6-YqfS; containing
(lanes 3 to 7) or not containing (lanes 1 to 2) different concentrations of NaCl. The reaction mixtures were loaded onto an agarose gel which was
developed at 70 V in 1⫻ Tris-acetate buffer and after drying subjected to autoradiography.

tion of the same AP site-containing 19-mer nucleotide (5). rhabditis elegans possess genes which potentially encode endo-
Evaluation of other enzymatic properties revealed that YqfS nuclease IV homologs. However, in the former case the
was capable of stimulating the nick translation activity of DNA SpApn1 gene is apparently not expressed, and no AP-endonu-
polymerase by catalyzing the cleavage of 3⬘ blocking phos- clease activity has been detected in S. pombe extracts (19). On
phates of DNA treated with micrococcal nuclease. This result the other hand, a CeApn recombinant gene did not express a
suggests that YqfS might play an important role in processing functional protein in E. coli; thus, the activities of this homolog
not only AP sites but also single strand breaks on DNA, par- are currently unknown (13). Therefore, YqfS of B. subtilis not
ticularly those which block the 3⬘-OH of a single DNA strand only represents the second homolog of bacterial origin but also
as a result of free radical attack (4). In addition, our results the fourth member of the family IV of AP-endonucleases with
revealed that YqfS lacks both 5⬘-phosphatase and 3⬘-exonucle- a demonstrated biochemical function.
ase activities, a common characteristic shared by the other Upon determination of other biochemical properties of
members of the type IV AP-endonuclease family (5, 12, 20). YqfS, our experiments revealed that in the presence of AP-
The type IV family of AP-endonucleases currently includes DNA, concentrations of EDTA as high as 500 mM were re-
three characterized members, namely, E. coli Nfo (12), S. cer- quired to inactivate this endonuclease. On the other hand, in
evisiae Apn (9, 20), and T. maritima endonuclease IV (5). the absence of DNA a concentration of EDTA 50 times lower
Eukaryotes such as Schizosaccharomyces pombe and Caeno- inhibited the AP-endonuclease activity of YqfS. The resistance
VOL. 185, 2003 B. SUBTILIS TYPE IV AP-ENDONUCLEASE 5389

of YqfS to EDTA inactivation is not unprecedented and is

actually a reported distinctive characteristic of type IV AP-
endonucleases (12, 20) which is not shared by Mg2⫹-depen-
dent endonucleases such as ExoIII and APE-1 (4, 6). Based on
these results as well as on the existence in YqfS of the nine
amino acids involved in forming the trinuclear Zn center in
Nfo (Fig. 1), we suggest that YqfS is a Zn-dependent enzyme.
It has been predicted that E. coli Nfo must bind and scan
normal DNA via electrostatic complementarity and hydrogen
bonding to the DNA phosphate backbone from ␤-barrel bonds
and ␣-helical dipoles ideally positioned by an ␣8␤8 framework
(6). Results of amino acid alignments revealed that YqfS

Downloaded from http://jb.asm.org/ on April 2, 2015 by UNIV OF MASS MED SCH

shares a high degree of amino acid identity with E. coli Nfo
(Fig. 1). Thus, we suspected that the structural properties re-
quired to bind DNA by Nfo should be also conserved in YqfS.
This suggestion was analyzed by EMSA. Results revealed that
YqfS was able to recognize and retard the electrophoretic
mobility of a 32P-labeled 19-mer nucleotide containing an AP
site, in a reaction which was dependent on the concentration of
the YqfS used in the assay. These results combined with the
high degree of sequence similarity between YqfS and Nfo tend
to support the notion that YqfS most probably adopts an ␣8␤8
TIM barrel which shares structural properties with E. coli Nfo
to bind DNA. Results of crystallographic analysis predicted
that the ␣8␤8 TIM barrel adopted by Nfo should be capable of
binding to DNA and discriminate between nondamaged DNA
from DNA containing AP sites (6). Thus, in a first level of
interaction Nfo should be capable of binding nondamaged
DNA to scan it in search of AP sites. It is also expected that a
complex of this nature should be very unstable. Our results
confirmed this prediction for YqfS. Essentially, the enzyme
was able to form a complex with nondamaged DNA which was
disrupted at a low ionic strength. On the other hand, in a
second level of interaction Nfo should be able the recognize an
AP site on DNA and establish a highly stable complex. Such a
complex was indeed formed between YqfS and substrate DNA
containing a single AP site. Disruption of this complex re-
quired concentrations of salt as high as 1 M. Taking these
results together, we conclude that YqfS is an enzyme which
possess structural properties not only to bind undamaged DNA
but also to strongly interact with DNA containing AP sites.
Although E. coli mutants deficient in either exoIII or nfo
show few biological abnormalities (3, 14), it has been reported
that the combination of both mutations generates cells which
exhibit not only a high sensitivity to MMS, H2O2, and tert-butyl
hydroperoxide but also an enhanced mutation rate by MMS
(33). Therefore, we used the exoIII nfo double mutant of E.
coli, RPC501, to investigate whether yqfS can genetically com-
plement the high sensitivity of this strain to H2O2 and MMS.
Our results demonstrated that expression of the His-tagged

the concentrations indicated, and incubation was continued for 1 h at

37°C with shaking. The cell suspensions were diluted serially 10-fold
and plated on solid LB containing the appropriate selective antibiotics.
FIG. 8. Survival curves generated in response to MMS (B) and The viable colonies were counted after 1 to 2 days of incubation at
H2O2 (C) for strains E. coli RPC501 (xth/nfo) (■), PERM398 (RPC501 37°C to estimate survival. (A) SDS-PAGE analysis of His6-YqfS syn-
⫹ pQE30-yqfS) (Œ), and PERM399 (RPC501 ⫹ pQE30) (F). The E. thesis in cell extracts of strains E. coli PERM398, in the absence (lane
coli strains were grown in LB to an OD600 of 0.5. The cells were 2) or presence (lane 3) of 0.1 mM IPTG. Lane 1, 5 ␮g of purified
collected by centrifugation, washed once and suspended in buffer B. His6-YqfS; lanes 4 and 5, cell extracts of E. coli RPC501 and E. coli
The cultures of each strain were treated with either H2O2 or MMS at PERM399, respectively.
5390 SALAS-PACHECO ET AL. J. BACTERIOL.

yqfS complements the DNA repair-deficient phenotype of E. A. Brans, M. Braun, S. C. Brignell, S. Bron, S. Brouillet, C. V. Bruschi, B.
Caldwell, V. Capuano, N. M. Carter, S. K. Choi, J. J. Codani, I. F. Conerton,
coli RPC501. Although yqfS was shown to be more efficient in A. Danchin, et al. 1997. The complete genome sequence of the Gram pos-
complementing the sensitivity to H2O2, it was evident that yqfS itive bacterium Bacillus subtilis. Nature 390:249–256.
was also proficient in reverting the DNA damaging effects 11. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 227:680–685.
produced by the alkylating agent MMS. Taking into account 12. Levin, J. D., A. W. Johnson, and B. Demple. 1988. Homogeneous Escherichia
that H2O2 treatment of cells induces the formation of single coli endonuclease IV. Characterization of an enzyme that recognizes oxida-
strand breaks whereas MMS indirectly generates AP site on tive damage in DNA. J. Biol. Chem. 263:8066–8071.
13. Mason, J. Y., S. Tremblay, and D. Ramotar. 1996. The Caenorhabditis el-
DNA, it is appropriate to conclude that YqfS possesses the egans gene CEAPN1 encodes a homolog of Escherichia coli and yeast
ability to correct, in vivo, both types of DNA lesions. Based on apurinic/apyrimidinic endonuclease. Gene 179:291–293.
14. Milcarek, C., and B. Weiss. 1972. Mutants of Escherichia coli with altered
these results, we conclude that YqfS not only possesses amino deoxyribonucleases. I. Isolation and characterization of mutants for exonu-
acid sequence similarity to functional members of the type IV clease III. J. Mol. Biol. 68:303–318.
AP-endonuclease family but also fulfills similar physiological 15. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor

Downloaded from http://jb.asm.org/ on April 2, 2015 by UNIV OF MASS MED SCH

Laboratory Press, Cold Spring Harbor, N. Y.
functions by conferring protection to cells against the delete- 16. Nicholson, W. L., N. Munakata, G. Horneck, H. G. Melosh, and P. Setlow.
rious effects of oxidative promoters and alkylating agents. Fur- 2000. Resistance of Bacillus endospores to extreme terrestrial and extrater-
thermore, the enzymatic activity of YqfS is in agreement for a restrial environments. Microbiol. Mol. Biol. Rev. 64:548–572.
17. Nicholson, W. L., and P. Setlow. 1990. Sporulation, germination, and out-
role for this protein in the repair of the damage done to the growth, p. 391–450. In C. R. Harwood and S. M. Cutting (ed.). Molecular
DNA of spores and promoting the successful process of ger- biological methods for Bacillus. John Wiley and Sons, Sussex, England.
18. Ramotar, D. 1997. The apurinic-apyrimidinic endonuclease IV family of
mination (26, 27, 31). DNA repair enzymes. Biochem. Cell. Biol. 75:327–336.
19. Ramotar, D., J. Vadnais, J. Mason, and S. Tremblay. 1998. Schizosaccharo-
myces pombe apn1 encodes a homologue of the Escherichia coli endonucle-
ACKNOWLEDGMENTS
ase IV family of DNA repair proteins. Biochim. Biophys. Acta 1396:15–20.
This work was supported by grant 31767-N from the Consejo Na- 20. Ramotar, D., S. C. Poppoff, E. B. Gralla, and B. Demple. 1991. Cellular role
of yeast Apn1 apurinic endonuclease /3⬘-diesterase:repair of oxidative and
cional de Ciencia y Tecnología (CONACYT) of México to Mario
alkylation DNA damage and control of spontaneous mutation. Mol. Cell.
Pedraza-Reyes. José M. Salas-Pacheco and Norma Urtiz-Estrada were Biol. 11:4537–4544.
supported by a doctoral fellowship from CONACYT. R.E.Y. was sup- 21. Rebeil, R. Y., Y. Sun, L. Chooback, M. Pedraza-Reyes, and W. L. Nicholson.
ported by MCB-9975140 from the National Science Foundation. 1998. Spore photoproduct (SP) lyase from Bacillus subtilis spores is a novel
We thank Edmundo Chavez-Cosio for facilities provided during the iron-sulfur enzyme which shares features with class III anaerobic enzymes
obtaining of the anti His6-YqfS antibodies and thank Juan A. Rojas for such as ribonucleotide reductase and pyruvate-formate lyases. J. Bacteriol.
technical assistance. 18:4879–4885.
22. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a
laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring
REFERENCES
Harbor, N.Y.
1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, 23. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with
and D. J. Lipman. 1997. Gapped blast and PSI-blast: a new generation of chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467.
protein database search programs. Nucleic Acids Res. 25:3389–3402. 24. Saporito, S. M., and R. P. Cunningham. 1988. Nucleotide sequence of the
2. Chan, E., and B. Weiss. 1987. Endonuclease IV of Escherichia coli is induced nfo gene of Escherichia coli K-12. J. Bacteriol. 170:5141–5145.
by paraquat, DNA damage by oxygen-derived species. Proc. Natl. Acad. Sci. 25. Schon, U., and W. Schumann. 1994. Construction of His6-tagging vectors
USA 84:3189–3193. allowing single-step purification of GroES and other polypeptides produced
3. Cunningham, R. P., S. M. Saporito, S. G. Spitzer, and B. Weiss. 1986. in B. subtilis. Gene 141:91–94.
Endonuclease IV (nfo) mutant of Escherichia coli. J. Bacteriol. 168:1120– 26. Setlow, B., and P. Setlow. 1994. Heat inactivation of Bacillus subtilis spores
1127. lacking small, acid-soluble spore proteins is accompanied by generation of
4. Friedberg, E. C., G. C. Walker, and W. Siede. 1995. DNA repair and mu- abasic sites in spore DNA. J. Bacteriol. 176:2111–2113.
tagenesis. American Society for Microbiology, Washington, D.C. 27. Setlow, P. 1988. Small acid-soluble spore proteins of Bacillus species: struc-
5. Haas, B. J., M. Sandigursky, J. A. Tainer, W. A. Franklin, and R. P. Cun- ture, synthesis, genetics, function, and degradation. Annu. Rev. Microbiol.
ningham. 1999. Purification and characterization of Thermotoga maritima 42:319–338.
endonuclease IV, a thermostable apurinic/apyrimidinic endonuclease and 28. Setlow, P. 1995. Mechanisms for the prevention of damage to DNA in spores
3⬘-repair diesterase. J. Bacteriol. 181:2834–2839. of Bacillus species. Annu. Rev. Microbiol. 49:29–54.
6. Hosfield, D. J., Y. Guan, B. J. Haas, R. P. Cunningham, and J. A. Tainer. 29. Setlow, B., K. J. Tautvydas, and P. Setlow. 1998. Small acid-soluble spore
1999. Structure of the DNA repair enzyme endonuclease IV and its DNA proteins of the ␣/␤-type do not protect the DNA in Bacillus subtilis spores
complex: double-nucleotide flipping at abasic sites and three-metal-ion ca- against base alkylation. Appl. Environ. Microbiol. 64:1958–1962.
talysis. Cell 98:397–408. 30. Shida, T., T. Ogawa, N. Ogasawara, and J. Sekiguchi. 1999. Characterization
7. Izumi, T., K. Ishizaki, M. Ikenaga, and S. Yonei. 1992. A mutant endonu- of Bacillus subtilis exoA protein: a multifunctional DNA-repair enzyme sim-
clease IV of Escherichia coli loses the ability to repair lethal DNA damage ilar to Escherichia coli exonuclease III. Biosci. Biotechnol. Biochem. 9:1528–
induced by hydrogen peroxide but not that induced by methyl methanesul- 1534.
fonate. J. Bacteriol. 174:7711–7716. 31. Slieman, T. A., and W. L. Nicholson. 2000. Artificial and solar UV radiation
8. Johnson, A. W., and B. Demple. 1988. Yeast DNA 3⬘-repair diesterase is the induces strand breaks and cyclobutane pyrimidine dimers in Bacillus subtilis
major cellular apurinic/apyrimidinic endonuclease: substrate specificity and spore DNA. Appl. Environ. Microbiol. 66:199–205.
kinetics. J. Biol. Chem. 263:18017–18022. 32. Uriz-Estrada, N., J. M. Salas-Pacheco, R. E. Yasbin, and M. Pedraza-Reyes.
9. Kunz, B. A., E. S. Henson, H. Roche, D. Ramotar, T. Nunoshiba, and B. 2003. Forespore-specific expression of Bacillus subtilis yqfS, which encodes
Demple. 1994. Specificity of the mutator caused by deletion of yeast struc- type IV apurinic/apyrimidinic endonuclease, a component of the base exci-
tural gene (APN1) for the major apurininc endonuclease. Proc. Natl. Acad. sion repair pathway. J. Bacteriol. 185:340–348.
Sci. USA 91:8165–8169. 33. Yajko, D. M., and B. Weiss. 1975. Mutations simultaneously affecting endo-
10. Kunst, F., N. Ogasawara, I. Moszer, A. M. Albertini, G. Alloni, V, Azevedo, nuclease II and exonuclease III in Escherichia coli. Proc. Natl. Acad. Sci.
M. G. Bertero, P. Bessieres, A. Bolotin, S. Borchert, R. Borriss, L. Boursier, USA 72:688–692.