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Journal of Bacteriology, October 1999, p. 6210-6213, Vol. 181, No. 19
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Functional Expression of hMYH, a Human Homolog of
the Escherichia coli MutY Protein
Malgorzata M.
Slupska,
Wendy
M.
Luther,
Ju-Huei
Chiang,
Hanjing
Yang, and
Jeffrey H.
Miller*
Department of Microbiology and Molecular
Genetics and the Molecular Biology Institute, University of
California, Los Angeles, California 90095
Received 1 June 1999/Accepted 25 July 1999
 |
ABSTRACT |
We have previously described the hMYH cDNA and genomic
clones (M. M. Slupska et al., J. Bacteriol. 178:3885-3892, 1996).
Here, we report that the enzyme expressed from an hMYH cDNA
clone in Escherichia coli complements the mutator phenotype
in a mutY mutant and can remove A from an
A · 8-hydroxydeoxyguanine mismatch and to a lesser extent
can remove A from an A · G mismatch in vitro.
| |
TEXT |
Reactive oxygen species generated
either as a by-product of cellular respiration or by ionizing radiation
and other oxidizing agents can cause damage to DNA. Cells have
developed mechanisms to prevent mutations from different types of
oxidative damage. Among oxidatively damaged DNA bases,
7,8-dihydro-8-oxodeoxyguanine (also termed
8-hydroxydeoxyguanine [8-oxodG]) is particularly mutagenic. It
can easily mispair with adenine, and if it is not repaired before
replication, such a mispair can result in a G · C-to-T · A transversion at the next round of replication (19). Cells
evolved repair systems to prevent mutation from 8-oxodG. One of the
enzymes involved in that process in Escherichia coli is the
MutY protein (11), a simple glycosylase that removes A when
paired with 8-oxodG or G (1, 10) and to a lesser extent removes A from A · C and A · 7,8-dihydro-8-oxodeoxyadenine (A · 8-oxodA)
(21). Recently, it was reported that after prolonged incubation, and with a large excess of the enzyme, E. coli
MutY can also remove G from G · 8-oxodG mispairs (24).
The 39-kDa E. coli MutY protein belongs to the superfamily
of base excision repair glycosylases, in which a helix-hairpin-helix
motif is the most conserved structural element (15). The
crystal structure of the catalytic core of MutY was recently determined
(3). The resolution of crystal structures revealed that, as
in other related glycosylases (4), the target adenine is
flipped out of the double-stranded DNA helix and accommodated in a
positively charged substrate-binding pocket of the MutY protein
(3).
We have previously described the cloning and sequencing of
hMYH, a human homolog of the E. coli mutY gene
(20). Here, we report functional expression and purification
of the hMYH enzyme. The cloned hMYH gene, when
expressed in E. coli, complements the mutator phenotype of a
mutY mutant. hMYH is a 59-kDa protein that has
glycosylase activity but no AP-lyase activity. hMYH protein can remove
A from an A · 8-oxodG mismatch and to a lesser extent can
remove A from an A · G mismatch. After prolonged incubation, it
can also remove G from a G · 8-oxodG mispair.
Complementation of the E. coli mutY mutation by
hMYH.
The whole cDNA sequence of hMYH was
subcloned into the pCR-Script SKII(+) vector (Stratagene, La Jolla,
Calif.) vector from different clones described previously
(20). For expression, a polyhistidine (His6)
affinity tag was attached to the amino terminus of hMYH by subcloning
into pQE30 (Qiagen, Inc., Chatsworth, Calif.), and this plasmid was
used to check whether hMYH cDNA complements the mutY
deficit in E. coli. The expressed hMYH gene had a
toxic effect on E. coli cells, and transformation of the pQE30/hMYH plasmid into E. coli cells was
difficult. The pQE30 vector is based on the T5 promoter
transcription-translation system. It contains the phage T5 promoter and
two lac operator sequences (Qiagen, Inc.). Since pQE30 has a
high level of background transcription, transformation was improved by
using a recipient strain carrying an additional plasmid, pREP4, that
constitutively expresses the lac repressor. Transformation
of pQE30/hMYH into a strain carrying the pREP4 plasmid was
1,000 times more efficient than its transformation into a strain not
carrying pREP4 (data not shown). It was also easier to maintain
pQE30/hMYH in strains with pREP4. The addition of 1 to 10 µM IPTG (isopropyl-
-D-thiogalactopyranoside)
induced expression of hMYH, but induction with IPTG at
concentrations higher than 100 µM resulted in lysis of bacteria.
The E. coli CC104 strain carries the F' plasmid with the
lac gene engineered for detection of G · C-to-T
· A transversions (2). Previous work has shown that
strains with defects in mutY have increased G · C-to-T · A transversions (16), so we used the CC104
mutY::Tn10 (12) derivative
to check whether hMYH complements the mutY
mutator phenotype. The complementation experiment was performed as
described previously (14). Cells of strain CC104 mutY carrying both the pREP4 and pQE30/hMYH
plasmids showed suppression of the mutator phenotype. The suppression
was complete when cells were grown in the presence of 1 µM IPTG
(Table 1). Use of higher concentrations
of IPTG resulted in a very low survival rate for the CC104
mutY strain, loss of the hMYH insert from the
plasmid, or both (data not shown).
Expression and purification of hMYH.
The
hexahistidine-tagged hMYH protein was purified by
Ni2+ affinity chromatography, followed by
single-stranded DNA (ssDNA) affinity chromatography. Conditions
for Ni2+-agarose purification were set
according to the protocols of the manufacturer (Qiagen, Inc.), except
that a pH of 6.5 and no NaCl were used for binding and the column was
washed with buffer containing 100 mM NaCl and 30 mM imidazole at pH
6.5. Conditions for ssDNA-cellulose were as described for the E. coli MutY purification (6). The purified protein reacts
with antibodies against the histidine tag [anti-RGS(H)4;
Qiagen, Inc.] (Fig. 1).
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FIG. 1.
(A) Sodium dodecyl sulfate-polyacrylamide gel analysis
of hMYH purified by Ni2+-agarose affinity chromatography,
followed by ssDNA-cellulose chromatography (E). The proteins were
separated on a 10% polyacrylamide gel in the presence of sodium
dodecyl sulfate and stained with Coomassie blue. Lane S, molecular mass
standards. Molecular masses, in kilodaltons, are marked on the side.
Lane C+, lysate of cells of the CC104 mutY mutant containing
pQE30/hMYH; lane C , lysate of the same strain with pQE30.
(B) Western blot analysis of hMYH. Proteins were separated on a 10%
polyacrylamide gel, transferred onto a Hybond enhanced
chemiluminescence nitrocellulose membrane (Amersham Pharmacia Inc.,
Piscataway, N.J.), and reacted with antibodies against the histidine
tag [anti-RGS(H)4; Qiagen, Inc.]. Western blotting was
performed by enhanced chemiluminescence analysis (ECL; Amersham).
Arrows point to the position of full-length hMYH. Lanes C+,
C, and E are as described for panel A.
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|
The expressed hMYH was rapidly degraded, in both a CC104
mutY mutant (
12) (normally used for expression)
and a strain that
lacked the
E. coli outer membrane protease
(
ompT mutant) (BL 21;
Novagen Inc., Madison, Wis.). The
degrees of degradation were
similar during growth at different
temperatures (15 to 37°C).
Purified fractions contained other
proteins, some of which cross-reacted
with
anti-RGS(H)
4 antibody (Fig.
1).
The cloned
hMYH gene has seven AGA and AGG codons in the 5'
terminus of the mRNA. These are rare arginine codons in
E. coli and can result in poor expression in
E. coli
(
7), as was the
case for
hMYH. It has been shown
that cloning the
argU gene, encoding
the arginine tRNA that
reads AGA and AGG codons, can improve the
expression of some eukaryotic
genes with rare arginine codons
(
18). Therefore, we cloned
the
argU gene with its natural promoter
into the same
expression plasmid but did not observe any improvement
in hMYH levels.
We also cloned and expressed N-terminally truncated
clones of hMYH. We
expressed a 54-kDa clone (missing the first
41 amino acids), a 52-kDa
clone (missing the first 62 amino acids),
and a 45-kDa clone (missing
the first 120 amino acids). We observed
high-level expression only for
the 45-kDa clone (data not shown).
It turned out that the protein
expressed by this clone was inactive
(data not shown). Also, this was
the only clone that did not have
rare arginine codons in the
5'-terminal part of the
gene.
hMYH activity.
To determine the specificity of hMYH, we
investigated its glycosylase activity on various mismatches
placed in 96-mers. The sequence of 96-mers containing
8-oxodG or G was
5'AATTGTCCTTCCCTTCCTTTCTCGCC ACGTTCGCCGAATTGGGCTTTCCCCGTCAAGCTCTA AATCGGGGGCTCCCTTTAGGGTTCCGATCCGGCC3' (the
bold G marks the position of 8-oxodG). We tested A · 8-oxodG, A · G, A · C, A · A, A · T,
C · G, C · 8-oxodG, C · T, C · C, G · 8-oxodG, G · T, G · G, T · T, and
T · 8-oxodG mispairs. We detected activity for the A
· 8-oxodG and A · G mispairs but not for the A · C
mispair (Fig. 2). As for E. coli MutY, cleavage of the oligomer by hMYH was seen only for the
strand containing A (data not shown). After prolonged incubation and
strong overexposure of the film, we could also see a small degree of
cleavage for the G · 8-oxodG mispair but not for the A
· C mispair (data not shown). The same substrates were processed by
the mouse MutY homolog (data not shown). The only difference we
observed between the hMYH and the E. coli MutY enzymes was
the lack of glycosylase activity for the A · C mispair. This
activity was also undetectable in HeLa cell extracts (23)
and very weak for the Schizosaccharomyces pombe MutY homolog
(5), but this activity for a partially purified bovine MutY
homolog has been reported (8).
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FIG. 2.
Reaction of hMYH and E. coli MutY (EcMY) with
different mispair-containing 96-mer templates. Reactions were carried
out for 3 h at 37°C; 3 µl of the protein preparation described
in the legend to Fig. 1 and 6 ng of E. coli MutY protein
were used for all templates. E. coli MutY was purified as
described previously (6). The asterisks each indicate an A
from a radiolabelled strand. s, substrate; p, product.
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|
We also tested hMYH activity towards A · 8-oxodG and A
· G mispairs placed in shorter oligomers. We could easily detect the
activity for the A · 8-oxodG mispair, but surprisingly, we
did
not see any activity for the A · G mispair placed in a
23-mer.
We saw, however, the activity of hMYH with the A · G
mispair in
45-mer (Fig.
3) and 72- and
96-mer (data not shown) templates.
To detect hMYH glycosylase activity
on A · G mismatches we had
to use more enzyme and longer
reaction times (Fig.
3). The time
and concentration courses for the
A · 8-oxodG mispair in a 23-mer
template and the A · G mispair in a 42-mer template are shown
in Fig.
3. The lack of
activity of hMYH with an A · G mispair
in a 23-mer is an
unexpected observation. An
S. pombe MutY homolog
(
5), a partially purified calf MutY homolog (
8),
and a mouse
MutY homolog from our lab (data not shown) all cleave a 20- or
23-bp DNA containing an A · G mismatch. The A · 8-oxodG-containing
oligomers are, however, much better substrates
for both hMYH and
mMYH (mouse MutY homolog), and this difference is
particularly
obvious when short reaction times and smaller amounts of
enzyme
are used (data not shown). One explanation for this phenomenon
might be that both hMYH and mMYH have low affinity to the A ·
G
mismatch placed in shorter templates but the affinity of the
mouse MutY
homolog to A · G-containing short oligomers is higher
than that
of hMYH. The differences between DNA glycosylase homologs
of closely
related species have already been described for methylpurine-DNA
glycosylase from mice and humans (
17). It was also observed
that human methylpurine-DNA glycosylase has a 10-fold-lower binding
capacity for shorter oligomers (<15 bp) than for longer ones
(
9).
We also cannot exclude the possibility that the lack of
hMYH glycosylase
activity on 23-mer substrates is due to the
neighboring sequence
context. The investigated mispairs were placed in
a different
context in the 23-mers than those of the 45-, 72-, and
96-mers
used in this work, and the sensitivity to sequence context of
the MutY glycosylase activity towards A · G mispairs has been
noted for
E. coli (
22).
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FIG. 3.
Concentration dependence and time course for the
enzymatic reaction of hMYH with A · 8-oxodG placed in a
23-mer (A) and A · G placed in a 45-mer (B). Reaction conditions
were as described previously (13). The sequence of the
A-containing 23-mer was 5'AGAGGAAAGGAGAGAAGGGAGAG3'.
The sequence of the 45-mer for the A-containing strand was
5'TTAGAGCTTGACGGGGAAAGCCAAATTCGGCGAACGTGGCGAGAA3'
(bold A marks the position of the mispaired A for both
templates). In the variable-concentration experiment, reaction with
A · 8-oxodG was carried out for 10 min, whereas reaction
with A · G was carried out for 60 min. In the time course
experiment for the A · 8-oxodG substrate, 0.3 µl of the
enzyme solution was used, and for the A · G substrate, 1 µl of
the enzyme was used. The A-containing oligonucleotide was
radiolabelled. S and P, substrate and product, respectively.
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|
To determine whether hMYH has both glycosylase and AP-lyase activities,
we repeated conditions for a similar experiment with
E. coli
MutY, described previously (
25). In this experiment,
the
product of the same reaction was resolved on acrylamide gels
under
different, carefully controlled heating conditions. As can
be seen in
Fig.
4, for hMYH as well as
E. coli MutY, under the
"no-heat" conditions the product of DNA
nicking is barely visible,
whereas when the same reaction mixture was
heated at 95°C for
5 min, the
-elimination product is obvious.
Additional treatment
with piperidine (known to cleave AP sites)
resulted in a shift
of the
-elimination product into a
-elimination product (Fig.
4). These results indicate that, similar
to the
E. coli MutY protein,
hMYH is a simple glycosylase
with only residual, if any, AP-lyase
activity.
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FIG. 4.
Products generated by hMYH under different conditions of
heating. Reactions were stopped by freezing in a dry ice bath (lanes
A), loading buffer was added and reaction mixtures were heated for 5 min at 95°C (lanes B), and panels an equal volume of 20% piperidine
was added and the reaction mixture was heated for 30 min at 95°C,
dried under vacuum, and dissolved in formamide loading buffer (lanes
C). Products were resolved on a 15% polyacrylamide-8 M urea gel under
conditions described previously (25). Numbers 1 and 2 indicate the enzyme from two different preparations of hMYH. s,
migration of substrate; p, product of -elimination; p, product
of -elimination. Samples without enzyme were used for the control.
Reactions were carried out with an A · 8-oxodG mispair
placed in a 23-mer, with the A-containing strand radiolabelled, for 10 min at 37°C. The sequence of the 23-mer is in the legend to Fig. 3.
|
|
In summary, the data presented in this paper show that hMYH, cloned in
our laboratory on the basis of DNA sequence homology
to the
E. coli mutY gene, is the true homolog of the bacterial
MutY
glycosylase.
| |
ACKNOWLEDGMENTS |
This work was supported by a grant to J.H.M. from the National
Institutes of Health (GM 32184).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Molecular Genetics and the Molecular Biology
Institute, University of California, Los Angeles, CA 90095. Phone:
(310) 825-3578. Fax: (310) 206-5231. E-mail: jhmiller{at}mbi.ucla.edu.
| |
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Journal of Bacteriology, October 1999, p. 6210-6213, Vol. 181, No. 19
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Gabrovsky, V., Yamamoto, M. L., Miller, J. H.
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Bai, H., Jones, S.ân, Guan, X., Wilson, T. M., Sampson, J. R., Cheadle, J. P., Lu, A-L.
(2005). Functional characterization of two human MutY homolog (hMYH) missense mutations (R227W and V232F) that lie within the putative hMSH6 binding domain and are associated with hMYH polyposis. Nucleic Acids Res
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Sieber, O. M., Howarth, K. M., Thirlwell, C., Rowan, A., Mandir, N., Goodlad, R. A., Gilkar, A., Spencer-Dene, B., Stamp, G., Johnson, V., Silver, A., Yang, H., Miller, J. H., Ilyas, M., Tomlinson, I. P. M.
(2004). Myh Deficiency Enhances Intestinal Tumorigenesis in Multiple Intestinal Neoplasia (ApcMin/+) Mice. Cancer Res.
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Tao, H., Shinmura, K., Hanaoka, T., Natsukawa, S., Shaura, K., Koizumi, Y., Kasuga, Y., Ozawa, T., Tsujinaka, T., Li, Z., Yamaguchi, S., Yokota, J., Sugimura, H., Tsugane, S.
(2004). A novel splice-site variant of the base excision repair gene MYH is associated with production of an aberrant mRNA transcript encoding a truncated MYH protein not localized in the nucleus. Carcinogenesis
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Ma, H., Lee, H. M., Englander, E. W.
(2004). N-terminus of the rat adenine glycosylase MYH affects excision rates and processing of MYH-generated abasic sites. Nucleic Acids Res
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Xie, Y., Yang, H., Cunanan, C., Okamoto, K., Shibata, D., Pan, J., Barnes, D. E., Lindahl, T., McIlhatton, M., Fishel, R., Miller, J. H.
(2004). Deficiencies in Mouse Myh and Ogg1 Result in Tumor Predisposition and G to T Mutations in Codon 12 of the K-Ras Oncogene in Lung Tumors. Cancer Res.
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Cheadle, J. P., Sampson, J. R.
(2003). Exposing the MYtH about base excision repair and human inherited disease. Hum Mol Genet
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Enholm, S., Hienonen, T., Suomalainen, A., Lipton, L., Tomlinson, I., Karja, V., Eskelinen, M., Mecklin, J.-P., Karhu, A., Jarvinen, H. J., Aaltonen, L. A.
(2003). Proportion and Phenotype of MYH-Associated Colorectal Neoplasia in a Population-Based Series of Finnish Colorectal Cancer Patients. Am. J. Pathol.
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Yamane, A., Shinmura, K., Sunaga, N., Saitoh, T., Yamaguchi, S., Shinmura, Y., Yoshimura, K., Murakami, H., Nojima, Y., Kohno, T., Yokota, J.
(2003). Suppressive activities of OGG1 and MYH proteins against G:C to T:A mutations caused by 8-hydroxyguanine but not by benzo[a]pyrene diol epoxide in human cells in vivo. Carcinogenesis
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Jensen, A., Calvayrac, G., Karahalil, B., Bohr, V. A., Stevnsner, T.
(2003). Mammalian 8-Oxoguanine DNA Glycosylase 1 Incises 8-Oxoadenine Opposite Cytosine in Nuclei and Mitochondria, while a Different Glycosylase Incises 8-Oxoadenine Opposite Guanine in Nuclei. J. Biol. Chem.
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Barone, F., Dogliotti, E., Cellai, L., Giordano, C., Bjoras, M., Mazzei, F.
(2003). Influence of DNA torsional rigidity on excision of 7,8-dihydro-8-oxo-2'-deoxyguanosine in the presence of opposing abasic sites by human OGG1 protein. Nucleic Acids Res
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Sieber, O. M., Lipton, L., Crabtree, M., Heinimann, K., Fidalgo, P., Phillips, R. K.S., Bisgaard, M.-L., Orntoft, T. F., Aaltonen, L. A., Hodgson, S. V., Thomas, H. J.W., Tomlinson, I. P.M.
(2003). Multiple Colorectal Adenomas, Classic Adenomatous Polyposis, and Germ-Line Mutations in MYH. NEJM
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Gu, Y., Parker, A., Wilson, T. M., Bai, H., Chang, D.-Y., Lu, A-L.
(2002). Human MutY Homolog, a DNA Glycosylase Involved in Base Excision Repair, Physically and Functionally Interacts with Mismatch Repair Proteins Human MutS Homolog 2/Human MutS Homolog 6. J. Biol. Chem.
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Boldogh, I., Milligan, D., Lee, M. S., Bassett, H., Lloyd, R. S., McCullough, A. K.
(2001). hMYH cell cycle-dependent expression, subcellular localization and association with replication foci: evidence suggesting replication-coupled repair of adenine:8-oxoguanine mispairs. Nucleic Acids Res
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Gu, Y., Lu, A-L.
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Iida, T., Furuta, A., Kawashima, M., Nishida, J.-i., Nakabeppu, Y., Iwaki, T.
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Shinmura, K., Yamaguchi, S., Saitoh, T., Takeuchi-Sasaki, M., Kim, S.-R., Nohmi, T., Yokota, J.
(2000). Adenine excisional repair function of MYH protein on the adenine:8-hydroxyguanine base pair in double-stranded DNA. Nucleic Acids Res
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Parker, A., Gu, Y., Lu, A-L.
(2000). Purification and characterization of a mammalian homolog of Escherichia coli MutY mismatch repair protein from calf liver mitochondria. Nucleic Acids Res
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(2000). Identification of human MutY homolog (hMYH) as a repair enzyme for 2-hydroxyadenine in DNA and detection of multiple forms of hMYH located in nuclei and mitochondria. Nucleic Acids Res
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Parker, A., Gu, Y., Mahoney, W., Lee, S.-H., Singh, K. K., Lu, A-L.
(2001). Human Homolog of the MutY Repair Protein (hMYH) Physically Interacts with Proteins Involved in Long Patch DNA Base Excision Repair. J. Biol. Chem.
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