DNA Repair Protocols. Prokaryotic Systems

189 Pages·2010·0.941 MB·English

by  Pat Vaughan

MMeetthhooddss iinn MMoolleeccuullaarr BBiioollooggyy
TTMM
VOLUME 152
DDNNAA RReeppaaiirr
PPrroottooccoollss
PPrrookkaarryyoottiicc SSyysstteemmss
EEddiitteedd bbyy
PPaatt VVaauugghhaann
HHUUMMAANNAA PPRREESSSS
Repair of A/G and A/8-oxoG Mismatches 3
1
Repair of A/G and A/8-oxoG Mismatches
by MutY Adenine DNA Glycosylase
A-Lien Lu
1. Introduction
Cellular and organism aging have been correlated with accumulated DNA
damage (1,2). 8-oxo-7,8-dihydrodeoxyguanine (8-oxoG or GO) is one of the
most stable products of oxidative DNA damage. The formation of GO in DNA,
if not repaired, can lead to misincorporation of A opposite to the GO lesion and
result in G:C to T:A transversions (3–6). In Escherichia coli, a family of
enzymes, MutY, MutM, and MutT, is involved in defending against the
mutagenic effects of GO lesions (7–9). The E. coliMutY is an adenine glycosylase
active on DNA containing A/GO, A/G, and A/C mismatches (7,10–15)and also has
a weak guanine glycosylase activity on G/GO-containing DNA (15a,15b).
MutY removes misincorporated adenines paired with GO lesions and reduces
the GO mutational effects. The 39-kDa MutY protein from E. coli is an iron-
sulfur protein. The MutY protein was shown by Tsai-Wu et al. (16) to have
both DNA glycosylase and apurinic/apyrimidinic (AP) lyase activities. Recent
results show that MutY and the N-terminal catalytic domain can be trapped in
a stable covalent enzyme-DNA intermediate in the presence of sodium boro-
hydride (17–19) and support that MutY contains both DNA glycosylase and
AP lyase activities. The DNA glycosylase activity removes the adenine bases
from the A/GO, A/G, and A/C mismatches (16) and the AP lyase activity
cleaves the first phosphodiester bond 3' to the AP site (12,16). Apparent
dissociation constants are 0.066, 5.3, and 15 nM for A/GO-, A/G-, and A/C-
containing DNA, respectively (20).
MutY homologous (MYH) activities have been identified in human HeLa
(21), calf thymus (22), and fission yeast Schizosaccharomyces pombe (23).
From: Methods in Molecular Biology, vol. 152: DNA Repair Protocols: Prokaryotic Systems
Edited by: P. Vaughan © Humana Press Inc., Totowa, NJ
3
4 Lu
The recombinant human MYH from the cloned cDNA has been expressed and
partially characterized (24a–c). A human cDNA of putative hMYH has been
cloned (24). These MYH proteins share high-sequence homology and similar
mechanisms with the E. coli MutY protein (21,24). The high homology of
MutY homologs among different organisms suggests important roles in their
cellular functions.
Genetic mutations can be detected by MutY protein (25,26) based on its
specific binding and nicking of DNA heteroduplexes containing an A/G or
A/C mismatch. In this mismatch repair enzyme cleavage (MREC) method,
DNA fragments amplified from normal and mutated genes by polymerase chain
(PCR) are mixed and annealed to create base/base mismatches for cleavage by
repair enzymes. MutY can detect A:T–C:G transversions and G:C–A:T transi-
tions. The method is powerful and sensitive.
2. Materials
2.1. Reagents and Buffers
1. 10X MutY reaction buffer: 200 mM Tris-HCl, pH 7.6, 800 mM NaCl, 10 mM
dithiothreitol (DTT), 10 mMethylendiaminetetraacetic acid (EDTA), 29% (v/w)
glycerol.
2. 10X MYH reaction buffer: 100 mMTris-HCl, pH 7.6, 5 mMDTT, 5 mMEDTA,
15% (v/w) glycerol.
3. MutY storage/dilution buffer: 20 mMpotassium phosphate, pH 7.4, 1.5 mMDTT,
0.1 mMEDTA, 50 mMKCl, 200 µg/mL bovine serum albumin (BSA), and 50%
glycerol.
4. 10X hybridization buffer: 70 mMTris-HCl, pH 7.6, 70 mM MgCl , and 500 mM
2
NaCl.
5. 5X Klenow buffer: 250 mM Tris-HCl, pH 7.6, 25 mM MgCl , 25 mM
2
β-mercaptoethanol, 0.1 mM dGTP, and 0.1 mM dTTP.
6. 10X kinase buffer: 500 mM Tris-HCl, pH 7.6, 100 mM MgCl , 50 mM DTT,
2
1 mM spermidine, and 1 mM EDTA.
7. 10X DNA dye: 60% glycerol, 50 mM EDTA, 0.5% sodium dodecyl sulfate
(SDS), 0.05% xylene cyanol, and 0.05% bromophenol blue.
8. Sequencing dye: 90% formamide, 10 mMEDTA, 0.1% xylene cyanol, and 0.1%
bromophenol blue.
9. 5X SDS-polyacrylamide gel electophoresis (PAGE) dye: 155 mM Tris-HCl,
pH 6.8, 25% (v/v) glycerol, 5% (w/v) SDS, 0.5 mg/mL bromophenol blue, and
5% (v/v) β-mercaptoethanol.
10. Klenow fragment of DNA polymerase I (New England BioLabs).
11. Polynucleotide kinase (New England BioLabs).
12. Poly(dI-dC): 200 µL at 10 µg/mL (Parmacia Biotech).
13. TBE buffer: 50 mM Tris-borate, pH 8.3, and 1 mM EDTA.
14. SDS-PAGE running buffer: 25 mM Tris-base, 192 mM glycine, and 1% SDS.
Repair of A/G and A/8-oxoG Mismatches 5
15. TE buffer: 10 mM Tris-HCl, pH 7.6, 0.1 mM EDTA.
0.1
16. Quick-spin column (Boehringer Mannheim).
17. [α-P32] dCTP and [γ-P32] ATP at 3000 Ci/mmol from NEN.
18. Diethylaminoethyl (DEAE)-81 paper (Whatman, cut into 1.2 ×1.2-cm squares).
19. GF/C filter (Whatman, 2.4-cm circle).
20. Coomassie stain: 0.25% (w/v) Coomassie brillant blue R250 in 50% methanol
and 10% acetic acid.
21. Buffer T: 50 mMTris-HCl, pH 7.6, 0.1 mM EDTA, 0.5 mM DTT, and 0.1 mM
phenylmethylsulfonyl fluoride (PMSF).
22. Buffer A: 20 mM potassium phosphate, pH 7.4, 0.5 mM DTT, 0.1 mM EDTA,
and 0.1 mM PMSF.
23. Buffer B: 0.01 M potassium phosphate, pH 7.4, 10 mM KCl, 0.5 mM DTT,
0.1 mM EDTA, and 0.1 mM PMSF.
2.2. DNA Substrates
2.2.1. Synthesis and Purification
The 19-mer oligonucleotides (see sequences in Subheading 2.2.2.) were
synthesized at 0.2-µmol scale on an Applied Biosystems 381A automated
synthesizer by using standard procedures. Phosphoramidite of 8-oxo-dG was
purchased from Glen Research.
1. Load deprotected oligonucleotides (1 OD per 1 cm × 0.15-cm well) on a 14%
sequencing gel (27) and run the gel at 600 V for 40 min.
2. Put the gel over a Whatman TLC plate (cat. no. 4410222) and shine it with short-
wave UV from a hand UV lamp (UV shadowing). Excise the full-length bands
(up to 10 OD) in a 15-mL Falcon centrifuge tube.
3. Crush the gel with a clean glass rod and add 10 mL of 1 M triethylammonium
bicarbonate (TEAB), pH 7.0 to the tube, which is rotated overnight at 37°C.
4. Spin with a table-top centrifuge for 10 min and transfer the supernatant to a new tube.
5. Wash a C18 Sep-Pak column (Waters) with 10 mL each of 100% ethanol, 50%
ethanol/50% 25 mM TEAB, and then 25 mM TEAB.
6. Load the eluted DNA onto the C18 Sep-Pak column.
7. Wash the column with 10 mL 25 mMTEAB and elute DNA with 2 mL of 40%
ethanol/ 60% 25 mM TEAB.
8. Lyophilize the sample to dry and dissolve DNA with 1 mL of distilled water.
Determine its concentration by A quantitation (1 OD = 33 µg/mL).
260
2.2.2. Annealing
1. Mix two complementary oligonucleotides in hybridization buffer in a 1.5-mL
microtube (150 pmol each 15 µL of 10X hybridization buffer and water to 150 µL).
2. Heat at 90°C for 2 min and then the tube is placed on the top of a 25-mL beaker
with 90°C water and cooled gradually to room temperature over more than
30min. Heteroduplexes are constructed as follows.
6 Lu
5'-CCGAGGAATTAGCCTTCTG-3'
3'-GCTCCTTAAGCGGAAGACG-5'
5'-CCGAGGAATTAGCCTTCTG-3'
3'-GCTCCTTAAOCGGAAGACG-5'
5'-CCGAGGAATTAGCCTTCTG-3'
3'-GCTCCTTAACCGGAAGACG-5'
5'-CCGAGGAATTCGCCTTCTG-3'
3'-GCTCCTTAAGCGGAAGACG-5'
2.4. Apparatus
1. Sequencing gel apparatus (IBI STS 45i DNA sequencing unit cat. no. IB80000 or
BRL cat. no. 21070-016 for 0.8-mm-thick spacer).
2. SDS-PAGE aparatus (Novex cat. no. EI9001).
3. Gel-shifting apparatus (BRL cat. no. 21070-024 for 1.5-mm-thick spacer).
4. Power supplies.
5. Desiccator.
6. Gel dryer.
7. X-ray film cassettes.
8. Microcentrifuge.
9. Water bath.
10. Beckman 70.1 Ti rotor and centrifuge.
11. Waters or Pharmacia FPLC system.
12. Table-top IEC clinical centrifuge.
3. Methods
3.1. Preparation of Labeled DNA Substrates
3.1.1. 3'-End Labeling Reaction
Oligonucleotides with A/G, A/GO, or A/C mismatches are substrates for
MutY. Homoduplex with C:G is not a substrate and should also be used as a
negative control for MutY nicking and binding.
1. To a microcentrifuge tube, add the following in order:
Sterile dH O 5.5µL
2
5X Klenow buffer 3 µL
Duplex Oligonucleotide (1 pmol/mL) 1 µL
[α-32P] dCTP at 3,000 Ci/mmol 5 µL
Klenow fragment (5 U/µL) 0.5 µL
Total 15 µL
2. Incubate the reaction for 30 min at room temperature.
(At the same time, prepare G-25 column, see below.)
3. Then, add 1 µL of 0.5 MEDTA and 34 µL of TE to stop the reaction.
0.1
Repair of A/G and A/8-oxoG Mismatches 7
4. Spot 0.5 µL onto a piece of square DEAE paper and then wash as described in
Subheading 3.1.4.
5. Pass the rest of the sample through a Quick-Spin G-25 column as described in
steps 8-13 in Subheading 3.1.3.
3.1.2. 5'-End-Labeling Reaction
1. To a microcentrifuge tube, add the following in order:
Sterile dH O 6.8µL
2
10X kinase buffer 2 µL
Oligonucleotide (single-stranded) (1 pmol/µL) 1 µL
[γ-32P] ATP at 3,000 Ci/mmol 10 µL
T4 polynucleotide kinase (10 U/µL) 0.5 µL
Total 15 µL
2. Incubate the reaction for 30 min at 37°C.
3. Stop the reaction by heating at 65°C for 5 min.
4. Add 30 µL of TE .
0.1
5. Spot 0.5 µL onto a piece of square DEAE paper and then wash as described in
Subheading 3.1.4.
6. Add 2 µL of 10X hybridization buffer and 2 pmol of the complementary strand of
oligonucleotide.
7. Heat 90°C for 2 min and then cool gradually to room temperature over 30 min to
form heteroduplexes.
8. Add 0.2 µL 10 mMeach of the four dNTP and 0.5 µL of Klenow fragment. Incu-
bate for 30 min.
9. Load the sample onto a Quick-Spin column (seesteps 8–13 inSubheading 3.1.3.)
3.1.3. Removal of Free Nucleotides
These procedures are modified from the manufacturer’s manual.
1. Invert the Quick-Spin G25 column several times.
2. Remove the top and bottom caps.
3. Put one receiving tube in a 15-mL plastic tube and then the column.
4. Spin the assembly in a table-top IEC clinical centrifuge with swing buckets for 2 min.
5. Discard the solution.
6. Add 0.4 mL TE on the top of the column, repeat steps 4 and 5.
0.1
7. Spin again for 2 min without adding buffer. Discard the solution and replace a
new receiving tube.
8. Load 49.5 µL of labelled sample from Subheadings 3.1.1.or3.1.2.(Remember to spot
0.5µL of sample onto DEAE paper to check incorporation, seeSubheading 3.1.4.)
9. Spin for 4 min.
10. Carefully transfer the solution passed through the column into another tube and
measure its volume.
11. Spot 0.5 µL onto a piece of square DEAE paper and follow the washing steps (see
Subheading 3.1.4.) and spot 0.5 µL onto a GF/C filter paper, dry under a heat
lamp, and count.
8 Lu
12. Store the rest in –20°C and make proper dilution to 1.8 fmol/µL according to
Subheading 3.1.5.
3.1.4. Check Incorporation
1. Spot 0.5 µL of labeled DNA samples before and after the G-25 column onto
pieces of DEAE paper. (Mark paper squares with a pencil.)
2. Wash DEAE papers with 200 mL of 0.25 M ammonium bicarbonate contained
within a 1L beaker.
3. Shake 5 min with speed sufficient for the papers to float.
4. Carefully discard the washing solution into a radioactive waste jar.
5. Repeat steps 2–4 two more times.
6. Wash DEAE papers with 200 mL of 95% ethanol, similar to steps 2–4three times.
7. Put papers on a sheet of aluminum foil and dry 10 min under a heat lamp.
8. Add 5 mL of scintillation cocktail and count.
3.1.5. Determination of Specific Activity (see Note 1)
Pre-G-25 DEAE paper A cpm/0.5 µL
Post-G-25 DEAE paper B cpm/0.5 µL
Post-G-25 GF/C paper C cpm/0.5 µL
Total cpm = cpm/pmol (pre-column) T1 = A × 100
cpm/1.8 fmol S = T1 × 0.0018
Total cpm (postcolumn) T2 = 2B ↔ vol (post-column)
Recovery T2/T1
% of cpm in DNA post-G25 B/C
Dilution to 1.8 fmol/µL 2B/S
3.2. MutY Enzyme Purification
1. Grow 12 L (four 3 L media in 6 L flasks) of E. coliJM109 cells harboring over-
production plasmid pJTW10-12 (16) to A of 0.7 in LB broth containing
590
50 mg/mL of ampicillin at 37°C.
2. Induce MutY production by adding IPTG to 0.4 mMand the cultures are continu-
ously shaken overnight at 28°C (seeNote 2).
3. Harvest cells by centrifugation in a GS3 rotor at 11,000gfor 15 min. At the end,
remove as much media as possible and scrape the cell paste in a 50-mL plastic
tube that is stored at –80°C.
4. Before the enzyme purification, prepare all the required buffers (filtered through
45µ membrane and autoclaved) and pack the columns at 4°C. All column chro-
matography is conducted in a Waters 650 FPLC system at 4°C and centrifugation
is done at 65000g for 30 min (see Note 3).
5. Cells (40 g of cell paste) are resuspended in 120 mL of buffer T and disrupted
with a bead beater (Biospec Products, Bartlesville, OK) using 0.1-mm glass beads
(10 times 20 s blending and 10 s pulse).
6. Remove cell debris by centrifugation and carefully pour the supernatant to a
graduated cylinder. The supernatant is then treated with 5% streptomycin sulfate
in buffer T and stirred for 30 min (nucleic acids are precipitated out).
Repair of A/G and A/8-oxoG Mismatches 9
7. After centrifugation, the supernatant is collected as fraction I (235 mL).
8. Add ammonium sulfate (162 g) to fraction I. After stirring for 30 min, the protein
is precipitated overnight.
9. After centrifugation, resuspend the protein pellet in 12 mL of buffer T and the
sample is dialyzed against two changes of 1 L of buffer T for 3 h each.
10. The dialyzed protein sample is diluted four-fold with buffer A containing 50 mM
KCl as fraction II (100 mL). Fraction II is loaded at flow rate of 2 mL per min
onto a 30 mL phosphocellulose (Whatman P-11) column that has been equili-
brated with buffer A containing 0.05 M KCl.
11. After washing with 60 mL of equilibration buffer, proteins are eluted with a
300-mL linear gradient of KCl (0.05–0.5 M) in buffer A. Fractions containing the
A/G-specific nicking activity (seeSubheading 3.5.1.) are pooled (fraction III, 67 mL)
(those fractions should have brown color because MutY contains a Fe-S cluster).
12. Load fraction III onto a 20-mL hydroxylapatite column equilibrated with buffer
B. After washing with 40 mL of equilibration buffer, the flowthrough and early
elution fractions are pooled and dialyzed against buffer A containing 0.05 MKCl
and 10% (vol/vol) glycerol for 2 h (fraction IV, 63 mL).
13. Fraction IV is loaded onto a 5-mL heparin-agarose column equilibrated with
buffer A containing 0.05 MKCl and 10% glycerol. After washing with 10 mL of
equilibration buffer, the column is developed with a 50-mL linear gradient of
KCl (0.1–0.6 M) in buffer A with 10% glycerol. Fractions containing the MutY
nicking activity, which eluted at 0.3 M KCl, are pooled (fraction V, 17 mL), are
then divided into small aliquots and stored at –80°C. Protein concentration is
determined by the Bradford method (28).
3.3. Preparation of Crude Cell Extracts
If pure MutY is not required for the experimental purpose, small-scale crude
extracts can be obtained to check MutY repair activity (11) by the following
procedures.
1. Grow 1 L of E. coliJM109 cells harboring overproduction plasmid pJTW10-12
(16)to A of 0.7 in LB broth containing 50 mg/mL of ampicillin at 37°C. Add
590
IPTG to 0.4 mM to the culture and leave overnight at 28°C.
2. Harvest by centrifugation in a GSA rotor at 10,500gfor 15 min. Remove media
as much as possible.
3. The cells are resuspended in 2 mL of 0.05 M Tris-HCl, pH 7.6, 10% sucrose,
transferred to a centrifuge tube for Beckman 70.1 Ti rotor, quickly frozen in a dry
ice/ethanol bath, and stored at –80°C.
4. Cell suspensions are supplemented with 1.2 mMDTT, 0.15 MKCl, 0.23 mg/mL
of lysozyme, kept on ice for 1 h, and heated at 37°C for a time sufficient to yield
a final suspension temperature of 20°C.
5. Centrifuge at 100,000gin a Beckman 70.1 Ti rotor for 1 h at 4°C. Save the super-
natant in a 15-mL Corex glass tube with a very small stirring bar.
6. Add solid ammonium sulfate (0.42 g/mL) to the supernatant. Stir for 20 min.
10 Lu
7. Collect the precipitate by centrifugation at 19,000g in SS34 rotor for 25 min.
8. Resuspend the pellet in 0.3 mL of 25 mMHEPES, pH 7.6, 0.1 mmEDTA, 2 mM
DTT, 0.1 MKCl and dialyze the sample against the same buffer (2 ×250 mL) for
90 min. Check the conductivity of the sample by diluting 10 µL into 4 mL dis-
tilled water. The conductivity should be about 80 µS.
9. The protein sample is quickly frozen in small aliquots and stored at –80°C.
3.4. MutY Binding Assay
3.4.1. Binding Assay
The binding of MutY to DNA substrates is assayed by gel retardation (see
Notes 4 and5).
1. Prerun an 8% polyacrylamide nondenaturing gel in 1X TBE buffer at 150 V for
more than 30 min.
2. To each reaction, add the following in order to a microcentrifuge tube.
Sterile dH O 14µL
2
10X MutY reaction buffer 2 µL
10µg/mL poly (dI-dC) 2 µL
3' end-labeled DNA (1.8 fmol) 1 µL
MutY (72 nM) 1 µL
Total 20 µL
Assay both A/G- and C/G-containing DNA. A control incubation consisting
of DNA only (no MutY protein) should also be run. Dilute MutY enzyme
with storage/dilution buffer. Incubate all reactions at 37°C for 30 min.
3. Remove the reaction tubes from water bath and add 1.5 µL of 50% glycerol.
4. Load the entire reaction products onto the gel. Do not delay in loading the samples
onto the gel. Also load into an adjacent well with 1X DNA dye in TE .
0.1
5. Run the gel at 10 V/cm until bromophenol blue has migrated more than half-way
down the gel.
6. Remove the glass plates and transfer the gel onto 3MM filter paper.
7. Dry the gel in a gel dryer for 45 min and autoradiograph until the proper exposure
is achieved. It takes 16 h for 3000 cpm of DNA. The free DNA migrates below
the bromophenol blue and the MutY-bound complex migrates at a position near
xylene cyanol.
3.4.2. K Determination
d
The apparent dissociation constants (K ) of MutY and DNA can be
d
determined using a range of protein concentrations. Procedures are similar to
the one described above except samples were loaded on alternate lanes. Mark
the four corners of the filter containing the dried gel with fluorescence dye
(Scienceware high-energy autoradiography pen, cat. no. 13351) (to line up the
X-ray film with the gel). Following autoradiography, bands corresponding to
bound and unbound DNA are excised from the dried gel and quantified by liquid
Repair of A/G and A/8-oxoG Mismatches 11
scintillation counting. Alternatively, the bands can be quantified by a
phosphoimager. K values are obtained from a computer-fitted curve gener-
d
ated by the Enzfitter program (29).
3.5. MutY Nicking Assay
3.5.1. Nicking Assay
The nicking activity of MutY is the combined action of the glycosylase and
AP lyase activities. MutY nicks on the A-containing strand at the first
phosphodiester bond (seeNotes 5 and 6).
1.To each reaction, add the following in order to a microcentrifuge tube.
Sterile dH O 7 µL
2
10X MutY reaction buffer 1 µL
3' end-labeled DNA (1.8 fmol) 1 µL
MutY (72 nM) 1 µL
Total 10 µL
Assay both A/G- and C/G-containing DNA. A control incubation consisting
of DNA only (no MutY protein) should also be run. Incubate all reactions at
37°C for 30 min.
2. Stop the reactions in a dry ice-ethanol bath and dry the samples in a desiccator for
45 min (seeNote 7).
3. Resuspend each tube in 3 µL of sequencing dye. Heat samples at 90°C for
2 min.
4. Analyze the reaction products on a 14% polyacrylamide DNA sequencing gel
(IBI STS45i DNA sequencing unit), which has been prerun for more than 30 min
at 1800 V.
5. Run the gel at 2000 V until bromophenol blue has migrated approximately
half-way down the gel.
6. Remove the glass plates and transfer the gel onto a used X-ray film for support.
7. Cover the gel with plastic film and autoradiograph until the proper exposure is
achieved. The nicked product (9 nucleotides long) migrates just below the
bromophenol blue and the intact DNA (20 nucleotides long) migrates between
xylene cyanol and bromophenol blue (seeNote 7).
3.5.2. Kinetic Determination
Kinetic analyses are performed using a concentration range of 20-mer
DNAs with fixed protein concentrations. Reactions are performed as in the
nicking assay up to step 3, but the products are analyzed on a 14% poly-
acrylamide DNA sequencing gel (BRL cat. no. 21070-016 with 0.8-mm-thick
gel) that has been prerun at 600 V for 30 min. Run the gel at 600 V for 40 min
or until bromophenol blue has migrated approximately half-way down the
gel. Remove one glass plate, put the gel in a tray, and fix the gel with 5%