Cytotoxic and mutagenic properties of alkyl phosphotriester lesions in Escherichia coli cells

Article abstract of DOI:10.1093/nar/gky140

Exposure to many endogenous and exogenous agents can give rise to DNA alkylation, which constitutes a major type of DNA damage. Among the DNA alkylation products, alkyl phosphotriesters have relatively high frequencies of occurrence and are resistant to repair in mammalian tissues. However, little is known about how these lesions affect the efficiency and fidelity of DNA replication in cells or how the replicative bypass of these lesions is modulated by translesion synthesis DNA polymerases. In this study, we synthesized oligodeoxyribonucleotides containing four pairs (S_p and R_p) of alkyl phosphotriester lesions at a defined site, and examined how these lesions are recognized by DNA replication machinery in Escherichia coli cells. We found that the S_p diastereomer of the alkyl phosphotriester lesions could be efficiently bypassed, whereas the R_p counterparts moderately blocked DNA replication. Moreover, the S_p-methyl phosphotriester induced TT→GT and TT→GC mutations at the flanking TT dinucleotide site, and the induction of these mutations required Ada protein, which is known to remove efficiently the methyl group from the S_p-methyl phosphotriester. Together, our study provided a comprehensive understanding about the recognition of alkyl phosphotriester lesions by DNA replication machinery in cells, and revealed for the first time the Ada-dependent induction of mutations at the S_p-methyl phosphotriester site.

Full text of DOI:10.1093/nar/gky140

Nucleic Acids Research, 2018
1
doi: 10.1093/nar/gky140
Cytotoxic and mutagenic properties of alkyl
phosphotriester lesions in Escherichia coli cells
Jiabin Wu, Pengcheng Wang and Yinsheng Wang*
Environmental Toxicology Graduate Program and Department of Chemistry, University of California Riverside,
Riverside, CA 92521-0403, USA
Received December 08, 2017; Revised February 10, 2018; Editorial Decision February 13, 2018; Accepted March 01, 2018
ABSTRACT
and exogenous agents, which may result in mutagenesis and
carcinogenesis (2,3).
Exposure to many endogenous and exogenous
agents can give rise to DNA alkylation, which consti-
tutes a major type of DNA damage. Among the DNA
alkylation products, alkyl phosphotriesters have rel-
atively high frequencies of occurrence and are re-
sistant to repair in mammalian tissues. However, lit-
tle is known about how these lesions affect the ef-
ficiency and fidelity of DNA replication in cells or
how the replicative bypass of these lesions is mod-
ulated by translesion synthesis DNA polymerases.
In this study, we synthesized oligodeoxyribonu-
cleotides containing four pairs (Sp and Rp) of alkyl
phosphotriester lesions at a defined site, and exam-
ined how these lesions are recognized by DNA repli-
cation machinery in Escherichia coli cells. We found
that the Sp diastereomer of the alkyl phosphotri-
ester lesions could be efficiently bypassed, whereas
the Rp counterparts moderately blocked DNA repli-
cation. Moreover, the Sp-methyl phosphotriester in-
duced TT→GT and TT→GC mutations at the flanking
TT dinucleotide site, and the induction of these muta-
tions required Ada protein, which is known to remove
efficiently the methyl group from the Sp-methyl phos-
photriester. Together, our study provided a compre-
hensive understanding about the recognition of alkyl
phosphotriester lesions by DNA replication machin-
ery in cells, and revealed for the first time the Ada-
dependent induction of mutations at the Sp-methyl
phosphotriester site.
Alkylation is a common type of DNA damage (4), and
DNA alkylation constitutes the major mechanism of ac-
tion for many commonly prescribed cancer chemothera-
peutic agents (5). So far, the majority of studies about
DNA alkylation have focused on nucleobase modifica-
tions, which is attributed to the potential mutagenic ef-
fects of these lesions (4). Aside from forming nucleobase
adducts, alkylating agents can also attack one of the
non-carbon-bonded oxygen atoms of internucleotide phos-
phate group to yield backbone alkylation products, i.e.
the alkyl phosphotriester (alkyl-PTE) lesions (6). In this
vein, Me- and Et-PTEs (Figure 1) were found to account
for 12–17% and 55–57% of the total alkylation in duplex
DNA treated with N-methyl-N-nitrosourea and N-ethyl-
N-nitrosourea, respectively (7). In addition, a very recent
study showed that pyridylhydroxybutyl-PTE adducts con-
stitute 38–55% and 34–40% of all the measured pyridine-
containing DNA adducts in lung and liver tissues, re-
spectively, of rats treated through drinking water with a
tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-
pyridyl)-1-butanone (8). Depending on which of the two
non-carbon-bound oxygen atoms on backbone phosphate
is alkylated, alkyl-PTEs can form in the Sp or Rp configu-
ration (Figure 1) (6).
Alkyl-PTEs are known to persist in mammalian tissues. A
previous study showed that, in liver tissues of mice, Et-PTE
displayed longer half-life (t1/2, up to 32 days) than any nu-
cleobase ethylation products (9). Another study by Shooter
et al. (10) revealed that the t1/2 for Me-PTE was 7 days
in kidney and lung tissues of C57BL mice, which is much
shorter than the t1/2 values for Et-PTE in the same tissues
(10–15 weeks). The poorer repair of the alkyl-PTE lesions
in mammalian tissues suggest that they are more likely to
be encountered by DNA replication machinery than nucle-
obase alkylation products.
INTRODUCTION
The repair and biological consequences of alkyl-PTE le-
sions are under-investigated (6). In this respect, the forma-
tion of alkyl-PTEs results in neutralization of the negative
charge of the phosphate backbone, which interferes with
the binding of proteins to DNA, thereby resulting in an
DNA carries the genetic information, but it has limited
chemical stability, where spontaneous hydrolysis can result
in deamination of nucleobases and cleavage of N-glycosidic
bonds in DNA (1). The integrity of the human genome can
also be compromised upon exposure to many endogenous
*To whom correspondence should be addressed. Tel: +1 951 827 2700; Fax: +1 951 827 4713; Email: Yinsheng.Wang@ucr.edu
ꢀ
C
The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.
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2 Nucleic Acids Research, 2018
X= -CH3
-CH2CH3
Me-PTE
Et-PTE
-(CH2)2CH3 nPr-PTE
-(CH2)3CH3 nBu-PTE
Sp
Rp
Figure 1. Sp and Rp diastereomers of alkyl phosphotriester residues in DNA.
Chemical synthesis
inhibition of the normal functions of many DNA-binding
proteins. For instance, a single isopropyl-PTE could inhibit
SF2 family of DNA helicases, including RecQ and WRN
(11). It was also observed that the Sp, but not the Rp di-
astereomer of Me-PTE could be removed by Ada protein
in Escherichia coli (12,13). In addition, in vitro biochemical
studies revealed that a 50:50 mixture of Sp- and Rp-Et-PTE
could inhibit primer extension mediated by T4 DNA poly-
merase (14). However, it remains unclear how the alkyl-PTE
lesions affect DNA replication in cells.
In the present study, we employed a robust shuttle vector-
based method (15,16), together with mass spectrometry, to
assess how alkyl-PTE lesions with different sizes of the alkyl
group (from methyl to n-butyl) and stereochemical configu-
rations (Sp and Rp) affect the fidelity and efficiency of DNA
replication in E. coli cells, and how replication across these
lesions is influenced by translesion synthesis DNA poly-
merases and the Ada protein.
General synthetic procedures for alkyloxyphosphine. The
thymidine alkylphosphoramidites were synthesized follow-
ing previously published procedures (Scheme 1) (19,20).
Compound 1 (200 mg, 0.75 mmol) was dissolved in anhy-
drous diethyl ether (7 ml) in an ice bath under argon atmo-
sphere, to which solution were added the corresponding al-
cohol (140 ␮l) and triethylamine (4.0 eq., 306 mg). The solu-
tion was stirred at room temperature overnight, mixed with
8 ml petroleum ether, and filtered. The resulting precipitate
was washed with ether. The filtrate and the ether solution
were pooled and evaporated under reduced pressure to yield
2a–b, which were employed directly for the synthesis in the
next step.
For the preparation of compounds 2c–d, compound 1
(300 mg, 1.13 mmol) in 2 ml tetrahydrofuran, the corre-
sponding alcohol (140 ␮l) and triethylamine (230 ␮l, 1.63
mmol) were added to a round bottom flask, which was un-
der an argon atmosphere and placed in an ice water bath.
The solution was stirred at room temperature for 1 h and
filtered. The filtrate was concentrated in vacuo. Pentane (10
ml) was added to the resulting solid, and the mixture was fil-
tered. The solvent in the filtrate was removed under vacuum
to yield compounds 2c–d.
MATERIALS AND METHODS
Materials
All chemicals, unless specified, were from Sigma-Aldrich (St
Louis, MO, USA) or Thermo Fisher Scientific (Pittsburg,
PA, USA). Common reagents for solid-phase DNA syn-
thesis were from Glen Research (Sterling, VA, USA) and
all the unmodified oligodeoxyribonucleotides (ODNs) were
from Integrated DNA Technologies (Coralville, IA, USA).
1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was from Oak-
wood Products Inc. (West Columbia, SC, USA), and [␥-
32P]ATP was obtained from Perkin Elmer (Piscataway, NJ,
USA). All enzymes were purchased from New England Bi-
olabs (Ipswich, MA, USA).
M13mp7(L2) plasmid, wild-type AB1157 E. coli strains,
C215 E. coli strains and Ada-deficient E. coli strains were
kindly provided by Prof. John M. Essigmann (17,18).
Polymerase-deficient AB1157 strains [Δpol B1::spec (Pol II
deficient), ΔdinB (Pol IV-deficient), ΔumuC::kan (Pol V de-
ficient) and Δpol B1::spec ΔdinB ΔumuC::kan (Pol II, Pol
IV, Pol V-triple knockout)] were generously provided by
Prof. Graham C. Walker (17).
General procedures for dimethoxytrityl (DMTr) protec-
tion of the 5ꢀ-hydroxyl group. Compound 4 was pre-
pared following the previously published procedures (21).
Briefly, compound 3 (100 mg, 0.41 mmol) was dis-
solved in anhydrous pyridine (10 ml), and to the mix-
ture were added dimethoxytrityl chloride (1.5 eq.) and 4-
dimethylaminopyridine (0.5 mol%). After stirring at room
temperature for 10 h, the solution was concentrated in vacuo
and the residues were purified with silica gel column chro-
matography by using ethyl acetate as the mobile phase to
yield compound 4.
General procedures for phosphoramidite building block syn-
thesis. To a round bottom flask, which contained a
solution of compound 4 (70 mg, 0.13 mmol) in dry
dichloromethane (1.5 ml), were added compound 2a–b (1
eq.) and diisopropylamine hydrotetrazolide (1 eq.), and the
mixture was stirred at room temperature under argon. Af-
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Nucleic Acids Research, 2018 3
a
1
2a-d
c
b
5a-d
a:R=Me b:R=Et
c:R=nPr d:R=nBu
3
4
Scheme 1. Syntheses of alkylphosphoramidite building blocks. Reagents and conditions: (A) for methyl and ethyl: ROH, triethylamine, diethyl ether, ice
bath, 14 h; for n-propyl and n-butyl: ROH, triethylamine, THF, 0◦C,1 h; (B) DMTr-Cl, DMAP, pyridine, room temperature, 10 h; (C) for methyl and ethyl:
diisopropylamine hydrotetrazolide, CH2Cl2, room temperature, 1 h; for n-propyl and n-butyl: 0.45 M tetrazole in acetonitrile, CH2Cl2, room temperature,
5 h.
ter 1 h, TLC analysis indicated complete conversion of the
starting material. The solvent was removed in vacuo and iso-
lated by silica gel column chromatography with a mixture
of hexane, ethyl acetate, and triethylamine (49:49:2, v/v) to
yield the desired products 5a–b.
For the syntheses of compounds 5c–d, compound 2c–d
(1 eq.) and tetrazole in acetonitrile (0.3 ml, 0.135 mmol)
were added to a round bottom flask containing a solution
of compound 4 (70 mg, 0.13 mmol) in dry dichloromethane
(1.5 ml), and the mixture was stirred at room temperature
under argon atmosphere. The reaction was continued for 5
h, and the desired products 5c–d were purified following the
aforementioned procedures for compounds 5a–b.
the synthesized 12-mer ODNs, the mobile phases consisted
of 50 mM triethylammonium acetate (pH 7.0, Solution A)
and a mixture of solution A and acetonitrile (70/30, v/v,
Solution B). A gradient of 5–30% B in 5 min and 30–60% B
in 65 min was employed, and the flow rate was 0.7 ml/min.
The HPLC traces for the purification of the 12-mer lesion-
containing ODNs are shown in Supplementary Figure S7
and the electrospray ionization-mass spectra (ESI-MS) and
tandem MS (MS/MS) of these ODNs are displayed in Sup-
plementary Figures S8–S11.
Determination of stereochemical configurations of Me-PTE-
containing ODNs. The Me-PTE-containing dimer 5ꢁ-
T(Me)T-3ꢁ was first purified by HPLC using a Synergi
Fusion-RP column (10 × 150 mm, 4 ␮m in particle size and
The reaction yields and spectroscopic characterizations
of the above-synthesized products are provided in the on-
line Supplementary Materials. The nuclear magnetic reso-
nance (NMR) spectra for these compounds are shown in
Supplementary Figures S1–S4.
˚
80 A in pore size; Phenomenex Inc., Torrance, CA, USA).
The mobile phases were water (Solution A) and methanol
(Solution B). A gradient of 5–25% B in 5 min and 25–50%
B in 75 min was used, and the flow rate was 1.1 ml/min.
The HPLC traces are shown in Supplementary Figure S5
and the products were identified by MS and 31P-NMR.
For NMR analyses, the two purified diastereomers were
dissolved in 300 ␮l CD3OD, and triphenylphosphine was
added as the reference for chemical shift calibration. The
31P-NMR spectra were recorded on a Bruker Avance Neo
400 spectrometer at 80 MHz for 31P (Supplementary Figure
S5).
To determine the stereochemical configurations of the
Me-PTEs in 12 mer ODNs, we digested the lesion-
containing ODNs with a cocktail of four enzymes and an-
alyzed the digestion mixture by HPLC to examine whether
the two diastereomers in 12 mer ODNs exhibited the same
elution order as those in the dimers. The digestion was
conducted following previously published procedures (22).
Briefly, nuclease P1 (0.1 U/␮g DNA), phosphodiesterase 2
(0.000125 U/␮g DNA), and 1 nmol of 12 mer ODNs with
Sp- or Rp-Me-PTE were incubated in a 100-␮l solution con-
taining 300 mM sodium acetate (pH 5.6) and 10 mM ZnCl2.
After incubation at 37◦C for 24 h, alkaline phosphatase (0.1
U/␮g DNA), phosphodiesterase 1 (0.00025 U/␮g DNA),
and 50 ␮l of 0.5 M Tris–HCl (pH 8.9) were added to the di-
ODN synthesis. The 12-mer lesion-containing ODNs, 5ꢁ-
ATGGCT(X)TGCTAT-3ꢁ (‘X’ designates the location of
the alkyl-PTE), and Me-PTE-containing dimer 5ꢁ-T(Me)T-
3ꢁ were synthesized on a Beckman Oligo 1000M DNA
synthesizer (Fullerton, CA) at 1 ␮mol scale. The corre-
sponding phosphoramidite building block was dissolved in
anhydrous acetonitrile at a concentration of 0.067 mM.
Commercially available ultramild phosphoramidite build-
ing blocks were used for the incorporation of unmodified
nucleotides (Glen Research Inc., Sterling, VA, USA) fol-
lowing the standard ODN assembly protocol. The ODNs
were cleaved from the controlled pore glass support and de-
protected with concentrated ammonium hydroxide at room
temperature for 55 min. The solvent was removed by us-
ing a Speed-vac, and the resulting solid residues were re-
dissolved in water and purified by high-performance liquid
chromatography (HPLC).
HPLC. The HPLC separation was performed on an Ag-
ilent 1100 system with a Kinetex XB-C18 column (4.6 ×
˚
150 mm, 5 ␮m in particle size and 100 A in pore size; Phe-
nomenex Inc., Torrance, CA, USA). For the purification of
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4 Nucleic Acids Research, 2018
gestion mixture, and the mixture was incubated at 37◦C for
2 h. The enzymes were subsequently removed by chloroform
extraction and the solution was dried by lyophilization. The
resulting solid residue was reconstituted in doubly distilled
water (100 ␮l) and analyzed by HPLC. A Thermo Hyper-
sil Gold C18 column (4.6 × 250 mm, 3 ␮l in particle size,
purified by Qiaprep Spin M13 Kit (Qiagen) to obtain the
ssM13 DNA template for PCR amplification.
Quantification of bypass efficiencies and mutation frequen-
cies. A modified version of the competitive replication and
adduct bypass (CRAB) assay (15,16,23,24) was employed
to assess the bypass efficiencies and mutation frequencies of
the alkyl-PTE lesions in E. coli cells. The sequence region of
interest in the M13 template was amplified by PCR with the
use of Phusion high-fidelity DNA polymerase. The primers
were 5ꢁ-YCAGCTATGACCATGATTCAGTGAGTGGA-
3ꢁ and 5ꢁ-YTCGGTGCGGGCCTCTTCGCTATTAC-3ꢁ
(‘Y’ denotes the H2N(CH2)6- group conjugated to the 5ꢁ
phosphate group of the ODNs). The amplification started
from annealing at 98◦C for 30 s, followed by 35 cycles of
amplification, each of which consisted of 10 s at 98◦C, 30 s
at 65◦C and 15 s at 72◦C, and then with a final 5-min ex-
tension at 72◦C and ending at 4◦C. The PCR products were
purified by using Cycle Pure Kit (Omega). For determining
the bypass efficiencies and mutation frequencies, 100 ng of
PCR products were digested with 10 U BbsI-HF restriction
endonucleases together with 10 U recombinant shrimp al-
kaline phosphatase (rSAP) in 10 ␮l CutSmart buffer (New
England Biolabs) at 37◦C for 25 min, followed by heating
at 80◦C for 10 min to deactivate the phosphatase. To the
mixture were subsequently added 5 mM DTT, 1.66 pmol
[␥-32P] ATP, 10 U T4 polynucleotide kinase (T4 PNK), T4
PNK buffer and water to give a total volume of 15 ␮l. After
a 30-min incubation at 37◦C, the T4 PNK was deactivated
by heating the solution at 80◦C for 10 min. To the resulting
solution was added 10 U MluCI restriction endonuclease,
and the mixture was then incubated at 37◦C for 25 min. The
reaction was subsequently quenched by adding 15 ␮l for-
mamide gel loading buffer which contained xylene cyanol
FF and bromophenol blue dyes. The mixture was resolved
by using 30% native polyacrylamide gel (acrylamide:bis-
acrylamide = 19:1), and the gel band intensities were de-
termined by phosphorimager analysis on a Typhoon 9410
variable-mode imager.
˚
300 A in pore size, Thermo Scientific Inc., Waltham, MA)
was employed for the separation of the digestion mixture. A
solution of 10 mM ammonium formate and methanol were
used as mobile phases A and B, respectively. The gradient
profile was 0% B in 42 min, 0–2% B in 1 min, 2% B for 17
min, 2–5% B in 1 min, 5–25% B in 5 min, 25–35% B in 75
min. The HPLC traces are shown in Supplementary Figure
S6.
Construction of single-stranded lesion-containing and lesion-
free competitor M13 genomes. The 12-mer alkyl-PTE-
containing ODNs were 5ꢁ-phosphorylated and ligated with
a 10-mer ODN (5ꢁ-ACTGGAAGAC-3ꢁ) in the presence of
ligation buffer with T4 DNA ligase and ATP at 16◦C for 8
h. The resulting 22-mer ODNs were purified by denaturing
polyacrylamide gel.
The lesion-containing and lesion-free M13mp7 (L2)
genomes were prepared following previously reported
procedures (Supplementary Figure S12) (21,23). First,
20 pmol of M13 plasmid was digested with 40 U EcoRI-
HF at room temperature for 8 h to linearize the vector.
The linearized vector was mixed with 2 scaffolds, 5ꢁ-
CTTCCACTCACTGAATCATGGTCATAGCTTTC-3ꢁ
and
5ꢁ-AAAACGACGGCCAGTGAATTATAGC-
3ꢁ (25 pmol), each spanning one end of the lin-
ear vector. To the mixture was subsequently added
30 pmol of 5ꢁ-phosphorylated 22-mer lesion-
containing ODN or 25-mer competitor ODN (5ꢁ-
GCAGGATGTCATGGCGATAAGCTAT-3ꢁ).
The
resulting mixture was treated with T4 DNA ligase at 16◦C
for 8 h, followed by incubation with T4 DNA polymerase
(22.5 U) at 16◦C for 2.5 h to degrade the unligated vector
and excess scaffolds. The lesion-containing and lesion-free
plasmids were purified using Cycle Pure Kit (Omega) and
subsequently normalized against the competitor plasmid,
following published procedures (21,23).
The
digestion
two-step
gave
restriction
to
endonuclease
rise
a
10-mer
duplex
where
d(p*GGCMNGCTAT)/d(AATTATAGCY),
‘M’ and ‘N’ designate the nucleobases at the dinucleotide
sites initially flanking the alkyl-PTE lesions, ‘Y’ is the
complementary base of ‘N’ in the opposite strand, and
p* denotes the [5ꢁ-32P]-labeled phosphate. The bypass effi-
ciency was calculated by using following equation: Bypass
efficiency (%) = (lesion signal/competitor signal)/(control
signal/competitor signal) × 100%.
Transfection of lesion-free, lesion-containing and competi-
tor plasmids into E. coli cells. The lesion-free or lesion-
containing M13 genome was mixed with the competitor
genome at a 1:1 molar ratio. The mixtures were transfected
into SOS-induced, electrocompetent AB1157 E. coli strain
as well as the isogenic cells deficient in Pol II, Pol IV, Pol
V, or all three in combination. The lesion-free or lesion-
containing M13 genome was also mixed with the competi-
tor genome at a 4:1 molar ratio and transfected into electro-
competent Ada-proficient (C215) and Ada-deficient (C217)
E. coli cells, as described elsewhere (21). The SOS induction
was conducted by irradiating E. coli cells with 254 nm ul-
traviolet light at a dose of 45 J/m2 (17). The AB1157, C215
and C217 E. coli cells were then grown in lysogeny broth
(LB) medium at 37◦C for 5.5 h. The phage was recovered
from the supernatant by centrifugation at 13 200 rpm for
5 min and further transfected into SCS110 cells to increase
the progeny/lesion-genome ratio. The amplified phage was
Identification of mutagenic products by LC–MS and
MS/MS. The PCR products were digested with 50 U
BbsI-HF restriction endonuclease and 20 U rSAP in 250 ␮l
CutSmart buffer at 37◦C for 2 h, and the phosphatase was
subsequently deactivated by heating at 80◦C for 20 min.
To the mixture was added 50 U MluCI and the solution
was incubated at 37◦C for 1 h. The solution was then
extracted once with phenol/chloroform/isoamyl alcohol
(25:24:1, v/v). The aqueous layer was dried, desalted by
HPLC and redissolved in 20 ␮l water. A 10-␮l aliquot
was injected for LC–MS/MS analysis on an LTQ linear
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Nucleic Acids Research, 2018 5
ion trap mass spectrometer (Thermo Electron, San Jose,
CA, USA). An Agilent Zorbax SB-C18 column (0.5 × 250
mm, 5 ␮m in particle size) was employed, and the gradient
for LC–MS/MS analysis was 5 min of 5–20% methanol
followed by 35 min of 20–50% methanol in 400 mM HFIP
(pH was adjusted to 7.0 with triethylamine). The mass
spectrometer was set up for monitoring the fragmentation
of the [M–3H]3− ions of 10-mer d(GGCMNGCTAT) and
d(AATTATAGCY), with ‘M’, ‘N’, and ‘Y’ being ‘A’, ‘T’,
‘C’ or ‘G’. The fragment ions detected in MS/MS were
manually assigned.
A
BbsI
MluCI
5’-ATGGCMNGCTATAATTCA-3’
3’-TACCGXYCGATATTAAGT-5’
MluCI
BbsI
SAP
SAP
[γ-32P]ATP + PNK
BbsI
[γ-32P]ATP + PNK
MluCI
5’-AT pGGCMNGCTAT AATTCAC-3’
3’-TACCGXpYCGATATTAA GTG-5’
5’-AT GGCMNGCTAT pAATTCA-3’
3’-TACCGX YCGATATTAAp GT-5’
B
C
RESULTS
The objectives of this study were to assess how alkyl-PTE le-
sions with different stereochemical configurations and vari-
ous sizes of alkyl group compromise DNA replication, and
to define the respective roles of the three SOS-induced poly-
merases and Ada protein in bypassing and repairing these
lesions in E. coli cells.
We first synthesized 12-mer ODNs harboring a site-
specifically inserted alkyl-PTE lesion (Scheme 1) and char-
acterized these ODNs by ESI-MS and MS/MS analy-
ses (Supplementary Figures S8–S11). The ESI-MS and
MS/MS results support the site of the alkyl-PTE lesion
and the identity of the alkyl group being conjugated with
backbone phosphate. In addition, we established the con-
figurations of the two diastereomers of Me-PTEs based
on 31P-NMR and HPLC analyses (Supplementary Fig-
ures S5 and S6). Previously, Weinfeld et al. (13,25) showed
that, under the same solvent conditions, the 31P signal for
the Sp diastereomer of Me-PTE appeared at a lower field
than that for the Rp counterpart. We first determined the
stereochemistry of the Me-PTE-containing T(Me)T based
on 31P-NMR analyses, where the earlier and later eluting
T(Me)T-1 and T(Me)T-2 were found to contain the Sp and
Rp diastereomers of the Me-PTE, respectively. A chemical
shift difference of 0.16 ppm for the two diastereomers was
nearly identical to what was previously reported (26). We
next digested the HPLC-purified Me-PTE-containing 12-
mer ODNs with a cocktail of four enzymes, which release
Me-PTE as T(Me)T. By comparing the HPLC elution prop-
erties of the T(Me)T liberated from the 12-mer ODNs with
those of the standards, we demonstrated that the earlier-
and later-eluting 12-mer ODNs contained the Sp- and Rp-
Me-PTEs, respectively. Based on the observations that the
Sp- and Rp-Me-PTE lesions in a dimer and a 12-mer ODN
exhibit the same elution orders on reversed-phase HPLC
columns, we assign the stereochemistry of the purified Et-,
nPr- and nBu-PTEs based on their relative orders of elution
from the HPLC column, i.e., the Sp diastereomer elutes ear-
lier than the Rp diastereomer.
Figure 2. Restriction enzyme digestion and radiolabeling, followed by na-
tive PAGE (30%) analysis for quantifying the bypass efficiencies and mu-
tation frequencies of alkyl phosphotriester lesions in wild-type AB1157
E. coli cells. (A) Selective labeling of the original lesion-containing strand
and its complementary strand via sequential restriction digestion. ‘SAP’
and ‘PNK’ represent shrimp alkaline phosphatase and T4 polynucleotide
kinase, respectively. ‘MN’ in the sequence denotes the site where the TT
dinucleotide flanking the alkyl phosphotriester lesions were initially situ-
ated. (B) Gel image showing the 13 mer (competitor genome) and 10 mer
(control or lesion-containing genome) digestion products formed from the
original strand, where 10 mer TT, 10 mer GC and 10 mer GT designate
[5ꢁ-32P]-labeled standard ODNs 5ꢁ-GGCMNGCTAT-3ꢁ, with MN being
TT, GC and GT, respectively. (C) Gel image showing 13 mer (competi-
tor genome) and 10 mer (control or lesion-containing genome) digestion
products formed from the opposite strand, where 10 mer A and 10 mer G
designate the [5ꢁ-32P]-labeled standard ODNs 5ꢁ-AATTATAGCY-3ꢁ, with
Y being A and G, respectively.
2, we were able to radiolabel the 5ꢁ terminus of either the
original lesion-situated strand, i.e. d(p*GGCMNGCTAT),
or its complementary strand, i.e. d(p*AATTATAGCY), by
switching the orders of BbsI and MluCI digestions. Re-
sults from native PAGE analyses of the resulting radi-
olabeled fragments revealed that the Sp diastereomer of
Me-PTE could give rise to TT→GT and TT→GC mu-
tations at the flanking TT dinucleotide sites, whereas no
mutagenic products were detectable for any other alkyl-
PTE lesions. In this regard, when BbsI was added first, we
could resolve [5ꢁ-32P]-labeled d(p*GGCTTGCTAT) (non-
mutagenic product, 10 mer TT) from the products with
TT→GT or TT→GC mutation, i.e. d(p*GGCGTGCTAT)
We next ligated these lesion-containing ODNs into
single-stranded M13mp7 plasmid and measured the bypass
efficiencies and mutation frequencies of the alkyl-PTE le-
sions using a modified version of the competitive replica-
tion and adduct bypass assay (Figure 2 and Supplemen-
tary Figures S13-S20) (15,16). After PCR amplification and
restriction digestion, the released ODNs were analyzed by
LC-MS/MS and native PAGE to identify the replication
products, as described elsewhere (24). As shown in Figure
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6 Nucleic Acids Research, 2018
(10 mer GT) and d(p*GGCGCGCTAT) (10 mer GC).
Nonetheless, 10 mer GT and 10 mer GC could not be dis-
tinguished by native PAGE analysis (Figure 2B). To resolve
these two mutagenic products, we first added MluCI to radi-
olabel the opposite strand, which enabled us to separate the
product with TT→GT mutation, i.e. d(p*AATTATAGCA)
(10 mer A), from that with TT→GC mutation, namely
d(p*AATTATAGCG) (10 mer G), as shown in Figure 2C
and Supplementary Figures S13–S15.
A
We further confirmed the identities of the aforemen-
tioned restriction digestion products by LC–MS/MS
analysis (Supplementary Figure S16). We moni-
tored the [M–3H]3− ions of d(GGCMNGCTAT) and
d(AATTATAGCY), where ‘M’ and ‘N’ designate the
nucleotides inserted at the initial TT dinucleotide flanking
the alkyl-PTE site, and ‘Y’ represents the opposing nu-
cleotide of ‘N’ in the complementary strand, respectively
(Supplementary Figures S17–S20). The LC–MS/MS data
showed that only Sp-Me-PTE could induce TT→GT and
TT→GC mutations, which are in keeping with the results
obtained from the aforementioned PAGE analyses.
B
Having identified the replication products, we determined
the bypass efficiencies of the alkyl-PTE lesions by measur-
ing the ratio of combined intensities of all 10-mer bands
arising from the lesion-containing genome over that of the
13-mer band emanating from the lesion-free competitor
genome (see Materials and Methods). The results showed
that the Sp diastereomers of all four alkyl-PTEs do not ap-
preciably impede DNA replication in E. coli cells, whereas
the Rp diastereomers elicit moderate blockage effects on
DNA replication (Figure 3A).
We also examined the roles of the three SOS-induced
polymerases (i.e. Pol II, Pol IV and Pol V) in bypassing
the alkyl-PTE lesions by conducting the replication exper-
iments using the isogenic E. coli strains that are deficient
in these polymerases. The results showed that the bypass of
the alkyl-PTE lesions does not require any of the three TLS
polymerases (Figure 3A). Moreover, our results showed
that the bypass efficiencies of these lesions were not mod-
ulated by SOS induction, either in wild-type AB1157 cells
or the isogenic cells with all three SOS-induced DNA poly-
merases being knocked out (Figure 3A).
Figure 3. Bypass efficiencies of alkyl phosphotriester lesions (A) and mu-
tation frequencies of the Sp methyl phosphotriester lesion (B) in AB1157
E. coli strains that are proficient or deficient in SOS-induced DNA poly-
merases, Pol II, Pol IV, Pol V, alone or all three in combination. The data
represent the means and standard deviations of results from three inde-
pendent replication experiments. *, 0.01 < P < 0.05. The P values were
calculated by using an unpaired two-tailed t-test and referred to compar-
isons with the data obtained for control cells.
or deficient in Ada protein (Figure 4). Our results showed
that the deletion of Ada led to a significant decline in by-
pass efficiency for Sp-Me-PTE, which is accompanied with
the abrogation of both TT→GT and TT→GC mutations
for the lesion, suggesting the involvement of Ada protein in
repairing the Sp-Me-PTE and inducing mutations for this
lesion. Similar as what we found for AB1157 cells, replica-
tion across Sp-Me-PTE and the two diastereomers of Et-
PTE lesions are error-free in Ada-deficient C217 cells, and
deletion of Ada protein did not appreciably alter the bypass
efficiencies of these three lesions (Figure 4).
We further determined the mutation frequencies of the
Me-PTE in wild-type and polymerase-deficient AB1157 E.
coli strains (Figure 3B). It turned out that the Sp-Me-PTE is
moderately mutagenic, resulting in TT→GT and TT→GC
mutations. Additionally, deletion of any of the three SOS-
induced polymerases alone did not confer any significant
alterations in mutation frequencies, though simultaneous
removal of the three polymerases in SOS-induced or unin-
duced cells led to a slight, albeit statistically significant de-
crease in TT→GT mutation (Figure 3B).
DISCUSSION
We systematically investigated the cytotoxic and mutagenic
properties of alkyl-PTE lesions in E. coli cells and our re-
sults led to several important conclusions. First, we found
that the Sp and Rp diastereomers of the alkyl-PTE lesions
exerted distinct effects on DNA replication in E. coli cells.
The lesions in the Sp configuration displayed higher replica-
tion bypass efficiencies than the corresponding unmodified
DNA, whereas those in the Rp configuration suppress DNA
replication in AB1157 cells. The replication bypass efficien-
cies for the Sp-alkyl-PTEs, except for the methyl adduct
The unique mutagenic property of Sp-Me-PTE parallels
the previous finding that Ada protein removes the methyl
group exclusively from the Sp-Me-PTE at an efficiency that
is much higher than the corresponding removal of larger
alkyl groups from alkyl-PTE lesions (13). Thus, we next
asked whether Ada protein assumes any role in modulating
the cytotoxic and mutagenic properties of the two diastere-
omers of Me- and Et-PTEs by conducting replication exper-
iments with the use of isogenic E. coli cells that are proficient
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Nucleic Acids Research, 2018 7
certained owing to the lack of stereochemically pure DNA
substrates. It is worth noting that, due to the differential ac-
cessibilities of the two non-carbon-bound oxygen atoms on
backbone phosphate to DNA alkylating agents, the Sp di-
astereomer is produced at a higher yield than the Rp coun-
terpart (27). Hence, the assessment about overall biological
consequences of alkyl-PTEs should also take into account
the relative frequencies of occurrence of these lesions.
Second, our results demonstrated that the bypass efficien-
cies of the alkyl-PTE lesions were not affected by deletion of
any of the three SOS-induced TLS polymerases, alone or all
three in combination. Additionally, the mutation frequen-
cies induced by Sp-Me-PTE were not influenced by the dele-
tion of any of the three TLS polymerases alone, though the
absence of all three polymerases resulted in a slight, yet sta-
tistically significant decrease in the frequency of TT→GT
mutation.
A
C
***
WT
Ada
160
140
120
100
80
60
40
20
MluCI
BbsI
Δ
5’-ATGGCMNGCTATAATTCA-3’
3’-TACCGXYCGATATTAAGT-5’
BbsI
SAP
[γ-32P]ATP + PNK
MluCI
5’-AT pGGCMNGCTAT AATTCAC-3’
3’-TACCGXpYCGATATTAA GTG-5’
Me, Sp Et, Sp Me, Rp Et, Rp
B
ΔAda
WT
Third, Ada protein, which could repair the Sp-Me-PTE,
is required for the TT→GT and TT→GC mutations in-
duced by this lesion. The removal of Me-PTE by Ada pro-
tein takes time. In this vein, we also measured the bypass
efficiencies and mutation frequencies of the two diastere-
omers of Me-PTEs at 1, 3 and 6 h following transfection,
and we observed that at 1 h, the bypass efficiency of Sp-
Me-PTE at 1 h is lower than that at 3 and 6 h (Supplemen-
tary Figure S21). Ada protein plays a crucial role in bacte-
rial tolerance toward exposure to alkylating agents in a pro-
cess known as adaptive response, where Ada acts both as a
DNA repair protein and a transcription activator (4,28,29).
In that process, the 39 kDa Ada protein undergoes proteol-
ysis at the sole Lys-Gln linkage to yield a 19 kDa C-terminal
fragment (C-Ada) and a 20 kDa N-terminal fragment (N-
Ada); while Cys321 in C-Ada is a methyltransferase for
the repair of O6-methylguanine and O4-methylthymine (30),
Cys38 in N-Ada could remove the methyl group specifically
from the Sp-Me-PTE (31). Moreover, He et al. (31) illus-
trated, from detailed solution (NMR) and crystal (X-ray)
structure studies, that this latter methyl group transfer stim-
ulated the binding of N-Ada with DNA through an electro-
static switch mechanism. The heightened DNA affinity led
to promoter binding and transcriptional activation of sev-
eral genes that are important in repairing alkylated DNA
lesions including Ada, Aid, AlkA and AlkB (6). Thus, Ada
protein is instrumental in protecting the genomic integrity
of bacterial cells by promoting the repair of alkylated DNA
lesions. Here we made an unexpected finding that, while
the Ada-stimulated repair of the Sp-Me-PTE results in en-
hanced replicative bypass, it comes at an expense by induc-
ing TT→GT and TT→GC mutations at the flanking nucle-
oside sites. Further studies are warranted for understand-
ing how the selective interaction between Ada and Sp-Me-
PTE would interfere with Watson-Crick base pairing during
DNA replication and trigger specific mutations at the lesion
site.
Figure 4. Ada protein promotes the replicative bypass of the Sp-Me-PTE,
but not the Rp-Me-PTE or the Sp-/Rp-Et-PTE lesion, and Ada is required
for the mutations induced by Sp-Me-PTE. (A) Selective labelling of origi-
nal lesion-containing strand and its complementary strand via sequential
restriction digestion. ‘SAP’ and ‘PNK’ represent shrimp alkaline phos-
phatase and T4 polynucleotide kinase, respectively. (B) Gel image show-
ing 13 mer (from the competitor genome) and 10 mer (from the control or
lesion-containing genome) digestion products formed from the strand ini-
tially containing the lesion, where 10 mer TT, 10 mer GC and 10 mer GT
designate [5ꢁ-32P]-labeled standard ODNs 5ꢁ-GGCMNGCTAT-3ꢁ, with
MN being TT, GC and GT, respectively. (C) The bypass efficiencies of the
two diastereomers of Me- and Et-PTE lesions in E. coli cells that are profi-
cient in DNA repair or deficient in both Ada and Ogt (ΔAda, ΔOgt). The
data represent the means and standard deviations of results from three in-
dependent replication experiments. ***P <0.001. The P values were cal-
culated by using an unpaired two-tailed t-test and referred to comparisons
with the data obtained for control cells.
which is removed by Ada protein, decrease with the size of
alkyl group, which might be attributed to the elevated steric
hindrance imposed by larger alkyl groups. In duplex DNA,
the alkyl group in the Sp diastereomer is known to point
perpendicularly out from the DNA double helix, whereas
that of the Rp diastereomer projects into the major groove
(6). Hence, the alkyl groups in the Sp configuration may as-
sume some favorable interaction with DNA polymerases to
promote translesion synthesis, where steric hindrance im-
posed by larger alkyl groups may diminish this enhance-
ment effect. However, the Rp-alkyl-PTE lesions display an
opposite trend, with nPr- and nBu-PTEs exhibiting higher
bypass efficiencies than Me- and Et-PTEs. This result sup-
ports that the difference in stereochemical configurations of
the alkyl group conjugated with the phosphate backbone
can modulate the replicative bypass of these lesions. By em-
ploying a DNA template containing an equimolar mixture
of Sp and Rp diastereomers of Et-PTE at a defined site, Tsu-
jikawa et al. (14) found that the lesions could inhibit par-
tially primer extension catalyzed by T4 DNA polymerase,
but could be bypassed by E. coli DNA polymerase I. The
results from that study also suggested that one of the two
diastereomers was a major block to DNA replication (14),
though the identity of the diastereomer could not be as-
A recent genome-wide mutation analysis of 32 E. coli
strains treated with N-methyl-N’-nitrosoguanidine showed
that, among the 4099 identified mutations, 96.6%, 2.17%
and 0.46% were G→A, T→C and T→G substitutions,
respectively (32). While G→A and T→C mutations are
thought to arise from methylation at the O6 of guanine and
the O4 of thymine (21,33), respectively, our results suggest
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8 Nucleic Acids Research, 2018
11. Suhasini,A.N., Sommers,J.A., Yu,S., Wu,Y., Xu,T., Kelman,Z.,
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13. Weinfeld,M., Drake,A.F., Saunders,J.K. and Paterson,M.C. (1985)
Stereospecific removal of methyl phosphotriesters from DNA by an
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17. Neeley,W.L., Delaney,S., Alekseyev,Y.O., Jarosz,D.F., Delaney,J.C.,
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that T→G mutation may be attributed, in part, to the Me-
PTE formed on the 3ꢁ side of thymidine. In this context, it is
worth noting that the formation frequency of the Me-PTE
lesions was found to be affected by flanking sequences (34),
and flanking sequences may also modulate the mutagenic
properties of alkyl-PTE lesions. Thus, it will be important
to investigate how the cytotoxic and mutagenic properties of
the alkyl-PTE lesions are influenced by flanking sequences
in the future.
In summary, our study demonstrated that the two di-
astereomers of alkyl-PTEs exert distinct effects on DNA
replication; the Sp-alkyl-PTEs did not appreciably impede
DNA replication, whereas the Rp counterparts could mod-
erately inhibit this process. We also revealed that the mu-
tagenic properties of the Sp-Me-PTE could be substantially
modulated by Ada protein, but not by any of the three SOS-
induced polymerases. Hence, our systematic shuttle-vector
study on alkyl-PTE lesions offered new insights about how
this group of DNA alkylation products are recognized by
the E. coli DNA replication machinery. It will be important
to examine how the alkyl-PTE lesions compromise DNA
replication in mammalian cells in the future.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
FUNDING
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cells by nanoflow liquid chromatography-nanoelectrospray ionization
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24. Zhai,Q., Wang,P., Cai,Q. and Wang,Y. (2014) Syntheses and
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National Institute of Environmental Health Sciences [R01
ES025121]. Funding for open access charge: National Insti-
tute of Environmental Health Sciences [R01 ES025121].
Conflict of interest statement. None declared.
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