Genetic control of predominantly error-free replication through an acrolein-derived minor-groove DNA adduct

Article abstract of DOI:10.1074/jbc.RA117.000962

Acrolein, anα,β-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from metabolic oxidation of polyamines, and it is a ubiquitous environmental pollutant. The reaction of acrolein with the N2 of guanine in DNA leads to the formation of γ-hydroxy-1-N²-propano-2′ deoxyguanosine (γ-HOPdG), which can exist inDNAin a ring-closed or a ring-opened form. Here, we identified the translesion synthesis (TLS) DNA polymerases (Pols) that conduct replication through the permanently ring-opened reduced form of γ-HOPdG ((r) γ-HOPdG) and show that replication through this adduct is mediated via Rev1/Polη-, Poli/Polκ-, and Polθ- dependent pathways, respectively. Based on biochemical and structural studies, we propose a role for Rev1 and Poli in inserting a nucleotide (nt) opposite the adduct and for Polsη and κ in extending synthesis from the inserted nt in the respective TLS pathway. Based on genetic analyses and biochemical studies with Polθ, we infer a role for Polθ at both the nt insertion and extension steps of TLS. Whereas purified Rev1 and Polθ primarily incorporate a C opposite (r)γ-HOPdG, Poli incorporates a C or a T opposite the adduct; nevertheless, TLS mediated by the Poli-dependent pathway as well as by other pathways occurs in a predominantly error-free manner in human cells. We discuss the implications of these observations for the mechanisms that could affect the efficiency and fidelity of TLS Pols.

Full text of DOI:10.1074/jbc.RA117.000962

JBC Papers in Press. Published on January 12, 2018 as Manuscript RA117.000962
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA117.000962
Genetic control of predominantly error-free replication through an acrolein-derived minor-groove
DNA adduct
1
2
2
1
1
Jung-Hoon Yoon , Richard P. Hodge , Linda C. Hackfeld , Jeseong Park , Jayati Roy Choudhury ,
1
1,*
Satya Prakash , and Louise Prakash
1
2
From the Department of Biochemistry and Molecular Biology, and the Sealy Center for
Environmental Health and Medicine, the University of Texas Medical Branch at Galveston, 301
University Blvd., Galveston, TX 77555-1061, USA
Running title: Genetic control of replication through (r) -HOPdG adduct
*To whom correspondence should be addressed: Department of Biochemistry and Molecular Biology,
University of Texas Medical Branch, Galveston, TX 77555-1061, Telephone: (409) 747-8601; E-mail:
l.prakash@utmb.edu
Keywords: -HOPdG, (r) -HOPdG phosphoramidite synthesis, translesion synthesis in human cells
ABSTRACT
opposite (r) -HOPdG, Pol incorporates a C
Acrolein, an , unsaturated aldehyde, is
generated in vivo as the end product of lipid
peroxidation and from metabolic oxidation of
polyamines, and it is an ubiquitous
environmental pollutant. The reaction of
or a T opposite the adduct; nevertheless, TLS
mediated by the Pol dependent pathway as
well as by other pathways occurs in a
predominantly error-free manner in human
cells. We discuss the implications of these
observations for the mechanisms that could
affect the efficiency and fidelity of TLS Pols.
2
acrolein with the N of guanine in DNA leads
2
to the formation of -hydroxy-1-N -propano-
2’ deoxyguanosine (-HOPdG) which can exist
in DNA in a ring-closed or a ring-opened form.
Here we identify the translesion synthesis
Acrolein, an ,-unsaturated aldehyde, is a
ubiquitous environmental pollutant formed by
incomplete combustion of organic materials and
it occurs in the environment as a component of
tobacco smoke and automobile exhaust.
Moreover, acrolein is generated endogenously as
the end product of lipid peroxidation and during
the metabolic oxidation of polyamines (1-5).
Acrolein adducts have been detected in DNA
from a variety of tissues in rats, mice, and
humans, indicating that this DNA adduct is
generated in vivo from cellular reactions (1-
(
TLS) DNA polymerases (Pols) which conduct
replication through the permanently ring-
opened reduced form of -HOPdG [(r) -
HOPdG] and show that replication through
this adduct is mediated via Rev1/Pol,
Pol/Pol, and Pol dependent pathways,
respectively. Based upon biochemical and
structural studies, we propose a role for Rev1
and Pol in inserting a nucleotide (nt) opposite
the adduct and for Pols  and  in extending
synthesis from the inserted nt in the respective
TLS pathway. Based upon genetic analyses
and biochemical studies with Pol, we infer a
role for Pol at both the nt insertion and
extension steps of TLS. Whereas purified
Rev1 and Pol primarily incorporate a C
3,6,7).
2
The reaction of acrolein with the N of
guanine in DNA followed by ring closure results
in the formation of the cyclic adduct -hydroxy-
2
1
, N –propano-2’-deoxyguanosine (-HOPdG).

-HOPdG can exist in DNA in a ring-closed or a
Copyright 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

ring-opened form (8-10). -HOPdG presents a
strong block to synthesis by replicative DNA
polymerases and it is also inhibitory to synthesis
by yeast and human Pol, particularly at the
nucleotide (nt) incorporation step (11,12). DNA
synthesis opposite -HOPdG, however, can be
mediated by the sequential action of Pols  and ,
in which Pol incorporates a nt opposite -
HOPdG and Pol performs the subsequent
extension step (12). In the presence of a reducing

-HOPdG adducted DNA with sodium
borohydride (11). Here we describe a new
method for the direct synthesis of the reduced
form of -HOPdG rather than having to first
synthesize -HOPdG and then converting it to the
reduced form. However, site specific direct
alkylation of the
2
N
position of 2’-
deoxyguanosine in DNA is particularly difficult
because of multiple competing reactions (14).
The method chosen here, though similar to  -
HOPdG modifications synthesized earlier by
others (8-10), provides a more direct route for
2
agent, -HOPdG can be trapped as the N -(3-
hydroxy propyl) 2’-deoxyguanosine adduct,
which permanently stays in the ring-opened
configuration (Fig. 1A). We refer to this reduced
ring-opened form of -HOPdG as (r)-HOPdG.
Biochemical studies with (r) -HOPdG have
indicated that both yeast and human Pol can
carry out proficient synthesis opposite this adduct
by inserting the correct nt and by extending
synthesis (11,13); Pol also performs proficient
TLS opposite this adduct by inserting the correct
nt and by extending synthesis (13). Pol
incorporates a nt opposite (r) -HOPdG; however,
it incorporates a T with only an ~ 3-fold reduced
catalytic efficiency than the correct C (13).
2
forming site-specific N -(r)  -HOPdG adduct
during solid phase DNA synthesis. This method
is described in detail in Experimental Procedures.
Briefly, based upon the earlier method of Hofman
et al. (15) using 2’-deoxy-4-desmethylwyosine
2
2
for direct N alkylation and conversion to N
alkylated 2’-deoxyguanosine, this synthesis
method was modified for alkylation with (3-
bromopropoxy)-t-butyldimethylsilyl ether. t-
butyldimethylsilyl protected -N -(r)  -HOPdG
nucleoside was then converted to t-
2
2
butyldimethylsilyl-N -(r)-HOPdG
phosphoramidite for use in solid phase DNA
synthesis. The t-butyldimethylsilyl protecting
group of the propano ether side chain is stable to
deprotection conditions during DNA synthesis
and is easily converted to the free alcohol by
tetrabutlyammonium fluoride treatment prior to
final reverse phase HPLC purification of the (r)
In this study, we identify the TLS Pols
required for replicating through the (r) -HOPdG
adduct in human cells and show that TLS
opposite this adduct is mediated via three
independent pathways that involve Rev1 and
Pol in one pathway, Pol and Pol in another
pathway, and Pol in the third pathway, and TLS
by all these pathways is mediated in a
predominantlly error-free manner. We discuss
the possible implications of these observations
for TLS opposite (r) -HOPdG and other minor
groove DNA lesions.

-HOPdG DNA sequence.
TLS Pols Required for Replicating through the
(
r) -HOPdG Adduct in Human Cells
To identify the TLS Pols required for
replicating through the (r) -HOPdG adduct (Fig.
A), we examined the effects of siRNA
1
depletions of TLS Pols on the frequency of TLS
opposite this lesion carried on the template for
leading strand replication in the SV40-based
duplex plasmid (Fig. 1B). In this plasmid,
Results
Synthesis of (r)  -HOPdG Phosphoramidite
The reduced form of  -HOPdG needed for
these studies was introduced into synthetic DNA
sequence during automated solid phase DNA
bidirectional replication initiates from
a
replication origin and TLS through the DNA
lesion generates Kan+ blue colonies (16,17). The
frequency of Kan+ blue colonies among total
Kan+ colonies give a highly reliable and
repeatable estimate of TLS frequencies (16-18).
synthesis
phosphoramidite form of N – hydroxypropanol-
’-deoxyguanosine. Previously, the reduced form
of -HOPdG has been generated by treatment of
via
a
suitably
protected
2
2
2

In normal human fibroblasts (HFs) treated
with control (NC) siRNA, TLS occurs with a
frequency of ~35%, and siRNA depletion of the
Rev3 catalytic or Rev7 accessory subunit of Pol
confers no reduction in TLS frequencies,
indicating that Pol plays no role in TLS opposite
2). In XPV cells treated with control siRNA, TLS
opposite (r) -HOPdG occurs with a frequency of
~25%. As expected from the involvement of
Pol, Pols /, and Pol, in three independent
pathways respectively, TLS is reduced to ~12%
upon depletion of Pol, Pol, or Pol or upon co-
depletion of Pols  and , and co-depletion of Pol
or Pol with Pol reduced TLS frequencies to
~5% (Table 2). The residual level of TLS that
remains in XPV cells co-depleted for Pol or Pol
with Pol likely results from the low levels of
TLS Pols (~2-3%) that persist in siRNA treated
cells (18,19) (and data not shown). Regardless of
this consideration, the requirement of Pol/Pol
and Pol for TLS through (r) -HOPdG in XPV
cells provides further confirmatory evidence for
the involvement of three independent pathways
for replicating through this minor groove DNA
adduct (Fig. 2).
(
r) -HOPdG (Table 1). siRNA depletion of
Pol, Pol, Pol, or Pol reduced TLS frequency
to ~22%, and siRNA depletion of Rev1 reduced
TLS frequency to ~11% (Table 1). To determine
which of the Pols function together in the same
TLS pathway and which Pols function in
different pathways, we examined the effects of
their simultaneous depletion on TLS frequency.
Our observation that co-depletion of Pols  and 
conferred no further reduction in TLS frequency
than that observed upon their individual depletion
indicated that these Pols function together in the
same TLS pathway (Table 1). Co-depletion of
Pol with Pol or with Pol, however, led to a
further reduction in TLS frequency to ~12%
compared to that seen upon their individual
depletion (~22%), indicating that Pol functions
in a TLS pathway independently of Pols  and 
Catalytic and Non-catalytic Roles of Rev1 in
TLS Opposite (r) -HOPdG in Human Cells
We have shown previously that Rev1
promotes replication through UV lesions together
with Pols , , and  (18). To verify that for TLS
opposite (r) -HOPdG also, Rev1 functions
together with Y-family Pols, we analyzed the
effects of simultaneous depletion of Rev1 with
Pol, Pol, or Pol on TLS frequencies. TLS
frequency in Rev1 depleted cells is reduced to
(
Table 1).
To determine whether Pol functions
together with Pol or whether it functions in an
independent pathway,we examined the effects of
co-depletion of Pols  and . Our observation
that co-depletion of these Pols confers a greater
reduction in TLS frequency that that seen upon
their individual depletion indicated that Pol and
Pol function in different TLS pathways (Table
~11%, a level similar to that in cell co-depleted
for Pol with Pol, or Pol with Pol (Table 1).
Our results that simultaneous depletion of Rev1
with Pol, Pol, or Pol confers no further
reduction in TLS frequency than that observed
upon Rev1 depletion alone indicate that Rev1
functions together with Pols , , and  for TLS
opposite (r) -HOPdG (Table 3). Since co-
depletion of Rev1 with Pol leads to a much
greater reduction in TLS frequency than observed
upon Rev1 depletion, Pol mediated TLS occurs
independently of Rev1 (Table 3).
Among the TLS Pols, Rev1 is highly
specialized for incorporating a C opposite
template G (20,21). Crystal structures of yeast
and human Rev1 have shown that template G is
evicted from the DNA helix into a solvent filled
cavity, and an Arg residue in Rev1 forms
hydrogen bonds with the incoming C (22,23).
1). To verify that Pol/Pol mediated TLS
operates independently of Pol, we examined the
effects of co-depletion of Pol or Pol with Pol.
The observation that co-depletion of these Pols
causes a greater reduction in TLS frequencies
than that observed upon their individual depletion
(
Table 1) indicated that Pol/Pol and Pol
function in different TLS pathways. From these
observations we infer that TLS opposite the (r) -
HOPdG adduct is mediated by three independent
pathway dependent upon Pol/Pol, Pol, and
Pol, respectively.
To further verify this inference, we examined
the effects of depletion of TLS Pols on the
frequency of TLS opposite (r) -HOPdG in
human XPV fibroblasts defective in Pol (Table
3

This protein-template directed mechanism of nt
incorporation is highly suited to allow Rev1 to
insert a C opposite the minor groove N -dG
Low mutagenicity of TLS opposite (r) -HOPdG
We find that TLS opposite (r) -HOPdG
occurs in a highly error-free manner as only ~1%
of TLS products harbor mutations. Among the ~
2
adducts. Therefore, to confirm that Rev1 DNA
polymerase activity was required for TLS
opposite (r) -HOPdG, we expressed the 3X Flag
siRNA sensitive wild type Rev1 (Fig. 3A) or the
siRNA resistant (siR) form of either the wild type
or the catalytic mutant D570A, E571A Rev1 in
human cells (Fig. 3B) and examined the effects of
this mutation on TLS opposite (r) -HOPdG. In
Rev1 siRNA treated cells expressing 3X Flag
wild type Rev1, Rev1 was efficiently depleted
400 TLS products sequenced from cells treated
with control (NC) siRNA, we observed 4
mutations wherein an A or T was incorporated
opposite the lesion site (Table 5). TLS opposite
(
r) -HOPdG incurred the same low mutagenicity
regardless of whether Pol, Pol, Pol, or Pol
was depleted, indicating that all these Pols
function in a predominantly error-free manner
opposite this DNA lesion (Table 5).
(
Fig. 3A) and the siRNA resistant form of wild
type Rev1 or catalytically inactive (AA) Rev1
was not affected (Fig. 3B). As shown in Table 4,
TLS in HF cells treated with Rev1 siRNA and
carrying the vector control occurs with a
frequency of ~12%, and TLS occurs with a
similar frequency in cells expressing the siRNA
sensitive wild type Rev1 protein. By contrast, in
cells expressing the siRNA resistant WT Rev1,
TLS is restored near to WT levels (~32%),
indicating that expression of WT Rev1
complements the TLS deficiency caused by Rev1
depletion. Expression of the siRNA resistant
Rev1 D570A, E571A catalytic mutant in cells
from which genomic Rev1 has been depleted,
however, reduced the TLS frequency to ~22%,
which suggested that in addition to its non-
catalytic role in TLS in Pol and Pol/Pol
pathways, Rev1 DNA polymerase activity is
required for TLS opposite (r) -HOPdG. To
determine if Rev1 DNA polymerase activity
contributes to TLS in the Pol dependent
pathway, we expressed the siRNA resistant Rev1
catalytic mutant in cells from which both Rev1
and Pol have been depleted. Our observation
that TLS frequencies remain the same in Pol
proficient or deficient cells (~22%) expressing
the Rev1 catalytic mutant indicates that Rev1
polymerase activity functions in TLS opposite (r)
Roles of TLS Pols in DNA Synthesis opposite (r)

-HOPdG
Rev1 inserts a C opposite the ring-closed
form of -HOPdG with the same catalytic
efficiency and fidelity as it inserts a C opposite
undamaged G (24). As expected, in the presence
of all 4 dNTPs, Rev1 inserts a C opposite (r) -
HOPdG and does not carry out extension of
synthesis from the inserted nt (data not shown).
For the Rev1 pathway, based upon this
biochemical observation, genetic data shown in
Table 4, and structural observations (22,23), we
suggest a role for Rev1 at the nt insertion step and
for Pol at the extension step of TLS opposite (r)

-HOPdG. For the Pol/Pol pathway, we have
previously reported biochemical evidence
indicating that Pol incorporates a C or a T
opposite this lesion (13).
Based upon
biochemical (13) and structural considerations
(
25,26), we suggest a role for Pol at the nt
insertion step of TLS opposite (r) -HOPdG and
for Pol in extending synthesis from the nt
inserted by Pol opposite the lesion site (27). By
contrast to the highly error-prone role of Pol in
incorporating a T nt opposite (r) -HOPdG with
only an ~ 3-fold reduced catalytic efficiency than
a C (13), Pol primarily incorporates a C opposite
(
r) -HOPdG (Fig. 4). Although Pol can carry

-HOPdG in conjunction with Pol (Table 4).
out the subsequent extension of synthesis, it is
considerably blocked in extending synthesis from
the nt inserted opposite the adduct (Fig. 4).
Altogether, the genetic data in Tables 1-4 support
the conclusion that replication through the (r) -
HOPdG adduct is mediated via three different
pathways in which Rev1 and Pol function in one
pathway, Pol and Pol function in another
pathway, and Pol mediates the third pathway
Discussion
Our genetic observations indicating that
replication through the (r) -HOPdG adduct is
(
Fig. 2).
4

mediated by three independent pathways,
comprised of Rev1/Pol, Pol/Pol, and Pol,
respectively (Fig. 2), differ strikingly from the
roles predicted for TLS Pols from biochemical
studies. Thus, from the proficient ability of Pol
and Pol to perform TLS opposite (r) -HOPdG
by both inserting a correct nt opposite the adduct
and then by extending synthesis from the inserted
nt, one would have expected these two Pols to
conduct replication through the adduct via two
independent pathways (11,13). However, rather
than acting by themselves in their respective
pathway, we find that in human cells TLS
opposite (r) -HOPdG is performed by Rev1 and
Pol in one pathway and by Pol and Pol in the
other pathway. To explain this discordance in the
roles for TLS Pols indicated from biochemical
studies vs. those inferred from genetic studies in
human cells, we suggest that replication through
DNA lesions in human cells is performed by TLS
Pols as components of multi-protein assemblies
and that the proficiency and fidelity of TLS Pols
is modulated in these assemblies.
and for Pol at the extension step of TLS. Pol
differs greatly from other DNA Pols in the ways
it synthesizes DNA opposite template purines and
pyrimidines (28-31). Pol incorporates nts with a
much higher catalytic efficiency and fidelity
opposite template purines than opposite template
pyrimidines; and among template purines, Pol
shows a higher efficiency and fidelity opposite
template A than opposite template G (28-31).
Pol incorporates nts opposite purine templates
by pushing the template A or G into a syn
conformation which then forms a Hoogsteen base
pair with the incoming T or C, respectively
(25,26,32). The ability of Pol to push the (r) -
HOPdG adduct into a syn conformation would
allow the adduct to form a Hoogsteen base pair
with the correct C or the incorrect T nt and our
previous biochemical studies with Pol have
shown that it proficiently incorporates a C or a T
opposite (r) -HOPdG (13). Biochemical and
structural studies have shown that Pol is highly
adapted for extending synthesis opposite from
minor groove DNA lesions (13,27); hence, Pol
could extend synthesis from the nt inserted by
Pol.
The propensity of Pol for incorporating a T
opposite (r) -HOPdG (13) would suggest that
TLS mediated by the sequential action of Pols 
and  would occur in a highly error-prone
manner. However, TLS opposite this adduct by
this pathway as well as by the other two
pathways, operates in a predominantly error-free
manner in human cells. Thus, the inference
derived from biochemical studies for the highly
error-prone role of Pol in TLS opposite (r) -
HOPdG does not extend to TLS that occurs in
human cells.
Roles of TLS Pols in Rev1/Pol and Pol/Pol
pathways:
(
a) Rev1/Pol pathway –The protein-template
directed mechanism of nt incorporation provides
Rev1 the ability to insert a C opposite N -dG
2
adducts which protrude into the DNA minor
groove (22,23). We find that in the presence of
all 4 dNTPs, Rev1 selectively incorporates a C
opposite (r) -HOPdG but it fails to extend
synthesis from the inserted nt (data not shown).
Based upon the ability of Rev1 for nt insertion
and upon the ability of Pol to extend synthesis
from the inserted nt, we suggest that TLS in the
Rev1/Pol pathway is mediated by the sequential
action of Rev1 and Pol, in which following the
incorporation of a C opposite (r) -HOPdG by
Rev1, Pol would extend synthesis from the
inserted nt. As judged from the proficient ability
of Rev1 for incorporating a C and of Pol for
extending synthesis from it, Rev1/Pol mediated
TLS opposite (r) -HOPdG would occur in an
error-free manner in human cells.
(c) Non-Catalytic Role of Rev1 in TLS -
Previously we showed that for TLS opposite UV
induced CPDs and (6-4) photoproducts, Rev1
functions as an indispensable scaffolding
component of Y-family Pols , , and , and that
it does not function together with Pol (18). For
TLS opposite (r) -HOPdG, we find that Rev1
functions together with Y-family Pols , , and 
and not with Pol. Presumably, Rev1 bound to
Pol, Pol, or Pol plays a crucial role in the
formation of multi-protein assembly. It would be
of much interest to identify the components of
(
b) Pol/Pol pathway -
Based upon
biochemical and structural considerations, we
suggest a role for Pol at the nt incorporation step
5

such assemblies, to determine the roles they play
in the efficiency and fidelity of TLS Pols opposite
DNA lesions, and to see whether the composition
of multi-protein assemblies for Y-family Pols and
for other TLS Pols differs for different types of
DNA lesions. For elucidating the highly error free
role of Pol in TLS opposite (r) -HOPdG, it
would be important to decipher how Rev1
together with other components of the multi-
protein assembly modulates Pol active site
opposite this lesion such that its proficiency for
the insertion of the incorrect nt T is greatly
reduced and its ability to insert the correct nt C is
highly enhanced.
base pair with the incoming C, and Pol then
extends synthesis from this base pair. An
alternative possibility is that this minor groove
DNA adduct is accommodated in a syn
conformation in Pol active site and it forms a
Hoogsteen base pair with the incoming C residue.
Such a mode of accommodating minor groove
DNA lesions in its active site could provide Pol
a greater latitude in its ability to replicate through
the diverse array of DNA adducts that form at the
2
highly reactive N group of a deoxyguanine.
(
d) Possible Roles of Multi-protein Assemblies in
The High Fidelity of TLS in Human Cells
In human cells, TLS opposite (r) -HOPdG
occurs with a high fidelity, generating only ~1%
mutations, similar to that observed for TLS
opposite DNA lesions such as cis-syn TT dimer
Activation or Inhibition of TLS Pols in the
Rev1/Pol and Pol/Pol pathways –We surmise
that in the Rev1/Pol and Pol/Pol pathways, in
which Rev1 and Pol would act at the nt
incorporation step of TLS and Pol and Pol
would carry out the extension step in the
respective pathway, the proficient ability of Pol
or Pol to insert a nt opposite (r) -HOPdG would
be inhibited in the multi-protein ensemble of
these Pols. Such inhibitory effects of multi-
protein assemblies on TLS Pols such as Pol and
Pol which can replicate through (r) -HOPdG
with a high catalytic efficiency and fidelity
(
(
16), (6-4) TT photoproduct (17), thymine glycol
33), N1-methyl adenine (34), and N3-methyl
adenine (35) where at most ~1-2% of the TLS
products harbor mutations. This is in spite of the
fact that TLS Pols synthesize DNA opposite
DNA lesions with a poor fidelity. For example,
even though Pol incorporates a T opposite the (r)

-HOPdG adduct with only a somewhat reduced
catalytic efficiency than a C (13), TLS opposite
this adduct occurs in a predominantly error-free
manner in human cells. The evidence for
predominantly error-free TLS opposite a number
of DNA lesions in human cells strongly suggests
that TLS mechanisms have been adapted to act in
highly specialized and predominantly error-free
ways.
(
11,13) raise the intriguing possibility that multi-
protein assemblies of TLS Pols have evolved to
have become highly specialized for replicating
through particular types of DNA lesions. Thus,
for replication through the large variety of minor
2
groove N -dG adducts, the Y-family Pols may
employ an identical or a very similar multi-
protein assembly in which the various
components coordinate TLS in a highly efficient
and relatively error-free manner.
Experimental Procedures
2
Synthesis
of
5’-DMT-N -(3-(tert-
butyldimethylsilyloxy)-propyl)-2'-
deoxyguanosine-3’-(N,N-diisopropyl--
cyanoethyl-phosphoramidite) [[(r)-HOPdG]
phosphoramidite]
Role of Pol in TLS
By contrast to the requirement of different
inserter and extender Pols for TLS mediated by
Rev1/Pol and Pol/ Pol opposite (r) -HOPdG,
our genetic studies indicate that Pol would act at
both the nt insertion and the subsequent extension
step of TLS opposite this DNA lesion. How
could Pol incorporate the correct nt opposite this
minor groove adduct? One possibility is that in
All solvents and reagents were purchased
from either Fisher or Sigma Aldrich with the
exception of bromine which was purchased from
Fluka. Anhydrous solvents were additionally
treated with molecular sieves and measured to be
less than 50ppm water content by Karl-Fischer
titration prior to use.
2
the Pol active site, the N -dG adduct stays in the
anti conformation and forms a Watson-Crick
6

All NMR data was obtained on a Bruker
Ultrashield 300, Avance II, 300 MHz. Positive
mode mass spectroscopy data was obtained on a
Sciex 5800 Maldi TOF/TOF.
mmol) was coevaporated with 20mL anhydrous
pyridine twice then dissolved in anhydrous
dimethylsulfoxide (120 mL) under argon with
stirring. Sodium hydride (60% suspension)
(
1.472 g, 36.8 mmol) was added and the mixture
was stirred at room temperature under argon for
hr. Bromoacetone (5.04 g, 36.8 mmol) was
added and stirring continued under argon for 1 hr
the solution immediately turned brown].
The method of Hofmann et al. (15) via 2’-
deoxy-4-desmethylwyosine as the protected
intermediate is preferred for the direct alkylation
1
2
of the N position of guanosine by 3-bromo-1-(t-
[
butyldimethylsilyl)-propano-ether.
For this
Ammonium hydroxide (60 mL, 889 mmol) was
added and the mixture was stirred an additional 2
hr at room temperature then evaporated to ~60
mL. Acetone (60 mL) was added and the mixture
was poured into to a solution of acetone (800 mL)
and ether (200 mL). The mixture was stirred at
synthesis, bromoacetone was freshly prepared
using the method of Levene et al. (36). 3-Bromo-
1-(t-butyldimethylsilyl)-propano-ether
was
prepared as described (37).
We use the following abbreviations in the
description below: thin layer chromatography
0
°C for 3 hr (monitored by TLC using 10%
MeOH / CH Cl , and developed using 1%
2
2
(
TLC),
dimethylformamide
(DMF),
anisaldehyde / ethanol spray with heating). When
the reaction showed no further progress, the
mixture was transferred to a 2L separatory funnel
and the layers separated. The resulting lower
layer of oil was washed further with ether (2x500
mL), separated and evaporated. Water (900 mL)
was added and the crude product was pre-
absorbed onto silica gel (10 g) by evaporation
followed by coevaporation three times with
tetrahydrofuran (THF), dimethoxytrityl (DMT),
t-butyldimethylsilyl (t-BDMSi).
All NMR data was obtained on a Bruker
Ultrashield 300, Avance II, 300 MHz. Positive
mode mass spectroscopy data was obtained on a
Sciex 5800 Maldi TOF/TOF.
Synthesis
phosphoramidite comprised the six steps [1] to
6] described below and outlined in Figure 5.
of
(r)
-HOPdG
200mL
ethanol.
The
2'-deoxy-4-
[
desmethylwyosine absorbed silica was added to a
silica gel column (200g) and eluted using a step
Bromoacetone [1]: Following the method of
Levene (36), acetone (50 mL, 0.676 mol) was
added to a 3-neck flask containing water (160
mL, 8.855 mol). Acetic acid (38 mL, 0.661 mol)
was added and the mixture was heated to 70-
gradient elution of 100% CH
2
Cl
2
2
to 90% CH Cl
2
/
MeOH to yield pure product (6.62 g, 21.68
1
mmol, 61.9 % yield). H NMR (DMSO-d6) 
ppm): 2.23 (d, J = 1.2 Hz, 3H, CH ); 2.57 (m,
H, H2'); 3.53 (m, 2H, H5'); 3.83 (m, 1H, H4');
(
3
2
4
5
6
1
80°C. Bromine (37.4 mL, 0.730 mol) was added
.35 (m, 1H, H3'); 4.93 (t, J = 5.4 Hz, 1H, 5'-OH);
.27 (dd, J = 4.2, 2.1 Hz, 1H, 3'-OH); 6.21 (t, J =
Hz, 1H, H1'); 7.30 (s, 1H, H9); 8.07 (s, 1H, H2);
2.30 (s, 1H, NH). ES-MS+ 306.0 g/mol, exact
dropwise over 1-1.5 hr. When the solution was
completely decolorized it was diluted with cold
water (80 mL). The mixture was cooled to 10°C
and neutralized with sodium carbonate (turns
yellow/orange and there are two layers). The oil
was separated and dried with anhydrous calcium
chloride. The product was distilled using house
vacuum, fraction at 65-70°C (brown) was the
mass = 305.11 g/mol
(
3-Bromopropoxy)-t-butyldimethylsilyl ether:
Following the preparation method of Choi et al
37), fresh 3-Bromo-1-propanol (2 mL, 22.12
mmol) was dissolved in dry CH Cl (8 mL).
Triethylamine (3.39 mL, 24.33 mmol) and 4-
dimethylamino)pyridine (0.29 g, 2.433 mmol)
(
1
main fraction (25.88 g). H NMR showed mostly
2
2
product, with some dibromo side products
present. A second distillation gave pure product
(
1
as a pale yellow liquid (22.54g, 24.34%).
H
were added under argon with stirring. The
solution was cooled to 0°C under argon and t-
butyldimethylsilyl chloride (t-BDMSi) (3.33 g,
NMR (CDCl ),  (ppm): 3.87 (CH
3
2
); 2.34 (CH
3
).
2’-Deoxy-4-desmethylwyosine [2]: Using the
22.12 mmol) was added. The solution was
protection method of Hofmann et al. (15), 2'-
deoxyguanosine monohydrate [1] (10 g, 35.1
allowed to come to room temperature while
7

stirring for 3 hr under argon (monitored by TLC
using 15% ethyl acetate / hexane with
2H, NHCH
(m, 2H, NHCH
2
CH
2
CH
CH
2
O); 3.87 (m, 1H, H4’); 4.15
CH O); 4.77 (m, 1H, H-3');
2
2
2
anisaldehyde detection).
quenched with water (5 mL) and allowed to stir
5 minutes. The crude product was extracted
from the aqueous solution with CH Cl , (3 x 10
mL). The combined organic extracts were dried
over Na SO , filtered and evaporated. Crude (3-
The reaction was
6.37 (t, J=6.6Hz, 1H, H-1'); 7.32 (s, 1H, H9); 8.04
(s, 1H, H-2). ES-MS+: 478.2 /mol, exact mass =
477.24 g/mol
1
2
2
5
2
’-DMT-(3-(t-butyldimethylsilyloxy)propyl)-
’-deoxy-4-desmethylwyosine [4]:
2
4
bromopropoxy)-t-butyldimethylsilyl ether was
purified on a silica gel column using 15% ethyl
3
-(t-Butyldimethylsilyloxy)propyl-2'-deoxy-4-
desmethylwyosine (700 mg, 1.47 mmol, from
two prepared batches) was coevaporated twice
with anhydrous pyridine (5 mL) then redissolved
in anhydrous pyridine (15 mL) under argon with
stirring. Dimethoxytrityl chloride, (DMT-Cl)
acetate / hexane to yield 4.52 g, 17.85 mmol, 81
1
%
(
SiCH
yield, as a yellow oil. H NMR (CDCl
3
) 
); 0.094 (s, 3H,
); 0.91 (s, 9H, t-Bu); 2.06 (m, 2H,
ppm): 0.089 (s, 3H, SiCH
3
3
BrCH
BrCH
BrCH
2
CH
CH
CH
2
CH
CH
CH
2
2
2
O); 3.53 (t, J = 5.7Hz, 2H,
O); 3.75 (t, J = 5.7Hz, 2H, ,
O).
(
0.546 g, 1.61 mmol) was added and the reaction
was stirred at room temperature 3 hr under argon
monitored by TLC 10% MeOH / CH Cl
2
2
2
2
(
2
2
containing 0.1% triethylamine, anisaldehyde
detection). A second addition of fresh DMT-Cl
3
-(t-butyldimethylsilyloxy)propyl-2’-deoxy-4-
desmethylwyosine [3]: Using the alykylation
method of Hofmann et al. (15), 2'-deoxy-4-
desmethylwyosine (1 g, 3.28 mmol) was
dissolved in anhydrous N,N-dimethylformamide
(
0.25 g, 0.74 mmol) was added and after an
additional 1 hr reaction TLC showed completion.
The reaction was evaporated of solvent and
redissolved in 15 mL CH
2
Cl
2
.
The organic
(
13 mL) under an argon atmosphere. Anhydrous
solution was washed with saturated NaHCO (10
potassium carbonate (0.475 g, 3.44 mmol) and (3-
bromopropoxy)-t-butyldimethylsilyl ether (2.074
g, 8.19 mmol) were added. The mixture was
stirred at 37 C for 19 hr (turns dark reddish
brown) and followed by TLC (10% MeOH /
3
mL), followed by water (10 mL), and the organic
layer dried over Na
2
SO
4
, filtered and evaporated.
o
The
5’-DMT-(3-(tert-
butyldimethylsilyloxy)propyl)-2’-deoxy-4-
desmethylwyosine was purified on a silica gel
CH
addition
2
Cl
2
, anisaldehyde detection). A second
of (3-bromopropoxy)-t-
column using a step gradient of CH
triethylamine to 10% MeOH / CH
2
Cl
Cl
2
with 0.1%
with 0.1%
butyldimethylsilyl ether (0.83 g, 3.28 mmol) was
added and the reaction stirred at room
temperature for an additional 65 hr (followed by
2
2
triethylamine to give pure product (0.794 g, 1.01
1
mmol, 68.7 % yield). H NMR (CDCl )  (ppm):
3
TLC using 15% MeOH / CHCl ). The reaction
3
0
2
.08 (s, 6H, SiCH
3
); 0.92 (s, 9H, t-Bu); 1.86 (m,
O); 2.45 (s, 3H, CH ); 2.5
mixture was filtered through celite and the celite
pad washed with additional warm DMF until the
washings showed no UV absorbance at 254 nm.
The filtrates and washings were combined and
evaporated to give crude product which was
purified on a silica gel column using an elution
gradient of chloroform with 0.2% triethylamine
to 30% methanol / chloroform with 0.2%
triethylamine
butyldimethylsilyloxy)propyl-2'-deoxy-4-
desmethylwyosine (550 mg, 1.152 mmol, 35.2 %
yield). H NMR (CDCl
CH ); .87 (s, 9H, t-Bu); 1.96 (m, 2H,
NHCH
H, NHCH CH CH
2
2
2
3
(
m, 1H, H-2'); 2.68 (m, 1H, H-2'); 3.29 (m, 1H,
NHCH
NHCH
2
CH
2
CH
2
O);
O ); 3.62 (t, J = 5.7 Hz, 2H, H5’);
); 4.1 (m,
O ); 4.64 (m, 1H, H-3');
.39(t,J = 6.3 Hz, 1H, H-1'); 6.78 (dd, J = 2.4, 4.5
3.4
(m,
1H,
2
CH
2
CH
2
3
2
6
.72 (m, 1H, H4’); 3.77 (s, 6H, OCH
H, NHCH CH CH
3
2
2
2
Hz, 4H, DMT-meta H's); 7.26 (m, 7H, DMT-
ortho H's, Bz & H9); 7.38 (m, 2H, DMT-ortho
H's); 7.7 (s, 1H, H-2). ES-MS+: 780.2 g/mol,
exact mass = 779.37 g/mol
to
give
pure
3-(t-
1
3
)  (ppm): .03 (s, 6H, Si-
5
2
’-DMT-(3-(t-butyldimethylsilyloxy)propyl)-
’-deoxyguanosine [5]: Deprotection was
3
2
CH
2
CH
2
O); 2.33 (s, 3H, CH
3
); 2.44 (m,
performed following the methods of Casale et al.
38), Hofmann et al. (15), and Boryski and Ueda
1H, H-2'); 2.81 (m, 1H, H-2'); 3.55 (m, 2H,
(
NHCH
2
CH
2
CH
2
O); 3.64 (m, 2H, H-5'); 3.71 (m,
8

(
39).
5’-DMT-(3-(t-
(0.128mL, 0.40 mmol) and tetrazole solution
(0.938 mL, 0.40 mmol) were added, again
simultaneously with stirring, and the reaction was
continued for 30 minutes. The reaction was
butyldimethylsilyloxy)propyl)-2'-deoxy-4-
desmethylwyosine (0.79 g, 0.99 mmol) was
dissolved in a mixture of THF (27 mL) and 0.2 M
aqueous potassium acetate pH 5.7 (11 mL). N-
Bromosuccinimide (0.216 g, 1.19 mmol) was
added and the mixture was stirred at room
temperature for 40min. (solution turns light blue
then pale yellow as reaction proceeds).
Ammonium hydroxide (5.7 mL) was added and
stirring was continued 30 minutes (monitored by
diluted with CH
2
Cl (11 mL) and the product
2
washed with 5% aqueous sodium bicarbonate
(10mL) followed by washing with an aqueous
brine solution (10mL). The organic layer was
separated, dried over sodium sulfate and filtered.
Triethylamine 20 uL was added to the filtrate and
the solution evaporated. The crude 5’-DMT-(3-
(t-butyldimethylsilyloxy)propyl)-2’-
TLC using 5% MeOH / CH
2
Cl with 0.1%
2
triethylamine). When the reaction was complete,
solvent was evaporated and the crude product
purified by silica gel column using a step gradient
deoxyguanosine phosphoramidite mixture was
purified on a silica gel column using a step
gradient of 100% ethyl acetate with 0.1%
triethylamine to 10% MeOH / ethyl acetate with
0.1% triethylamine as eluant. TLC using pre-run
ethyl acetate / 0.1% triethylamine TLC plates,
dried, spotted and eluted with same solvent gave
two isomeric phosphoramidite products which
were detected with anisaldehyde. Dittmer reagent
was utilized to detect any excess phosphytilating
agent separated from the product isomers. The
of 100% CH
.1% TEA to give pure 5’-DMT-(3-(t-
butyldimethylsilyloxy)propyl)-2'-
deoxyguanosine (226 mg, 0.305 mmol, 30%
2
Cl
2
to 5% MeOH / CH
2
Cl with
2
0
1
yield). H NMR (CDCl
3
)  (ppm): 0.010 (s, 6H,
Si-CH ); 0.85 (s, 9H, t-Bu); 1.78 (m, (2H,
3
NHCH2CH
H, H2'); 3.33 (m, 2H, NHCH
t, J = 5.7 Hz, 2H, H5’); 3.67 (m, 1H, H4'); 3.70
s, 6H, OCH ); 4.08 (m, 2H, NHCH CH CH O);
.68 (m, 1H, H3'); 6.23 (t, J = 6 Hz, 1H, H1'), 6.73
m, 4H, DMT meta H’s); 7.16 (m, 7H, DMT-
2
CH2O); 2.51 (m, 1H, H2'); 2.70 (m,
1
2
CH CH O); 3.61
2
2
phosphoramidite products were combined to
(
1
yield 120 mg, (0.127 mmol, 47.2% yield)
H
(
3
2
2
2
NMR (CD
0
1
3
CN)  (ppm): 0.04 (s, 6H, SiCH
.89 (s, 9H, t-Bu); 1.20 (m, 12H, NCH(CH
.74 (m, 2H, NHCH CH CH
CH CN); 2.63 (t, J = 6Hz, 2H,
); 2.94 (m, 1H, H2’); 3.30 (m, 4H,
H5’ & NHCH CH CH O); 3.67 (m, 4H,
CH CH CN & NHCH CH CH O); 3.73 (s, 6H,
OCH ); 4.16 m, 1H, H4’); 4.74 (m, 1H, H3’);
3
);
);
4
(
3 2
)
2
2
2
O); 2.52 (m, 3H,
ortho H’s & Benzyl H’s); 7.33 (m, 2H, DMT-
ortho H’s); 7.64 (s, 1H, H2) ES-MS+: 742.2
g/mol; exact mass 741.36 g/mol
H2’ & CH
NCH(CH
2
2
3 2
)
2
2
2
DMT-5-(3-(tert-
2
2
2
2
2
butyldimethylsilyloxy)propyl)-2’-
deoxyguanosine-3’-(N,N,-diisopropyl--
cyanoethyl-phosphoramidite, [(r)-HOPdG
3
6.21 (t, J = 6.6Hz, 1H, H1’); 6.78 (m, 4H, DMT
meta H’s); 7.24 (m, 7H, DMT ortho H’s & bz
H’s); 7.39(m, 2H, DMT ortho H’s); 7.66 (s, 1H,
phosphoramidite]
[6]:
5’-DMT-(3-(t-
3
1
butyldimethylsilyloxy)propyl)-2'-
H2). P NMR (CD
148.88.
3
CN)  (ppm): 148.79;
deoxyguanosine (0.30 g, 0.40 mmol) was
dissolved in dry methylene chloride (11 mL) in
an argon atmosphere in a sealed round bottom
Oligonucleotide syntheses
2
Oligonucleotides containing N --hydroxy-
propano-deoxyguanosine [(r)-HOPdG] were
synthesized on a Model 8909 Expedite DNA
flask with rubber stopper.
2-Cyanoethyl-
N,N,N’,N’-tetraisopropyl-phosphine (0.177 mL,
0
0
.55 mmol) and tetrazole / THF solution (1.2 mL,
.51 mmol tetrazole) were added simultaneously
synthesizer using standard DNA synthesis
2
chemistry.
The N --hydroxy-propano-
via syringes under argon. The solution was
stirred at room temperature for 1 hr (monitored
by TLC using pre-run ethyl acetate / 0.1%
triethylamine TLC plates, dried, spotted and
eluted with same solvent and detected with
deoxyguanosine [(r) -HOPdG] phosphoramidite
was incorporated using an offline coupling
method to conserve reagent. The oligos were
deprotected using standard conc. ammonia
deprotection, with an additional deprotection step
using 0.1M tetrabutylammonium fluoride / THF
anisaldehyde).
A second addition of 2-
cyanoethyl-N,N,N’,N’-tetraisopropyl-phosphine
9

for removal of the t-BDMSi group from the
propano hydroxyl side chain. Oligonucleotides
were purified and analyzed by reverse phase
HPLC on a Beckman System Gold HPLC.
Purified oligonucleotides were analyzed and
confirmed by MALDI-MS on a Bruker Autoflex
MALDI mass spectrophotometer.
mixture of primer/template at a 1:1.5 molar ratio
to 95 °C and allowing it to cool to room
temperature for several hours. The template 75-
mer oligonucleotide contained the sequence
5΄AGC AAG TCA CCA ATG TCT AAG AGT
TCG TAT GAT GCC TAC ACT GGA GTA
CCG GAG CAT CGT CGT GAC TGG GAA
AAC-3΄ and was either undamaged G or harbored
an (r) -HOPdG at the underlined position. For
examining the incorporation of dATP, dTTP,
dCTP, or dGTP nucleotides individually, or of all
4 dNTPs, a 44 mer primer 5΄ GTT TTC CCA
GTC ACG ACG ATG CTC CGG TAC TCC
AGT GTA GGC AT-3΄ was annealed to the
above mentioned 75 mer template.
Construction of plasmid vectors containing (r)

-HOPdG
The in-frame target sequence of the lacz’ gene
containing (r) -HOPdG is shown in Fig. 1B.
Since the lacz’ sequence in the (r) -HOPdG -
containing DNA strand is in-frame, it encodes
functional -galactosidase (-gal); the opposite
DNA strand harbors an SpeI restriction site
containing a +1 frameshift which makes it non-
functional for -gal. The (r) -HOPdG -
containing strand carries the kanamycin gene
The standard DNA polymerase reaction (5
μl) contained 25 mM Tris·HCl (pH 7.5), 5 mM
MgCl2, 1 mM dithiolthreitol, 100 μg/ml BSA,
10% glycerol, and 10 nM DNA substrate, and 1
nM Polθ. For nucleotide incorporation assays, 10
μM dATP, dTTP, dCTP, or dGTP (Roche
Biochemicals, Indianapolis) were used, and for
examining synthesis through the (r)-HOPdG
lesion all 4 dNTPs (10 μM each) were used.
Reactions were carried out for 10 min at 37 °C.
Reaction products were resolved on a 12%
polyacrylamide gel containing 8M urea and
analyzed by a PhosphorImager.
(
Kan+), whereas the other DNA strand has the
-
kan gene (Fig. 1B). The detailed methods for the
construction of lesion-containing SV40-based
duplex plasmids have been published previously
(
16,17).
Assays for translesion synthesis and mutation
analyses of TLS products in human cells
The detailed method for TLS assays have been
described previously (16,17). Briefly, human
fibroblast GM637 cells are transfected with the
particular siRNA and after 48 h of incubation, the
target vector DNA and siRNA (second
transfection) are co-transfected. After 30h of
incubation, plasmid DNA is transfected into E.
coli XL1-Blue super competent cells (Stratagene)
and cells plated on LB/kan plates containing
isopropyl-1-thio--D-galactopyranoside
Acknowledgments – This work was supported
by National Institutes of Health Grants ES022948
and ES020833, and in part by NIEHS Center
Grant P30 ES06676.
Conflict of Interest – The authors declare that
they have no conflicts of interest with the
contents of this article.
Author Contributions – J. H.Y. performed and
analyzed the experiments on the genetic control
of TLS and mutagenicity; R.P.H. and L.C.H.
synthesized the (r)-HOPdG phosphoramidite,
constructed oligonucleotides containing this
adduct, and wrote that section; J. P. contributed
to the genetic experiments; J. R. C. performed the
biochemical experiment; S.P. and L. P. designed
and co-ordinated the study and wrote the paper.
All authors reviewed the results and approved the
final version of the manuscript.
(
(
GenDEPOT)
GenDEPOT). TLS frequency is determined
and
100g/ml
X-Gal
from the number of blue colonies among total
colonies growing on LB/Kan plates and mutation
frequencies and mutational changes are analyzed
by DNA sequencing.
DNA polymerase assays
DNA substrates consisted of a radiolabeled
oligonucleotide primer annealed to a 75nt
oligonucleotide DNA template by heating a
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4

Table 1
Effects of siRNA knockdowns of TLS polymerases on the replicative bypass of the (r) -
HOPdG lesion carried on the leading strand template in human fibroblasts
No. Kan+
colonies
No. blue colonies
siRNA
TLS (%)
among Kan+
NC
669
642
486
548
623
523
547
412
396
308
326
278
317
302
235
248
185
130
141
106
123
46
35.1
38.6
38.1
23.7
22.6
20.3
22.5
11.2
24.2
12.7
12.6
11.5
11.4
11.9
Rev3
Rev7
Pol
Pol
Pol
Pol
Rev1
Pol + Pol
Polη + Pol
Polη + Pol
Polη + Polθ
Pol + Polθ
Pol + Polθ
96
39
41
32
36
36
1
5

Table 2
Effects of siRNA knockdowns of TLS polymerases on the replicative bypass of the (r) -
HOPdG lesion carried on the leading strand template in XPV human fibroblasts
No. Kan+
colonies
No. blue colonies
siRNA
TLS (%)
among Kan+
NC
238
272
286
236
245
204
196
60
33
30
34
30
10
9
25.2
12.1
10.5
12.8
12.2
4.9
Pol
Pol
Polθ
Pol + Pol
Pol + Polθ
Pol + Polθ
4.6
1
6

Table 3
Effects of co-depletion of Rev1 with Y-family Pols or with Pol on TLS opposite
(r) -HOPdG carried on the leading stand template in human fibroblasts
No. of Kan+
colonies
No. blue colonies
siRNA
TLS (%)
among Kan+
NC
669
412
381
394
426
468
235
46
43
45
47
20
35.1
11.2
11.3
11.4
11.0
4.3
Rev1
Rev1 + Polη
Rev1 + Pol
Rev1 + Pol
Rev1 + Polθ
1
7

Table 4
Effect of siRNA knockdown of Rev1 alone or together with Pol on TLS opposite (r) -
HOPdG present on the leading strand template in wild type human fibroblasts carrying a
vector expressing the siRNA resistant (siR) form of wild type or catalytically inactive Rev1
No. blue
Vector
expressing
No. Kan+
colonies
siRNA
colonies +
TLS (%)
among Kan
Rev1
Vector control
Flag-WT-Rev1
325
384
326
358
39
45
12.0
11.7
31.9
22.1
“
“
“
Flag-WT-siR-Rev1
104
79
Flag-D570A, E571A-
siR-Rev1
Pol +
Flag-WT-Rev1
348
316
372
40
75
81
11.5
23.7
21.8
Rev1
“
“
Flag-WT-siR-Rev1
Flag-D570A, E571A-
siR-Rev1
1
8

Table 5
Effects of siRNA knockdowns of TLS polymerases on the frequencies of nucleotides inserted
opposite (r) -HOPdG carried on the leading strand template in human fibroblasts
No. kan+
blue
colonies
sequenced
Mutation
Nucleotide inserted
frequency
%)
siRNA
(
A
2
2
2
1
0
G
0
0
0
0
0
C
T
NC siRNA
Polη
386
384
288
196
196
382
381
284
194
194
2
1.0
0.8
1.4
1.0
1.0
1
2
1
2
Polι
Polκ
Polθ
1
9

FIGURE LEGENDS
Figure 1. Assay for TLS opposite (r) -HOPdG. A, Structure of reduced -HOPdG. B, The
target 16-mer sequence containing (r) -HOPdG nt at G* is shown on top and the lacz’ sequence
in the leading strand in the pBS vector containing the adduct at G* is shown below. The (r) -
HOPdG-containing DNA strand is in frame and it carries the Kan+ gene. TLS through the adduct
generates Kan+ blue colonies.
Figure 2. Pathways for replicating through the (r) -HOPdG adduct in human cells.
Replication through the (r) -HOPdG adduct is mediated by three independent pathways in a highly
error-free manner. See Text for roles of the TLS Pols in these pathways.
Figure 3. Stable expression of wild type Rev1 and catalytic mutant of Rev1 in human
fibroblasts. Normal human fibroblasts (GM637) expressing A, 3x-Flag –siRNA sensitive wild
type (WT) Rev1; B, 3x-Flag-siRNA resistant (siR) WT Rev1 or 3x-Flag-siR D570A, E571A (AA)
mutant Rev1 were treated with siRNA for 48 h. The efficiency of Rev1 depletion and of Rev1
expression were determined by western blot analysis with anti-Flag antibody. Actin was used as
the loading control.
Figure 4.
Deoxynucleotide incorporation
opposite undamaged G and (r) -
DNA substrate (10 nM) and 10 M each
HOPdG by Pol. Pol (1 nM) was incubated with
of a single deoxynucleotide dATP, dTTP, dGTP, or dCTP, or all four deoxynucleotides (N) for 10
o
min at 37 C. DNA substrate is shown above the gel where denotes an undamaged G or an (r) -
HOPdG template base.
2
Figure 5. Synthesis of (r) -HOPdG phosphoramidite. Synthesis of N --hydroxy-propano-
2
’-deoxyguanosine [(r)-HOPdG] phosphoramidite comprised six steps beginning with protection
1
2
of the N -N positions of deoxyguanosine [1] with bromoacetone to give 2’-deoxy-4-
desmethylwyosine [2]. Alkylation of 2 with (3-bromopropoxy)-t-butyldimethylsilyl ether gave 3-
(t-butyldimethylsilyloxy)propyl-2’-deoxy-4-desmethylwyosine [3]. Protection of the 5’-hydroxyl
group of 3 using dimethoxytrityl chloride gave 5’-DMT-(3-(t-butyldimethylsilyloxy)propyl)-2’-
1
2
deoxy-4-desmethylwyosine [4]. Deprotection of the N -N positions of 4 gave 5’-DMT-(3(t-
butyldimethylsilyloxy)propyl)-2’-deoxyguanosine [5]. Phosphoramidite [6] was prepared using
the bis-N,N,N’N’-diisopropyl--cyanoethyl phosphitylation method.
2
0

OH
A
B
O
H
N
N1 6
5
4
7
2
8
3
N
9
N
N
H
reduced γ-HOPdG
2
(
N -(-3-hydroxypropyl)-2’-deoxyguanosine
*
5
’-GGA AGC AAT G GTACGG-3’
Spe I (+1 nt)
BamH I
Sbf I
TA
C
G
-
5
’- ATG ACC ATG ATT ACG GGA TCC CGT A
TT GCT TCC TGC AGG TCA CTG
Kan
Kan
+
3’-TAC TGG TAC TAA TGC CCT AGG GCA TG G T AA CGA AGG ACG TCC AGT GAC
*
M
T
M
I
T
G
S
L
T
N
A
S
C
R
S
L
Leading strand (pBS vector)
Figure 1

(r) γ-HOPdG
Inserter Pol Rev1
ι
θ
Extender Pol
η
κ
θ
Mutagenicity of TLS
Predominantly error-free
Figure 2

A
B
siR-Rev1
WT AA
WT-Rev1
_
+
Rev1 siRNA
Rev1 siRNA
+
+
kDa
kDa
1
50
150
3
x Flag-Rev1
3x Flag-Rev1
Actin
100
100
5
0
7
50
37
Actin
3
Figure 3

dNTP
primer
44 nt 5’
template 75 nt 3’
GGCAT
CCGTAGTA
5’
*
G
(r) γ-HOPdG
primer
dNTPs
-
A
T
G
C
N
-
A
T G C N
Figure 4

Figure 5

Genetic control of predominantly error-free replication through an acrolein-derived
minor-groove DNA adduct
Jung-Hoon Yoon, Richard P. Hodge, Linda C. Hackfeld, Jeseong Park, Jayati Roy
Choudhury, Satya Prakash and Louise Prakash
J. Biol. Chem. published online January 12, 2018
Access the most updated version of this article at doi: 10.1074/jbc.RA117.000962
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