High-efficiency bypass of DNA damage by

The EMBO Journal (2004) 23, 4484–4494
www.embojournal.org
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2004 European Molecular Biology Organization | All Rights Reserved 0261-4189/04
THE
EMBO
JOURNAL
High-efficiency bypass of DNA damage
by human DNA polymerase Q
Mineaki Seki1, Chikahide Masutani2, Lee
Wei Yang3, Anthony Schuffert1, Shigenori
Iwai4, Ivet Bahar3 and Richard D Wood1,*
1
University of Pittsburgh Cancer Institute, Hillman Cancer Center,
Research Pavilion, Pittsburgh, PA, USA, 2Graduate School of Frontier
Biosciences, Osaka University and CREST, Japan Science and
Technology Corporation, Suita, Osaka, Japan, 3Department of Molecular
Genetics & Biochemistry, Center for Computational Biology and
Bioinformatics, School of Medicine, University of Pittsburgh, Pittsburgh,
PA, USA and 4Graduate School of Engineering Science, Osaka University,
Toyonaka, Osaka, Japan
Endogenous DNA damage arises frequently, particularly
apurinic (AP) sites. These must be dealt with by cells in
order to avoid genotoxic effects. DNA polymerase h is a
newly identified enzyme encoded by the human POLQ
gene. We find that POLQ has an exceptional ability to
bypass an AP site, inserting A with 22% of the efficiency of
a normal template, and continuing extension as avidly as
with a normally paired base. POLQ preferentially incorporates A opposite an AP site and strongly disfavors C. On
nondamaged templates, POLQ makes frequent errors, incorporating G or T opposite T about 1% of the time. This
very low fidelity distinguishes POLQ from other A-family
polymerases. POLQ has three sequence insertions between
conserved motifs in its catalytic site. One insert of B22
residues into the tip of the polymerase thumb subdomain
is predicted to confer considerable flexibility and additional DNA contacts to affect enzyme fidelity. POLQ is the
only known enzyme that efficiently carries out both the
insertion and extension steps for bypass of AP sites,
commonly formed as endogenous genomic lesions.
The EMBO Journal (2004) 23, 4484–4494. doi:10.1038/
sj.emboj.7600424; Published online 21 October 2004
Subject Categories: proteins; genome stability & dynamics
Keywords: AP site; DNA damage; DNA polymerase; POLQ;
thymine glycol
Introduction
Mammalian cells have about 15 distinct DNA polymerases,
but the functions of only a few of them are well defined
(Goodman, 2002; Hübscher et al, 2002). Some of these DNA
polymerases help cells tolerate endogenous DNA damage.
Such enzymes are of particular importance, because DNA
damage can block progression of replication forks. About
10 000 apurinic (AP) sites arise per cell per day through
*Corresponding author. University of Pittsburgh Cancer Institute,
Hillman Cancer Center, Research Pavilion, Suite 2.6, 5117 Centre
Avenue, Pittsburgh, PA 15213, USA. Tel.: þ 1 412 623 7762;
Fax: þ 1 412 623 7761; E-mail: [email protected]
Received: 17 June 2004; accepted: 27 August 2004; published online:
21 October 2004
4484 The EMBO Journal VOL 23 | NO 22 | 2004
endogenous hydrolytic loss of purine bases (Lindahl, 1993;
Nakamura et al, 1998). Many further AP sites arise as
intermediates during removal by base excision repair of
dUTP incorporated into DNA (Guillet and Boiteux, 2003).
Enzymes that could efficiently bypass AP sites encountered
during DNA replication could be valuable in promoting
cellular survival and minimizing chromosomal breakage.
DNA polymerases are divided into families denoted A,
B, X, and Y based on amino-acid sequence relationships.
Members of the A-family have a wide distribution in nature.
The most extensively studied A-family polymerase,
Escherichia coli pol I, is a high-fidelity enzyme that assists
in maturation of Okazaki fragments during DNA replication
and also participates in gap-filling during base excision
repair, nucleotide excision repair, and repair of DNA interstrand crosslinks. We recently identified the DNA polymerase
activity of an A-family enzyme found in mammalian cells,
POLQ (Seki et al, 2003). There are no yeast homologs, but a
POLQ homolog in Drosophila nuclei called Mus308 apparently functions in a pathway of DNA crosslink repair or
tolerance. This is suggested because fly mus308 mutants
are sensitive to DNA crosslinking agents (Harris et al,
1996). POLQ and Mus308 have a characteristic three-domain
organization with a helicase-like domain in the N-terminal
portion, a less conserved central domain, which is largely
derived from one large exon, and a C-terminal A-family
polymerase domain (Seki et al, 2003). Mouse Polq was
recently proposed as a candidate gene defective in the
chaos1 mouse mutant, which displays chromosome segregation abnormalities and radiation sensitivity in reticulocytes
(Shima et al, 2003). The identity of Polq and chaos1 has been
confirmed by direct disruption of Polq in the mouse and by
correction of the phenotype with the Polq gene (Shima et al,
2004).
To understand the functions of human POLQ, we have
explored its fidelity in DNA synthesis and ability to perform
translesion synthesis when encountering DNA lesions.
Although residues in the polymerase catalytic site are highly
homologous to high-fidelity A-family DNA polymerases, we
report here that POLQ has extremely low fidelity, comparable
to some Y-family polymerases. Moreover, POLQ can perform
translesion DNA synthesis at an AP site and a thymine glycol
(Tg). Unusually, POLQ both inserts a base opposite an AP site
and efficiently extends the misincorporated nucleotide, making it the most efficient known polymerase for AP-site bypass.
Results
Low fidelity of POLQ
These experiments utilized full-length recombinant POLQ, a
2590-amino-acid protein including both the DNA polymerase
and helicase-like domains (Figure 1A). As POLQ is an
A-family polymerase, it was anticipated that it would perform
relatively accurate DNA synthesis. Fidelity was analyzed on
a 30-mer template with a 16-mer DNA primer (Figure 1B).
& 2004 European Molecular Biology Organization
Bypass of damage by low-fidelity human POLQ
M Seki et al
A
1
kDa
2
B
POLQ
250
5´-[32P]-CACTGACTGTATGATG
GTGACTGACATACTACXTCTACGACTGCTC
160
105
75
50
Lane
Template
C
1
2
3
4
5
6
7
8
9
10
11 12
A
A
A
A
T
T
T
T
C
C
C
C
T
T
T
T
T
A
G
C
dNTP
: A
incorporated
C
G
X=T
T
A
C
G
X=A
T
A
C
G
X=G
T
13
14
15
16
A
C
G
T
X=C
Figure 1 POLQ is an error-prone enzyme. (A) Full-length recombinant human POLQ was purified as described and 75 ng was electrophoresed
on an SDS–polyacrylamide gel and silver-stained (lane 2). Molecular weight markers are shown in lane 1. (B) Template used for the primer
extension assay. 50 -32P-labeled 16-mer was annealed to 30-mer DNA. The first template base is denoted by X. (C) POLQ frequently
misincorporates residues. The first template base (X) was changed from T (lanes 1–4) to A (lanes 5–8), G (lanes 9–12), and C (lanes 13–
16). Template bases are indicated on the left.
The results suggested an unexpected tendency of POLQ to
misincorporate bases and extend from mispaired termini.
There was frequent misincorporation when the first template
base was a T (Figure 1C, lanes 1–4). With dATP as the only
deoxynucleotide present, there was not only efficient incorporation of A opposite the first two T bases in the template,
but further extension by incorporation opposite noncomplementary bases (Figure 1C, lane 1). Incorporation of G and T
across from template T and further extension past noncomplementary bases was also observed (Figure 1C, lanes 3
and 4), but incorporation of C was not apparent (Figure 1C,
lane 2). When the first template base was changed to A, G, or
C, other misincorporation events took place (Figure 1C, lanes
5–16). With template C, only a low level of incorporation was
catalyzed by POLQ (Figure 1C, lanes 13–16), with the correct
nucleotide dGTP the best utilized and extended (Figure 1C,
lane 15).
These experiments indicated that POLQ catalyzes considerable misincorporation, dependent on sequence context.
This was surprising because most DNA polymerases with
very low fidelity are classified in the Y-family and include
human polymerases Z, i, k, and Rev1 (Goodman, 2002).
Consequently, we determined the fidelity of POLQ by making
kinetic measurements (Table I). As a control, the values for
human pol Z and exonuclease-deficient E. coli pol I Klenow
fragment (exo-free pol I Kf) were measured. For pol Z and pol
I, the Km and Vmax values for incorporation of dATP opposite
& 2004 European Molecular Biology Organization
template dT were similar to values previously reported
(Polesky et al, 1990; Kusumoto et al, 2002) and were similar,
within a few fold, to the corresponding values for human
POLQ (Table I). Measurements were obtained with other
incoming nucleotides, and from the ratio Vmax/Km
(Creighton et al, 1995), a misincorporation frequency (finc)
was calculated for POLQ. Compared to a value of 1.0 for
correct utilization of dATP opposite template T, these were
1.7 103 for dCTP, 2.0 102 for dGTP, and 7.5 103 for
dTTP (Table I). These frequencies are remarkably high for an
A-family polymerase, and about 10–100 times higher than the
misincorporation frequency of B105–104 found for exonuclease-defective E. coli pol I or Thermus aquaticus (Taq)
polymerase (Minnick et al, 1999). The misincorporation
frequencies are similar to those found for Y-family DNA
polymerases (Goodman, 2002).
One possible mechanism of misincorporation is primer–
template slippage, as has been observed for the Y-family
enzyme DNA pol k (Ohashi et al, 2000a). In this mechanism,
a template base loops out and allows a second, complementary template base to align with incoming nucleotide (see
footnote to Table II). To examine this possibility, the first
template base was fixed as T and the second template base
was varied systematically. Each of the four incoming nucleotides was then tested separately. If a primer–template slippage mechanism is in operation, an incoming nucleotide
would be best ‘misincorporated’ if the second template base
The EMBO Journal
VOL 23 | NO 22 | 2004 4485
Bypass of damage by low-fidelity human POLQ
M Seki et al
Table I Fidelity of POLQ on normal DNA and at an AP site
DNA substrate
dNTP
Km (mM)
Vmax (nM/min)
Vmax/Km
finc
Insertion opposite T
50 -ATG
-TACTTC
dATP
dCTP
dGTP
dTTP
7.071.4
2207110
110722
6607150
7.570.3
0.4270.13
2.470.1
5.571.7
1.1
1.9 103
2.2 102
8.3 103
1
1.7 103
2.0 102
7.5 103
Pol Z (insertion opposite T)
Exo-free pol I Kf (insertion opposite T)
dATP
dATP
4.470.2
3.170.9
3.470.7
1.770.2
0.77
0.55
Insertion opposite AP site (X)
50 -ATG
-TACXTC
dATP
dCTP
dGTP
dTTP
9.571.8
UDa
5273.0
510722
2.370.6
0.24
0.22
1.470.2
1.370.5
2.7 102
2.5 103
2.5 102
2.3 103
Extension with dATP from N
opposite AP site (X)
Base (N) opposite AP site:
Km (mM)
Vmax (nM/min)
Vmax/Km
fext
A
C
G
T
2.870.6
4170.5
3507140
8.271.6
3.270.4
1.570.1
0.470.1
2.970.8
1.1
3.7 102
1.1 103
0.35
1.0
3.4 102
1.0 103
0.32
50 -ATGN
-TACXTC
a
UD: undetectable.
were complementary. For example, with incoming dGTP,
apparent incorporation across from the first template T
would arise from slippage and pairing when the second
template base was C, and would be much more efficient
than if the second template base were A, G, or T. We found,
however, that misincorporation was not influenced by complementarity with the second template base (Table II). Total
extension with dGTP was B12% when the second template
base was C, but was similar or higher (11–21%) when the
second base was A, G, or T. An analogous result was obtained
with the other incoming dNTPs (Table II). It therefore
appeared that frequent misincorporation was due to low
insertion fidelity of the enzyme, not mediated by a slippage
mechanism.
POLQ efficiently bypasses an AP site and Tg in DNA
The error-prone DNA synthesis and the lack of 30 to 50
exonuclease proofreading activity (Seki et al, 2003) prompted
us to test the ability of POLQ to replicate past DNA adducts.
The bypass activity of POLQ was assessed using a cyclobutane pyrimidine dimer (CPD), a 6-4 photoproduct (6-4 PP), an
abasic (AP) site, 5R- and 5S-diastereoisomers of Tg, and the
major adduct produced by cis-diaminedichloroplatinum (II)
(cisplatin) (Figures 2 and 3). A primer with a 30 terminus
located just before the lesion was used. As controls, the
translesion activities of exo-free pol I Kf and pol Z were
also tested.
All three enzymes could extend the primer to the end of a
nondamaged 30-mer template (Figures 2A and 3A). There
were several notable features of POLQ extension on nondamaged DNA. POLQ extended 1 nt further than did pol Z or
exo-free pol I Kf, indicating that the enzyme can perform
efficient nontemplated addition at a blunt end in a way
similar to the A-family enzyme Taq polymerase (Clark et al,
1987; Clark, 1988). Second, many of the bands representing
extension with POLQ were doublets, particularly apparent
after extension past C25 (Figure 3A). Some bands resulting
from extension with pol Z were also doublets. This reflects
frequent misincorporation, giving rise to a mixture of DNA
4486 The EMBO Journal VOL 23 | NO 22 | 2004
Table II Primer/template slippage does not determine misincorporation by POLQ
Incoming dNTP
Second template base
% of primer extended
dGTP
A
C
G
T
11–16
9.2–14
11–15
15–21
dTTP
A
C
G
T
8.5–14
4.6–10
8.3–12
11–17
dCTP
A
C
G
T
1.4–2.1
1.4–1.5
2.1–2.5
1.0–1.4
dATP
A
C
G
T
26–31
24–25
27–29
36–38
To test whether primer/template slippage was involved in misincorporation, the second template base, denoted X below, was
varied. The fraction (%) of primer extended in two separate
experiments is shown in the table. The second template base
complementary to incoming deoxynucleotide is highlighted in
bold type.
5 0 -½32 P-CACTGACTGTATGATG
3 0 -GTGACTGACATACTACTXCTACGACTGCTC
As an example for incoming dGTP, where X ¼ C, only part of the
substrate is shown:
& 2004 European Molecular Biology Organization
Bypass of damage by low-fidelity human POLQ
M Seki et al
A
B
No damage (−dT)
N4
A
C
G
T
AP analog
N4
A
C
G
T
C
T
C
G
T
C
A
G
C
A
T
C
T
T
AP
Figure 2 Nucleotide preference for bypass of an AP site. A 50 -32Plabeled 16-mer primer was annealed to (A) 30-mer template DNA or
(B) a template containing an AP site. Either all four dNTPs (N4) or
each deoxynucleotide separately at 100 mM was utilized in reactions
as indicated.
fragments with slightly different mobilities. Third, in the
extension reaction with POLQ, several bands were more
intense than the rest (Figure 3A), and these occurred each
time the polymerase incorporated a base opposite template C
(positions 19, 22, 25, 28). This suggests that POLQ tends to
stall after incorporation at template C.
POLQ was able to bypass an AP site, by both inserting a
base opposite the site and extending from the inserted base to
the end of the template (Figures 2B and 3B). The efficiency of
AP site bypass was much higher than that of pol Z, which can
incorporate a deoxynucleotide opposite an AP site but stalls
and has only a weak ability to extend after incorporation
(Figure 3B) (Masutani et al, 2000; Haracska et al, 2001).
POLQ also bypassed an AP site when a shorter 14-mer primer
was used that allowed incorporation opposite two normal
template bases before the enzyme encountered the AP site
(not shown).
POLQ could also bypass Tg lesions in DNA, common
adducts produced by reactive oxygen species (Figure 3C
and D). As reported before, pol Z extended efficiently from
the 5R-Tg diastereoisomer (Figure 3C), and somewhat less
well from the 5S-Tg diastereoisomer (Figure 3D) (Kusumoto
et al, 2002). POLQ bypassed both types of Tg lesions with
similar efficiency. Exo-free pol I Kf could bypass 5S-Tg with
low efficiency but not 5R-Tg (Figure 3C and D). POLQ was
unable to insert deoxynucleotides opposite a UV-induced
CPD (Figure 3E). Human pol Z, on the other hand, is defined
by its ability to bypass this adduct. POLQ also did not insert a
deoxynucleotide opposite a 6-4 PP, although pol Z was able
to insert one dNTP opposite this adduct as reported previously (Figure 3F) (Masutani et al, 2000). Neither a 1,2d(GpG) adduct (Figure 3G) nor a 1,3-d(GpTpG) adduct (not
shown) was bypassed by POLQ.
As efficient AP-site bypass activity is a unique feature of
POLQ, we investigated the incorporation of deoxynucleotides
at an AP site and found that A was the most efficiently
& 2004 European Molecular Biology Organization
inserted and extended base (Figure 2B). The Km for incorporation of A opposite the AP site was 9.5 mM, close to the Km
for incorporation of A opposite template T (Table I). The
relative efficiency of incorporation (finc) of A against an AP
site was 22% of that of a nondamaged template. G and T were
also inserted, with 10- and 100-fold lower efficiency, respectively. Incorporation of C was too rare to be detected, even
with high concentrations of deoxynucleotide. POLQ was also
very efficient in extending a primer with a base opposite an
AP site (Table I). Remarkably, the Km and Vmax values for
POLQ extension from an A residue opposite an AP site were
within a few fold of the values for insertion of A opposite T
with normal base pairing (Table I). When the primer-terminal
base opposite the AP site was T, POLQ also worked well in
primer extension (fext ¼ 0.32). A primer-terminus with C
opposite the AP site was poorly extended on this template
(fext ¼ 0.034).
The Y-family enzyme DNA polymerase pol k can carry out
some bypass of an AP site via a misalignment mechanism,
resulting in a 1 deletion during extension and a shorter
DNA product than with nondamaged DNA (Ohashi et al,
2000b). In contrast, the bypass by POLQ took place directly
and not by misalignment, as shown by several observations.
First, the extension products formed by POLQ on a template
containing an AP site (or Tg) were of the same length as those
on a nondamaged template, extending to 31 nt (Figures 2
and 3). Second, POLQ could efficiently extend a T residue
opposite an AP site (fext ¼ 0.32) even when the next template
base was a T (Table I). This could not occur if extension
utilized misaligned pairing to the next template base as
occurs with pol k (Ohashi et al, 2000b). Finally, experiments
were performed on templates where the base following the
AP site was changed from T to G, A, or C (Figure 4).
Regardless of the sequence context, an A was also the bestincorporated residue opposite an AP site, a G the next best
incorporated, and a T or C incorporated very poorly or not at
all, consistent with the kinetic measurements in Table I. The
absence of a marked effect of sequence context shows that
misincorporation opposite the AP site takes place directly,
rather than by a misalignment mechanism involving pairing
with the template base immediately following the AP site.
Sequence inserts in the POLQ catalytic domain
Like other DNA polymerases, enzymes of the A-family have a
structure that can be envisioned as a clasping right hand with
palm, thumb, and finger domains. In the DNA polymerase
domain of POLQ, all six conserved motifs of A-family polymerases are present in the primary protein sequence, and are
well conserved with the same motifs in other A-family
members (Seki et al, 2003). Yet the other known A-family
polymerases are high-fidelity enzymes without significant
ability to bypass AP sites. How can the properties of POLQ
be rationalized with the presence of an A-family polymerase
catalytic site? A sequence alignment of POLQ/Mus308 family
polymerases with other A-family member polymerases reveals that POLQ from human and mouse cells has three extra
spans of sequence in the polymerase catalytic domain
(Figures 5A and 6B). One sequence of B22 amino acids
(designated Insert 1 in Figures 4 and 5) lies between Motifs 1
and 2. A longer sequence, designated Insert 2, is located
between Motifs 2 and 3. Insert 3, close to the C-terminus, is
located between Motifs 5 and 6.
The EMBO Journal
VOL 23 | NO 22 | 2004 4487
Bypass of damage by low-fidelity human POLQ
M Seki et al
A
B
No damage
Q
η
K
C
AP site
Q
η
K
D
5R -Tg
Q
5S-Tg
K
η
G
Cisplatin (GG)
Q
K
η
N31
C30
T29
C28
G27
T26
C25
A24
G23
C22
A21
T20
C19
T18
T17
CPD
E
Q
K
6-4 PP
F
η
Q
η
K
Q
K
η
Figure 3 POLQ bypasses AP sites and Tg. (A–G) A 50 -32P-labeled 16-mer was annealed with 30-mer template DNA containing lesions and
incubated with either human POLQ (Q), exonuclease-defective pol I Klenow fragment (K), or pol Z (Z). (A) Undamaged control, (B) AP analog,
(C) 5R-Tg, (D) 5S-Tg, (E) T–T CPD, (F) T–T 6-4 PP, and (G) 1,2-d(GpG) cisplatin adduct. The position of the adduct is indicated by an arrow (B–
D) or bracket (E–G).
Template
Lane
1
2
3
4
5
6
7
8
9
T
T
T
C
C
C
T
A
G
AP
AP
AP
10
11
12
13
14
15
16
A
C
G
T
T
C
dNTP supplied:
Template base
following AP site:
A
C
G
T
T
A
C
G
A
T
C
AP
A
C
G
T
G
C
0
32
Figure 4 Incorporation opposite an AP site takes place directly rather than by template slippage. A 5 -[ P]-labeled 16-mer was annealed to 30mer templates containing an AP site at the next template position as in Figures 2A and 3A. Four templates were used, having either T, A, G, or C
as the next template base following the AP site. Each of the four possible reaction mixtures containing only a single dNTP was tested. After a
10 min incubation at 371C, an A was preferentially incorporated opposite the AP site, regardless of the following sequence context.
4488 The EMBO Journal VOL 23 | NO 22 | 2004
& 2004 European Molecular Biology Organization
Bypass of damage by low-fidelity human POLQ
M Seki et al
Tip of thumb
Insert 1
HsPOLQ
MsPOLQ
Mus308
EcPolI
Haein
TaqPolI
Deinorad
Rhod
Trepon
Ricketts
BstPolI
Anaero
Myctu
Helico
hsPOLN
mmPOLN
CeMus1
V E MP SQYCL ALLE LN GI G F S T A E C E S Q K H IM Q A K LDAI E T Q AY Q L AG HS F S FTSSDD IA E VL FL E LK L PPNREMKNQGSKKTLGSTRRGIDNGRKLRLGRQFST SKDV L N KL KA..L HP L PG LILEW R RI TNAI TK
V E MP SQYCL ALLE LN GI G F S T A E C E S Q K H VM Q A K LDAI E T Q AY Q L AG HS F S FTSADD IA Q VL FL E LK L PPNGEMKTQGSKKTLGSTRRGNESGRRMRLGRQFST SKDI L N KL KG..L HP L PG LILEW R RI SNAI TK
I E MP IQLTL CQME LV GF P A Q K Q R LQ Q L Y Q RM V A V MKKV E T K IY E Q HG SR F N LG SSQA VA K VL GLH R........KAKG......................RVTT SRQV L E KL N . . . . SP I SH LILGY R KL SG LL A K
I E MP LVPVL SRIE RN GV KI DPKV LH N H S E EL T L R LAEL E K K A H E I AG EE F N LS ST KQ LQ T IL FE K QG I KPLKK......................TP.GGAPST SEEV L E EL A L . . D YP L PK VILEY R GL AK LK ST
M E LP LLHVL SRME RT GV LI DSDA LF M Q S N EI A S R LTAL E K Q AY A L AG QP F N LA ST KQ LQ E IL FD K LE L PVLQK......................TP.KGAPST NEEV L E EL SY..S HE L PK ILVKH R GL SK LK ST
V E RP LSAVL AHME AT GV RL DVAY LR A L S L EV A E E IARL E A E VF R L AG HP F N LN SR DQ LE R VL FD E LG L PAIGK......................TEKTGKRST SAAV L E AL RE..A HP I VEKILQY R EL TK LK ST
M E KP LSGVL GRME VR GV QV DSDF LQ T L S I Q A G V R LADL E S Q I H E YAG EE F H IR SP KQ LE T VL YD K LE L ASSKK......................TKLTGQRST AVSA L E PL RD..A HP I IP LVLEF R EL DK LR GT
M E FP LIEVL ADME RT GI CI DRAV LR E I G K QL E A E LHEL E V K IY E V AG VE F N IG SP QQ LA D VL FK K LG L KPRAR......................TS.TGRPST KESV L Q EL AT..Q HP L PG LILDW R HL AK LK ST
I E MP LLPIL ARME EV GI FL RKDV VQ Q L T R S F S D L IQQYE H D IF S L AG HE F N IG SP KQ LQ T VL FQ E LH L PPGK........................KNTQGYST DHSV L K KL AR..K HP I AEKILLF R DL SK LR ST
I D LP TCFIL DKME KV GI KV DANY LN R L S D E F G T E ILKI E E E IF A L SG TK F N IG SQ KQ LG E IL FK K MQ L PSGNT......................LAKTSSYST RAGI L K KL SED.GYH I AT LLLRW R QL TK LK NT
L E QP LAGIL ANME FT GV KV DTKR LE Q M G A EL T E Q LQAV E R R IY E L AG QE F N IN SP KQ LG T VL FD K LQ L PVLK........................KTKTGYST SADV L E KL AP..H HE I VEHILHY R QL GK LQ ST
I E RP LIPVL YEME KT GF KV DRDA LI Q Y T K EI E N K ILKL E T Q IY Q I AG EW F N IN SP KQ LS Y IL FE K LK L PVIK........................KTKTGYST DAEV L E EL FD..K HE I VP LILDY R M Y TK IL TT
M E LP VQRVL AKME SA GI AV DLPM LT E L Q S Q F G D Q IR D A A E A AY G V IG K Q IN LG SP KQ LQ V VL FD E LG M PKTK........................RTKTGYTT DADA L Q SL FDKTG HP F L Q HLLAH R DV TR LK VT
I E TP FMKVL MGME FQ GF KI DA P Y F K R L E Q E F K N E LHVL E R Q I L E L IG VD F N LN SP KQ LS E VL YD K LG L PKNK............................SHST DEKS L L KI LD..K HP SIA LILEY R EL NK LF NT
L E LP LIPIL AVME SH AI QV NKEE ME K T S A LL G A R LKEL E Q E A H F V AG ER F L IT SN NQ LR E IL FG K LK L HLLSQRNSLP................RTGLQKYPST SEAV L N AL RD..L HP L PK IILEY R QV HK IK ST
L E LP LIPIL AVME N H KI PV DKEE ME R T S A LL G A R LKEL E Q E A H F V AG EQ F L IM SN NQ LR E IL FG K LK L HLLSQRKHLP................RTGLQNQLST SEAM L N SL QD..L HP L PK LILEY R QV HK IK ST
L E MESCQTV LNIFY S GI V F DQ A L C N S F I Y KI R K Q IENL E E N IW R L AY G KF N IH SS NE VA N VL FY R LG L IYPETSG...................CKPKLRHLPT NKLI L E QM NT..Q HP I VGKILEY R QI QH TL TQ
HsPOLQ
MsPOLQ
Mus308
EcPolI
Haein
TaqPolI
Deinorad
Rhod
Trepon
Ricketts
BstPolI
Anaero
Myctu
Helico
hsPOLN
mmPOLN
CeMus1
1
Insert 2
V V FP LQREKCLNPF L G M E R I Y P V SQ S HT A T G RIT FTEP N I QNV P R D F E I K M PTLVGESPPSQAVGKGLLPMGRGKYKKGFSVNPRCQAQMEERAADRGMPFSISMRHA F V PFP.G GS IL A AD YSQLE L R I L A H L SH
V V FP LQREKHLNPL L R M E R I Y P V SQ S HT A T G RIT FTEP N I QNV P R D F E I K M PTLVRESPPSQAP.KGRFPMAIGQDKKVYGLHPGHRTQMEEKASDRGVPFSVSMRHA F V PFP.G GL IL A AD YSQLE L R I L A H L SR
S I QP LME......C C QA D R I H G Q S IT YT A T G RIS MTEP N L QNV A K E F S I Q V G SDVVH.............................................ISCRSPF M PTDESRCLL S AD FCQLE M RIL A HM SQ
Y T DK LPLMI NP.. . K TG R V HT SY HQ A VT A T G RL S STDP N L QN I PV RN E EG R RI R....................................................QA F I A.PEDYVIV S AD YSQIE L R I MA H L SR
Y T DK LPQMV NS.. . Q TG R V HT SY HQ A VT A T G RL S SSDP N L QN I PI RN E EG R HI R....................................................QA F I A.REG YS IV A AD YSQIE L R I MA H L SG
Y I DP LPDLI HP.. . R TG R L HT RF NQ T AT A T G RL S SSDP N L QN I PV RT P LG Q RI R....................................................RA F I A.EEG WL LV A LD YSQIE L RV L A H L SG
Y L DP IPNLV NP.. . H TG R L HT TF AQ T AV A T G RL S SLNP N L QN I PI RS E LG R EI R....................................................KG F I A.EDG FT LI A AD YSQIE L RL L A HI AD
Y V DG LEPLI HP.. . E TG R I HT TF NQ T VT A T G RL S SSNP N L QN I PV RT E MG R EI R....................................................RA F V P.RPG WK LL S AD YVQIE LRIL A AL SG
Y T ES LAKLAD.... Q TG R V HT SF VQ I GT A T G RL S SRNP N L QN I PI KS T EG R KI R....................................................QA F QA.TVG HE LI S AD YTQIE L VV L A H L SQ
Y T DS LPKQI NN.. . I T KR I HT TF LQ T ST T T G RL S SQEP N L QNV P I RS S DG N KI R....................................................EA F I A.EEG YK LI S AD YSQIE L R I LS HI AN
Y I EG LLKVV HP.. . V TG K V HT MF NQ A LT Q T G RL S SVEP N L QN I PI RL E EG R KI R....................................................QA F V PSEPDWLIF A AD YSQIE L RV L A HI AE
Y CQG LLQAI NP.. . S SG R V HT TF IQ T GT A T G RLA SSDP N L QN I PV KY D EG K LI R....................................................KVF V P.EGG HV LI D AD YSQIE L R I L A HI SE
. V DG LLQAV A... . A DG R I HT TF NQ T IA A T G RL S STEP N L QN I PI RT D AG R RI R....................................................DA F V VGDGYAELM T AD YSQIE M RI MA H L SG
Y TTP LLRLKD.... K D DK I HT TF IQ T GT A T G RL S SHSP N L QN I PV RS P KG L LI R....................................................KG F I ASSKEYCLL G VD YSQIE L RL L A HF SQ
F V DG LLACM K... . . KG SI SSTW NQ T GT V T G RLS AKHP N I QGI S K HP I Q I T T P KNFKGKEDKIL......................................TISPRA MFVSSKG HTFL A AD FSQIE L R I LT H L SG
F I DG LLAYM K... . . KG SI SSTW NQ T GT V T G RLS AKHP N I QGI S K HP I K I S K PWNFKGKEEETV......................................TISPRTLFVSSEG HTFL A AD FSQIE L R I L A H L SG
C L MP LAKFI GR.. . . . . . . I H CW FE M CT S T G RIL TSVP N L QNV P K RI S S D G M SA....................................................RQLFIANSENLLI G AD YKQLE L RV L A H L SN
HsPOLQ
MsPOLQ
Mus308
EcPolI
Haein
TaqPolI
Deinorad
Rhod
Trepon
Ricketts
BstPolI
Anaero
Myctu
Helico
hsPOLN
mmPOLN
CeMus1
HsPOLQ
MsPOLQ
Mus308
EcPolI
Haein
TaqPolI
Deinorad
Rhod
Trepon
Ricketts
BstPolI
Anaero
Myctu
Helico
hsPOLN
mmPOLN
CeMus1
2
3
DRRL IQVLNTGA..DVF R S IAA E W K M IE P ES V G D D LR QQ A K QI C YG II Y G MGA K S L GEQ MG IK E ND AA C Y I DS F KSRY TGI NQ FM TET V KNCK R DG FV Q T I L G RRRYL P GI K D NN PYR KAHA E R Q AIN T I VQG S A A
DCRL IQVLNTGA..DVF R S IAA E W K M IE P DA V G D D LR QH A K QI C YG II Y G MGA K S L GEQ MG IK E ND AA S Y I DS F KSRY KGI NH FM RDT V KNCR K NG FV E T I L G RRRYL P GI K D DN PYH KAHA E R Q AIN T T VQG S A A
DKAL LEVMKSSQ..DLF I A IAA H W N K IE E S E V T Q D LR N S TK QV C YG IV Y G MG M R SL AES LN CS E Q E AR M I SDQ F HQAY KGI RD YTTRV V NFAR S KG FV E T I T G RRRYL E NI NSDVEHL KNQA E R Q AVN S T IQG S A A
DKGL LTAFAEGK. .DIHR A TAA E V F G LP L ET V T S E QR RS A K AI N FG LI Y G MSA F G L ARQ LN IP R KE AQ K Y M DL Y FE RY PGV LE YM ERTRAQAK E QG YV E T L D G RRLYL P DI K S SN GAR RAAA E R A AIN A P MQG T AA
DQGL INAFSQGK. .DIHR S TAA E I F G VS L DE V T S E QR RN A K AI N FG LI Y G MSA F G L SRQ LG IS R A D AQ K Y M DL Y FQ RY PSV QQ FM TDIREKAK A QG YV E T L F G RRLYL P DI N S SN AMR RKGA E R V AIN A P MQG T AA
DENL IRVFQEGR. .DIHT E TAS W M F G VP R EA V D P L MR RA A K TI N FG VL Y G MSA H R L SQE LA IP Y EE AQ A F I ER Y FQ SF PKV RA WI EKT L EEGR R RG YV E T L F G RRRYV P DL EARVKSV REAA E R M AFN M P VQG T AA
DPLM QQAFVEGA. .DIHR R TAA Q V L G LD E A T V D A N QR RA A K TV N FG VL Y G MSA H R L SND LG IP Y AE AA T F I EI Y FA TY PGI RR YI NHT L DFGR T HG YV E T L Y G RRRYV P GL S S RN RVQ REAEE R L AYN M P IQG T AA
DEAL RRAFLEGQ. .DIHT A TAA R V F K VP P EQ V T P E QR RR A K MV N YG IP Y G ISA W G L AQR LR CS T R E AQ E LI EE Y QRAF PGV TR YL HRV V EEAR Q KG YV E T L L G RRRYV P NI N S RN RAE RSMA E R I AVN M P IQG T QA
DRNL LNAFRQHI. .DIHA L TAA Y I F N VS I DD V Q P A MR RI A K TI N FG IV Y G MSA F R L SDE LK IS Q K E AQ S F I YR Y FE TY PGV YA FSTQVAEQTR K TG YV T S L A G RRRYI R TI D S RN TLE RARA E R M ALN T Q IQS S A A
IDVL KQAFINKE. .DIHT Q TAC Q I F N LK K H E L T S E HR RK A K AI N FG II Y G ISA F G L AKQ LN VSN S TAS E Y I KQ Y FA EY KGV QE YM TQTKACASRNG YV T N F FG RKCFI P LI HDKK..L KQFA E R A AIN A P IQG T NA
DDNL IEAFRRGL. .DIHT K TAM D I F H VS E ED V T A N MR RQ A K AV N FG IV Y G IS D Y GL AQN LN IT R K E AA E F I ER Y FA SF PGV KQ YM DNI V QEAK Q KG YV T T L L H RRRYL P DI T S RN FNV RSFA E R T AMN T P IQG S A A
DERL ISAFKNNV. .DIHS Q TAA E V F G VD I A D V T P E MR SQ A K AV N FG IV Y G IS D Y GL ARD IK IS R K E AA E F I NK Y FE RY PKV KE YL DNT V KFAR D NG FV L T L F N RKRYI K DI K S TN RNL RGYA E R I AMN S P IQG S PA
DEGL IEAFNTGE. .DLHS F VAS R A F G VP I DE V T G E LR R R VK AM S YG LA Y G LSA Y G L SQQ LK IS T E E AN E QM DA Y FA RF GGV RD YL RAV V ERAR K DG YT S T V L G RRRYL P EL D S SN RQV REAA E R A ALN A P IQG S A A
DKDL MEAFLKGR. .DIHL E TSK A L F G . . . EY L A K E KR SI A K SI N FG LV Y G MGS K K L SET LN IS L N E AK S Y I EA Y FK RF PSI KD YL NRMKEEILKTSKAFT L L G RYR.V F D F T G AN DYV KGNYLR E GVN A I FQG S AS
DPEL LKLFQESER DDVF S T L T S Q W K D VP V EQ V T H A DR E Q TK KV V YAVV Y G A G KERL AAC LG VP I QE AA Q F L ES F LQKY KKI KD FARAA I AQCH Q TG CV V S I M G RRRPL P RI H A HD QQL RAQA E R Q AVN F V VQG S A A
DPEL LKLFQESER DDVF S T L T S Q W K D IP I ER V T H M DR E Q TK KV V YS VV Y G A G KERL AAC LG VTV L E AT H F L ER F LQKY KKI KD FAQTV I GQCH S AG YV T S I L G RRRPL P RI C A QD QQL RAQA E R Q AVN F V VQG S A A
DSNL VNLITSDR..DLF E E LSI Q W N F P . . . . . . . . .R D A VK QL C YG LI Y G MGA K S L SELTRMSI E D AE K ML KA F FA MF PGV RS YI NETKEKVCKEEPI S T I I G RRTII KAS..GIGEE RARIE R V AVN Y T IQG S AS
4
6
Insert 3
* * **
DIVK IATVN IQ K QLE T F H S T F K S H G H R E G M L Q S D R T G L S R K R K L Q G M F C P I R GGFFILQ LHDEL L Y EV V E E D V V QV AQ IVK NE ME SAVKL S.......V K L K V KVKIG A SW GE LK DFDV.....
DIVK IATVN IQ K QLE T F R S T F K S H G H R E S M L Q N D R T G L L P K R K L K G M F C P M R GGFFILQ LHDEL L Y EV A E E D V V QV AQ IVK NE ME CAIKL S.......V K L K V KVKIG A SW GE LK DFDV.....
DIAK NAILK ME K NIE R Y R . . . . . . . . . E K L A L G D N S . . . . . . . . . . . . . . . . .VDLVMH LHDEL I F EV P T G K A K KI AK VLSLT ME NCVKL S.......V P LK V KLRIG R SW GE FK EVSV.....
DIIK RAMIA VD A WLQ A E Q . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P RVRMIMQ VHDEL V F EV H K D D V D AV A K QIH QL ME NCTRL D.......V P LL V EVGSG E NW D Q AH .........
DIIK RAMIK LD E VIR H D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P DIEMIMQ VHDEL V F EV R S E K V A F F R E QIK QH ME AAAEL V.......V P LI V EVGVG Q NW DE AH .........
DLMK LAMVK LF P RLE E M G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .AR M LLQ VHDEL V L EA P K E R A E AV AR LAK EV ME GVYPL A.......V P LE V EVGIG E DW L S AK E........
DIMK LAMVQ LD P QLD A I G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .AR M LLQ VHDEL L I EA P L D K A E QV AA LTK KV ME NVVQL K.......V P LA V EVGTG P NW FD TK .........
DMIK LAMVH IY H RLK R E G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Y RAK M LLQ VHDEL V F EM P P E E V E PV RQ LVEQE MKQALP L EG......V P I E V DIGVG D NW LD AH .........
DIVK IAMIA IQRAF A R R P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L RAQL LLQ VHDEL I F EA P A A E T A IV KE ILFAE ME HAVEL S.......I P L R I HVESG N SW GD FH .........
DIIK IAMIK LD K EIE E R K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L KTR L ILQ IHDEL L F EV P E I E V E IV IP IIK KI ME HSTNM N.......V P I I T EIKAG N NW KE IH .........
DIIK KAMID LS V RLR E E R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L QAR L LLQ VHDEL I L EA P K E E I E RL CR LVPEV ME QAVAL R.......V P LK V DYHYG P TW YD AK .........
DIMK LAMIK VY Q KLK E N N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L KSK I ILQ VHDEL L I EA P Y E E K D IV KE IVK RE ME NAVRL K.......V P LV V EVKEG L NW YE TK .........
DIIK VAMIQ VD K ALN E A Q . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L ASR M LLQ VHDEL L F EI A P G E R E RV EA LVR DK MGGAYP L D.......V P LE V SVGYG R SW DAAAH........
DLLK LGMLK VSERF K N N P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SVR L LLQ VHDEL I F EI E E K N A P EL Q Q EIQRI LN DEVYPLR......V P LETSAFIA K RW NE LK G........
DLCK LAMIH VF T AVA A S H T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L TAR L VAQ IHDEL L F EV EDPQIPECAA LVR RT ME SLEQV QALELQLQV P LK V SLSAG R SW GHLVPLQEAWGPP
DLCK LAMIR IS T AVA T S P T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L TAR L VAQ IHDEL L F EV EDTQVPEFAA LVR RI ME SLQQV QTLELQLQV P LK V NLSVG R SW GHLTPLQEILGSA
EIFK TAIVD IE S KIK E F G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .AQI VLT IHDEV L V EC P E I H V A A A S E SIENC MQ NALSHLLR.....V P M R V SMKTG R SW AD LK .........
5
Figure 5 Sequence insertions in the catalytic site of POLQ. Sequence alignment of the polymerase catalytic region of POLQ/Mus308 family
members and other prokaryotic A-family polymerases is shown. Identical amino acids are indicated with dark shading and similar amino acids
with light shading. The alignment was carried out using the Clustal X program. The locations of the inserts and the conserved DNA polymerase
motifs 1–6 are indicated. A putative PCNA binding motif is denoted by asterisks. EcPolI: E. coli pol I; TaqPolI: Thermus aquaticus pol I; BstPolI:
Bacillus stearothermophilus pol I; Anaero: Anaerocellum thermophilum pol I; Ricketts: Rickettsia prowazekii pol I; Haein: Haemophilus
influenzae pol I; Trepon: Treponema pallidum pol I; Deinorad: Deinococcus radiodurans pol I; Rhod: Rhodothermus marinus pol I; Myctu:
Mycobacterium tuberculosis pol I; Helico: Helicobacter pylori pol I; HsPOLQ: human POLQ; MsPOLQ: mouse POLQ; hsPOLN: human POLN;
mmPOLN: mouse POLN; Mus308: Drosophila melanogaster Mus308; CeMus1: Caenorhabditis elegans Mus1.
To gain some idea of the location of these inserts, an
energy-minimized structural model for the POLQ–DNA complex was obtained, using as modeling template the closed
form of Taq pol I bound to a 12-mer replicating DNA (Figure
6B and C). To model inserts, the Molecular Operating
Environment package utilizes a library of pentapeptide rotamers and sequence-specific structural motifs extracted from
the Protein Data Bank (PDB). The underlying method is a
combination of the segment-matching procedure of Levitt
(1992) and modeling of insertions and deletions according
to Fechteler et al (1995). The approach is to combine available structural motifs for generating the possible conformations of groups of residues in insert regions and to select the
conformers that minimize the intra- and intermolecular energy in the context of other segments of the protein and
substrate (double-stranded DNA and ddTTP in the case of the
Taq structure). The resulting model is not meant to be
interpreted as a detailed atomic structure, but it does provide
an indication of the location of the inserts in the protein and
their potential to interact with DNA.
& 2004 European Molecular Biology Organization
Insert 1 is located at the tip of the thumb subdomain. This
area of other A-family polymerases is highly flexible and
known to interact with the duplex template–primer (Beese
et al, 1993). The thumb appears to influence DNA binding,
processivity, and frameshift fidelity by tracking the minor
groove of the duplex (Minnick et al, 1996; Doublie et al, 1998;
Cannistraro and Taylor, 2004). Deletion of this region in
E. coli pol I causes significant reduction of DNA binding,
reduction of processivity, and enhanced usage of misaligned
primer/template (Minnick et al, 1996). The extended Insert 1
in POLQ is predicted to make close contacts with the major
groove of double-stranded DNA, adjacent to the nucleotide
incorporation site. One factor contributing to this prediction
is that within the 36 amino acids in the loop at the tip of the
thumb domain including Insert 1, there are six Arg and five
Lys residues, which favor interaction with the negatively
charged DNA backbone. The remaining 25 amino acids
contain 14 nonpolar residues, which may favor docking
into the major groove of DNA. Several residues in this
model of Insert 1 are close enough to form hydrogen bonds
The EMBO Journal
VOL 23 | NO 22 | 2004 4489
Bypass of damage by low-fidelity human POLQ
M Seki et al
A
2078
1
Insert 1
2
Insert 2
3(A)
4(B)
6
Insert 3 5(C)
2592
POLQ
427
865
POLN
2059
1653
Mus308
433
832
Taq pol I
Tip of thumb
Thumb
Structure
B
POLQ
Palm
Fingers
Palm
C
Taq pol I
Insert 3
Insert 2
Palm
Fingers
Insert 1
Thumb
Figure 6 Sequence insertions in the catalytic site of POLQ. (A) The DNA polymerase catalytic region of POLQ is shown in relation to the other
A-family polymerases human POLN, Drosophila Mus308, and Taq pol I. The locations of the inserts are shown in red and the conserved DNA
polymerase motifs 1–6 are indicated as gray regions. Nonconserved differences in the insert regions are indicated in green. The bottom line
shows coding regions for the main finger, palm, and thumb structural domains. (B, C) A computationally derived model for the polymerase
domain of POLQ–DNA (B) obtained with the Amber94 potential, as compared to the known structure of the closed form of Taq pol I (C). The
polymerase domain of Taq pol I (1QTM) is shown in white, while the modeled closed structure of human POLQ is colored blue to red, in the
increasing order of r.m.s. deviation from Taq pol I. In both diagrams, the DNA template strand is green, and the primer strand is red. Gray
spheres show the location of two Mg2 þ cations, and an incoming ddTTP is positioned nearby in the structure. Inserts 1–3 of POLQ are shown
in a ball-and-stick mode. The model is not meant to propose a detailed structure for the inserts, but instead to provide insight into the potential
of Insert 1 to make contact with template DNA.
with the sugar–phosphate backbone. All three inserts in the
model consist largely of coil, with a short helical region likely
in Insert 2. Regardless of their detailed conformation, the
residues of Inserts 2 and 3 seem likely to be located beyond
the range of probable interaction with DNA (Figure 6B and
C). They may serve, for example, as parts of interaction
domains with other proteins.
Discussion
POLQ has unusual properties of fidelity and ability
to bypass DNA damage
Although several human DNA polymerases in the Y-family
(pol Z, pol i, and REV1) have some capacity to incorporate
a base opposite an AP site, they cannot efficiently extend
4490 The EMBO Journal VOL 23 | NO 22 | 2004
the primer-terminus after doing so (Johnson et al, 2000;
Masutani et al, 2000; Zhang et al, 2001; Lawrence, 2002;
Vaisman et al, 2002). This has prompted the prevailing model
that a multistep reaction is necessary for efficient bypass,
where a switch to a second DNA polymerase, such as pol z, is
required to extend the primer after insertion (Johnson et al,
2000). Yeast pol z indeed extends primer ends opposite AP
sites, but with very low efficiency (Johnson et al, 2000).
However, POLQ is unusual among all known DNA polymerases in its ability to efficiently carry out both the insertion
and extension reactions of AP site bypass, so that a two-step
mechanism is unnecessary. The biochemical features of
POLQ result in preferential incorporation of A during APsite bypass (Table III). A is the most efficiently incorporated
base opposite an AP site and a primer ending with an A
& 2004 European Molecular Biology Organization
Bypass of damage by low-fidelity human POLQ
M Seki et al
Table III POLQ AP-site bypass and extension efficiency
dNTP
Insertion
Extension
Relative finc
dATP
dCTP
dGTP
dTTP
0.22
UD
2.5 102
2.3 103
1.0
3.4 102
3.7 104
0.32
0.22
—
9.3 106
7.4 104
The relative finc value was calculated as the product of the insertion
and extension values from Table 2. For POLQ, this calculation is
meaningful because the efficiency of extension from an incorporated A opposite an AP site is the same as extension of A opposite an
undamaged template. UD: undetectable.
residue opposite an AP site is the best primer extended by
POLQ. It is interesting that POLQ is extremely poor at
incorporating dCTP opposite an AP site, completely opposite
to the preferred reaction of REV1 at an AP site (Lawrence,
2002). Moreover, POLQ poorly inserts C opposite an AP site
and extends a primer end having C opposite such a site. This
might be termed an ‘Anti-C rule’ (Table III) and could ensure
that POLQ works for bypass of AP sites in situations quite
separate from those where REV1 may be employed.
Maga et al (2002) partially purified a DNA polymerase
activity from HeLa cell nuclear extracts, provisionally identifying it as pol y. This was based on reactivity of one of several
bands of o100 kDa in the preparation with an antibody
raised against a fragment of pol y. The fidelity of DNA
synthesis performed by this enzyme preparation was high
and a 30 –50 exonuclease activity was present (Maga et al,
2002). The features of this activity are quite different from
human POLQ, which is an B250 kDa protein (Seki et al,
2003; Shima et al, 2003) with low fidelity (this study) and no
exonuclease activity (Seki et al, 2003) or conserved exonuclease motifs in the sequence.
Role of POLQ-specific insertions in the catalytic fold
POLQ is distinguished from other A-family DNA polymerases
by its ability to efficiently bypass AP sites and Tg, and by the
presence of insertions in the DNA polymerase catalytic
domain. Although more detailed biochemical analyses and
structural studies are needed, it is possible that these two
features are connected. Insert 1 is somewhat analogous to the
loop on the tip of the thumb of T7 DNA polymerase that
binds thioredoxin and acts as a processivity element (Bedford
et al, 1997). It is possible that Insert 1 is important for the
ability of POLQ to bind and extend the primer end opposite
an AP site.
POLQ has relatively low fidelity, in contrast to related
enzymes such as pol I and Taq. Insert 1 may potentially
interfere with the nucleotide recognition process by directly
participating in DNA–protein interactions or indirectly altering the structure of the subdomain that accommodates
incoming nucleotides. A Gaussian network analysis (Bahar
et al, 1997, 1999) of the collective dynamics of POLQ polymerase domain reveals that in a low-frequency mode, Insert 1
is the most mobile region of the entire polymerase domain
(not shown). While high mobility is usually a property that
facilitates substrate recognition, it may also give rise to a
loose association at the binding site, thereby decreasing the
cooperativity and efficiency of functional changes in conformation. One possibility is that the low fidelity of POLQ is not
& 2004 European Molecular Biology Organization
due to a decrease in its DNA binding affinity, but to a lack of
cooperativity in transmitting signals between different parts
of the protein. It may be important to remember, however,
that full-length POLQ also contains a helicase-like protein
domain. The role of this domain has not yet been determined.
It may participate, for example, in binding to specific DNA
templates (Seki et al, 2003).
It has been assumed that vertebrate POLQ may be functionally similar to the Mus308 protein of Drosophila (Sharief
et al, 1999; Seki et al, 2003). However, the fly Mus308
sequence does not have the area identified as Insert 1
(Figures 5 and 6), and Inserts 2 and 3 in POLQ are also
distinct from Mus308. This raises the possibility that Mus308
and POLQ are not functional orthologs. Mus308 has been
genetically implicated in DNA crosslink repair, while this
study shows that the POLQ enzyme efficiently bypasses AP
sites. It is possible that the recently recognized POLN (Marini
et al, 2003) is instead the vertebrate ortholog of the DNA
polymerase portion of Mus308, as the sequence of POLN also
lacks Inserts 1–3.
Implications of the unusual properties of POLQ
for function in vivo
The preference of some DNA polymerases to incorporate A
opposite noncoding or miscoding lesions has been referred to
as the ‘A-rule’ (Strauss, 2002), and a recent study indicates
that human cells normally follow an A-rule when encountering an AP site (Avkin et al, 2002). POLQ may be a major
enzyme for such bypass in mammalian cells as it both inserts
a base opposite an AP site and efficiently extends the
misincorporated nucleotide, making it the most proficient
known polymerase for AP-site bypass. Although POLQ is an
error-prone DNA polymerase, the incorporation of A opposite
an AP site is 10- to 100-fold more efficient than misincorporation of a base opposite T. Insertion of A opposite an AP site
would be the correct choice if such a site arose as an
intermediate in base excision repair of incorporated dUTP,
as appears to occur frequently (Guillet and Boiteux, 2003).
Insertion of A opposite an AP site arising from spontaneous
hydrolytic depurination would be mutagenic, and is probably
not the most likely in vivo function for POLQ. The ability of
POLQ to bypass Tg adducts may also be of physiological
importance, as these major DNA adducts can block replicative DNA polymerases (Kusumoto et al, 2002).
POLQ is expressed in many human tissues, cell lines, and
cancer cells (Seki et al, 2003; Kawamura et al, 2004), and is
represented by ESTs from various tissue types. Thus, it may
have a function in maintaining normal genomic integrity by
allowing bypass of lesions. A need for such bypass may arise
in emergency situations, such as when a DNA replication fork
encounters an unrepaired lesion. This is underlined by the
identity of murine POLQ with the mouse chaos1 mutant,
noted in Introduction. These mice have elevated levels of
spontaneous and mutagen-induced micronuclei in red blood
cells, indicative of increased chromosome breakage or errors
in chromosome segregation (Shima et al, 2003). As we have
shown here, POLQ can efficiently bypass AP sites and Tg
lesions, and it is possible that lack of POLQ bypass activity
could cause replication fork collapse or breakage, leading to
the instability seen in the chaos1 model. As there are also
some indications for preferred expression of POLQ in lymphoid tissues (Kawamura et al, 2004), another potential
The EMBO Journal
VOL 23 | NO 22 | 2004 4491
Bypass of damage by low-fidelity human POLQ
M Seki et al
function is during the somatic hypermutation of antibody
genes (Neuberger et al, 2003), which generates AP sites as a
mutagenic intermediate.
Materials and methods
Purification of POLQ
Recombinant POLQ was purified as reported (Seki et al, 2003), with
minor changes. Instead of Buffer A, cells were lysed in 40 ml of
100 mM Tris–HCl (pH 8.0), 0.6 M (NH4)2SO4, 10% glycerol, 0.5%
Nonidet P-40, 10 mM EDTA, 5 mM DTT, 1 mM PMSF, 50 mM
N-acetyl-leucyl-leucyl-norleucinal (ALLN), and EDTA-free protease
inhibitor mix (Roche Diagnostics). After centrifugation, POLQ was
purified from the supernatant by FLAG and Ni2 þ -agarose affinity
chromatography (Seki et al, 2003).
DNA polymerase assays
Reaction mixtures for dNTP misincorporation experiments were
incubated at 371C for 10 min and contained 300 fmol of 50 -32Plabeled 16-mer primer annealed to 30-mer template DNA, 113 fmol
of POLQ, and 100 mM of each dNTP (unless otherwise noted) in
20 mM Tris–HCl (pH 8.75), 4% glycerol, 80 mg/ml BSA, 8 mM
MgCl2, and 0.1 mM EDTA. Assay conditions for recombinant POLQ
were optimized for pH and temperature before undertaking these
experiments. At pH 8.75 and 371C, the DNA polymerase activity of
POLQ was four times higher than the previously used conditions
(Seki et al, 2003) of pH 7.5 and 301C. The overall properties of
POLQ were not changed, as for example avid bypass activity for an
AP site was also observed at pH 7.5. For translesion synthesis, the
same amounts of templates containing specific lesions were used.
The amounts of pol Z (0.125 ng) and exo-free pol I Kf (2.5 105 U,
with pol I reaction buffer, New England Biolabs) were chosen to
yield activity similar to POLQ. Purification and assay of pol Z were
as described (Kusumoto et al, 2002). For steady-state kinetics
(Table I), 1 pmol of primer/template was used and the procedure
was as detailed elsewhere (Creighton et al, 1995; Kusumoto et al,
2002). This procedure utilizes extensive titration of nucleotide
concentration and time, and produces results valid for a considerable range of enzyme concentrations. For the primer–template
slippage assay (Table II), the amount of substrate was reduced to
150 fmol and the dNTP concentrations increased to 1 mM in order to
increase the possibility of slippage.
Oligonucleotide substrates
Primer oligonucleotides were purchased from Bio-Synthesis or
Sigma GenoSys, purified, and 50 -labeled using polynucleotide
kinase and [g-32P]dATP. Oligonucleotides containing a single
thymine–thymine CPD (T–T CPD), T–T 6-4 PP, AP site, or Tg were
synthesized as described (Kusumoto et al, 2002). The oligonucleotide containing a 1,2-d(GpG) cisplatin intrastrand adduct was as
described by Szymkowski et al (1992). Primers were annealed to
these oligonucleotides to create substrates for bypass assays
(lesions highlighted by bold letters) as follows:
CPD and (6-4)PP substrate:
Selection of a template from the PDB for 3D homology
modeling
The protein sequences of 27 pol I family members were aligned, all
containing the DNA polymerase domain with the previously
defined six conserved motifs (Marini et al, 2003). Among these 27
sequences, those whose crystal structures were available in the PDB
and whose sequence identity with respect to human POLQ was
higher than 35% within the polymerase domain were selected as
template candidates. E. coli pol I, Taq pol I, and Bacillus
stearothermophilus pol I were found to match these two criteria.
Taq pol I has 36% sequence identity with POLQ within the DNA
polymerase domain, and both open (native protein alone) and
closed (complexed with DNA double strand) structures available in
the PDB. Consequently, the ddTTP-trapped closed form of Taq pol I
(PDB code: 1QTM) (Li et al, 1999) with 12-mer double-stranded
DNA, and the open form of Taq pol I (PDB code: 1TAQ) (Kim et al,
1995) were chosen as templates for structural homology modeling
of the open and closed structures of POLQ. The 50 nuclease domains
of both chains were removed in silico. The polymerase domain of
human POLQ has 515 residues, while that of the template Taq pol I
has 400. The length difference is mainly contributed by the three
inserts connecting conserved motifs of HsPOLQ: Insert 1 between
Motifs 1 and 2 (22 residues), Insert 2 between Motifs 2 and 3 (52
residues), and Insert 3 between Motifs 6 and 5 (33 residues).
The MOE package (Molecular Operating Environment, Chem
Comp Group Inc.) (http://www.chemcomp.com/Corporate_Information/MOE_Bioinformatics.html) was used for modeling of the
inserts. For this purpose, MOE uses a library of pentapeptide
rotamers, or sequence-specific structural motifs, extracted from the
PDB. The underlying methodology is a combination of a segmentmatching procedure (Levitt, 1992) and modeling of insertions/
deletions (Fechteler et al, 1995). The approach is to combine the
structural motifs generating possible conformations of groups of
residues in the loop regions and to select the conformers that
minimize the intra- and intermolecular energy in the context of
other protein segments and substrate (here the double-stranded
DNA 12-mer and ddTTP). The method of Levitt has been applied to
a series of proteins ranging in size from 46 to 323 residues to obtain
an all-atom-root-mean-square deviation of 0.93–1.73 Å. Fechteler
et al (1995) have shown that the search of representative protein
fragments using geometric scoring criteria can assist in predicting
three-dimensional structures in insertion and deletion regions of
proteins. Among the three insertions of POLQ, Insert 1 is the
shortest (22 residues), and it is conceivable that its preferred
conformation(s) can be estimated to a reasonable approximation by
this segment-matching methodology, as used in MOE. The model
for Insert 1 shown in Figure 6B is one of 10 predicted by MOE,
which vary in detailed atomic structure with a root mean square
(r.m.s.) deviation (for Insert 1 only) of 5.77 Å. Notably, all of the
models show insertion into the DNA major groove, and this
tendency is consistent with the abundance of positively charged
residues in Insert 1. Interpretation of secondary structure used the
algorithm of SWISS-Model (Swiss-PDB Viewer) (Guex et al, 1999).
50 -[32P]-AGAAGAAGAAGG
TCTTCTTCTTCCGGATCTTCTTCT-50
Refinement by energy minimization
The MOE package was then used to build the models for the human
POLQ open and closed structures. Calculations were performed
using two different force fields, AMBER94 (Weiner et al, 1984,
1986) and MMFF94s (Halgren, 1996), to assess the sensitivity and
robustness of the results with respect to the choice of intramolecular potentials. For each target structure (open or closed form of
POLQ), 10 intermediate structures were generated by MOE. The
Cartesian average model was created by averaging the coordinates
of the 10 intermediates in each case, which led to two open and two
closed form structures using the two different force fields. The
structures found with the two sets of potentials were in close
agreement, except for the relative orientation of Insert 2 in the two
DNA-bound forms: the r.m.s. deviation in the backbone coordinates
of the two open forms was 2.3 Å, and that of the two closed forms
without Insert 2 was 1.7 Å. While the conformation of Insert 2
differed in the two models, none of its atoms was closer to doublestranded DNA and incoming nucleotides than 7.5 Å for both
models.
The resulting four structures were subjected to structural
refinement to correct any bond lengths and angles that violated
the conventional ranges of accessible values (e.g. if the peptide
bond significantly departed from the trans state). A constrained
4492 The EMBO Journal VOL 23 | NO 22 | 2004
& 2004 European Molecular Biology Organization
50 -[32P]- CACTGACTGTATGATG
GTGACTGACATACTACTTCTACGACTGCTC-50
AP analog substrate (AP analog is denoted by X):
50 -[32P]- CACTGACTGTATGATG
GTGACTGACATACTACXTCTACGACTGCTC-50
Tg substrate:
50 -[32P]- CACTGACTGTATGATG
GTGACTGACATACTAC(Tg)TCTACGACTGCTC-50
1,2-d(GpG) cisplatin intrastrand adduct substrate:
Bypass of damage by low-fidelity human POLQ
M Seki et al
energy minimization was performed for each structure for this
procedure, during which Inserts 1–3 and the corresponding regions
of the original templates were allowed to undergo conformational
changes while the remaining structural elements (essentially the
core and surrounding conserved secondary structural elements)
were held fixed at their original state. A total of 500 steps of steepest
descent and 100 steps of the conjugated gradient method were
performed for each energy minimization run. The procedure was
repeated with decreasing strengths of bond stretch, angle bend, and
torsion potentials that penalize the unrealistic bond lengths and
angles, until no further change in conformation or decrease in
energy was observed.
The computations performed for obtaining the closed structure
considered the complex formed with the bound DNA. The DNA
segment was incorporated into the models after structural alignment with the cognate template, 1QTM. The same energy
minimization procedure as the one described above for the open
structure was then applied to the DNA polymerase–DNA complex to
generate the most stable conformations. The r.m.s. deviation in the
backbone coordinates for the POLQ–DNA complex between the
initial homology model and its energy-minimized form was only
0.55 Å, indicating that the model was stable and closely retained its
conformation during energy minimization.
Acknowledgements
We thank Dr Birgitte Wittschieben for discussion and advice on
protein purification, Dr Dror Tobi for advice on protein modeling,
and Dr Tom Ellenberger, Dr Luis Breiba and Dr Patrick Moore for
comments on the manuscript. This work was supported by grants
NIH CA101980 (to RDW), NIH P20 GM065805 (to IB), and by a
grant to CM from the US–Japan Cooperative Cancer Research
Program of the Japan Society for the Promotion of Science (JSPS).
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