Báo cáo khoa học: The noncatalytic C-terminus of AtPOLK Y-family DNA polymerase affects synthesis fidelity, mismatch extension and translesion replication ppt

The noncatalytic C-terminus of AtPOLK Y-family DNA
polymerase affects synthesis fidelity, mismatch extension
and translesion replication
Marı
´
a Victoria Garcı
´
a-Ortiz, Teresa Rolda
´
n-Arjona and Rafael R. Ariza
Departamento de Gene
´
tica, Universidad de Co
´
rdoba, Spain
Cells are equipped not only with high-fidelity enzymes
that accurately replicate the genome, but also with spe-
cialized DNA polymerases that play essential functions
in repair and ⁄ or replication of damaged DNA [1–3].
Many of these enzymes are structurally related and
belong to the Y family of DNA polymerases, which
includes four subfamilies represented by Escherichia
coli DinB (Pol IV) and UmuC (Pol V), and Saccharo-
myces cerevisiae pol g (Rad30) and Rev1 [4]. UmuC-
like proteins have been identified exclusively in
bacteria, and the Rad30 and Rev1 subfamilies contain
only eukaryotic members. The DinB subfamily is the
most phylogenetically diverse, with bacterial, archaean
and eukaryotic proteins [4].
Members of the Y family contain 350–1200 amino-
acid residues, but share five conserved sequence motifs

distributed along the N-terminal part of the molecule.
Crystal structures of this region in several Y-family
polymerases have revealed a catalytic core with an
archetypal DNA polymerase fold including finger,
thumb and palm domains arranged in a classic ‘right
hand-like’ configuration, and an extra domain known
as little finger, polymerase-associated domain, or wrist
domain [5]. Unlike replicative DNA polymerases,
Y-family enzymes have an open solvent-accessible active
site but lack proofreading exonuclease activity [2].
Consequently, they have some remarkable biochemical
properties, such as low fidelity on undamaged DNA [6]
Keywords
Arabidopsis thaliana; base-pair mismatch;
DNA damage; DNA replication; translesion
DNA synthesis
Correspondence
R. R. Ariza, Departamento de Gene
´
tica,
Edificio Gregor Mendel, Campus de
Rabanales s ⁄ n, Universidad de Co
´
rdoba,
14071-Co
´
rdoba, Spain
Fax: +34 957 212 072
Tel: +34 957 218 979
E-mail:

(Received 10 April 2007, accepted 4 May
2007)
doi:10.1111/j.1742-4658.2007.05868.x
Cell survival depends not only on the ability to repair damaged DNA
but also on the capability to perform DNA replication on unrepaired or
imperfect templates. Crucial to this process are specialized DNA polym-
erases belonging to the Y family. These enzymes share a similar catalytic
fold in their N-terminal region, and most of them have a less-well-con-
served C-terminus which is not required for catalytic activity. Although
this region is essential for appropriate localization and recruitment
in vivo, its precise role during DNA synthesis remains unclear. Here we
have compared the catalytic properties of AtPOLK, an Arabidopsis
orthologue of mammalian pol j, and a truncated version lacking 193
amino acids from its C-terminus. We found that C-terminally truncated
AtPOLK is a high-efficiency mutant protein, the DNA-binding capacity
of which is not affected but it has higher catalytic efficiency and fidelity
than the full-length enzyme. The truncated protein shows increased pro-
pensity to extend mispaired primer termini through misalignment and
enhanced error-free bypass activity on DNA templates containing 7,8-di-
hydro-8-oxoGuanine. These results suggest that, in addition to facilita-
ting recruitment to the replication fork, the C-terminus of Y-family
DNA polymerases may also play a role in the kinetic control of their
enzymatic activity.
Abbreviations
PCNA, proliferating cell nuclear antigen, UBZ, ubiquitin-binding Zn-finger motif; 8-oxoG, 7,8-dihydro-8-oxoGuanine; edA, 1,N
6
-ethenoadenine.
3340 FEBS Journal 274 (2007) 3340–3350 ª 2007 The Authors Journal compilation ª 2007 FEBS
and the ability to synthesize DNA opposite-damaged
templates by translesion synthesis [7]. The current

hypothesis is that these enzymes act transiently at
arrested replication forks to copy any faulty nucleotides
and extend the resultant noncanonical primer–template
pairs with varying degrees of accuracy, but give way to
high-fidelity replication downstream of the arrest
point [8].
In addition to their conserved N-terminal catalytic
core, most Y-family DNA polymerases have a C-ter-
minal region with a low degree of sequence conserva-
tion between members of the family [9]. This region is
not essential for catalytic activity, but plays important
roles in vivo through protein–protein interactions that
mediate appropriate localization and recruitment [3].
Mammalian pols g, j and i contain a consensus prolif-
erating cell nuclear antigen (PCNA)-binding PIP motif
at their C-termini that is essential for their targeting to
the replication machinery in vivo [3]. This region also
contains novel ubiquitin-binding domains that are evo-
lutionarily conserved in all Y-family polymerases and
are required for their recruitment to replication factor-
ies [10]. It has been proposed that binding of Y-family
DNA polymerases to ubiquitinated PCNA via both
the PIP motif and the ubiquitin-binding domains facili-
tates their recruitment to stalled replication forks
and displacement of the replicative DNA polymerase
[3,11].
Despite its important in vivo functions, the role of
the C-terminal region of Y-family DNA polymerases
during DNA synthesis remains unclear. We have previ-
ously reported that the activity and processivity of

AtPOLK, an Arabidopsis orthologue of mammalian
pol j, are enhanced markedly upon deletion of 193
amino acids from its C-terminus [12]. AtPOLK is a
plant Y-family DNA polymerase belonging to the DinB
subfamily. The N-terminal half of AtPOLK shows the
five conserved motifs (I–V) present in all Y-family
DNA polymerases and an N-terminal extension unique
to eukaryotic DinB orthologues, whereas its C-terminal
half shows a much lower degree of sequence conserva-
tion (Fig. 1A). In the C-terminal region, AtPOLK con-
tains a putative ubiquitin-binding Zn-finger motif
(UBZ), a predicted bipartite nuclear localization signal,
and a candidate PCNA-interaction domain.
To better understand the role of the C-terminal
domain of this Y-family DNA polymerase during DNA
synthesis, we compared the catalytic properties of
full-length AtPOLK and the truncated version
(AtPOLKDC1–478). We found that deletion of the
C-terminus increased the catalytic efficiency and fidelity
of AtPOLK during synthesis on undamaged DNA
A
B
Fig. 1. DNA polymerase activity of AtPOLK and AtPOLKDC. (A) Schematic diagram of AtPOLK (wt) and AtPOLKDC(DC) showing the regions
of similarity to other DinB orthologues as shaded sections. The positions of motifs I–V, identified in all Y-family DNA polymerases, are
shown. Letters x, y and z designate motifs that are characteristic of the DinB subfamily. The motif N is an N-terminal extension unique to
eukaryotic DinB orthologues. The putative nuclear localization site (NLS), UBZ3 motif and PCNA-binding domain are also indicated. (B) DNA
polymerase activity of full-length AtPOLK (wt) versus its C-terminally truncated version (DC) on various templates. Increasing concentrations
(100, 300 and 500 n
M) of AtPOLK or AtPOLKDC were assayed for their ability to extend a 5¢-end-labelled 21-nucleotide primer annealed to a
40-nucleotide template (100 n

M). A portion of each substrate is shown on top. Lanes 1, 9, 17 and 25 contain no enzyme. Klenow (250 nM)
was used as a positive control in lanes 8, 16, 24 and 32.
M. V. Garcı
´
a-Ortiz et al. Role of C-terminus in AtPOLK DNA polymerase
FEBS Journal 274 (2007) 3340–3350 ª 2007 The Authors Journal compilation ª 2007 FEBS 3341
templates, enhanced its ability to extend mismatches
through misalignment, and strongly influenced its trans-
lesion activity through error-prone and error-free
bypass.
Results
DNA polymerase activity of wild-type and
truncated AtPOLK
The full-length and truncated AtPOLK were purified
to homogeneity as described by Garcı
´
a-Ortiz et al.
[12]. AtPOLKDC retains amino acids 1–478, which
encompass the five conserved motifs (I–V) present in
all Y-family DNA polymerases, the x, y and z motifs
that are unique to the DinB subfamily, and the N-ter-
minal extension characteristic of the eukaryotic DinB
orthologues (Fig. 1A). A structure-based alignment of
AtPOLK with human pol j, Sulfolobus solfataricus
Dpo4, Dbh, and E. coli DinB suggests that the
enzyme catalytic core is preserved in the C-terminally
truncated enzyme (Supplementary material, Fig. S1).
In our previous analysis of AtPOLK, we found that
the full-length enzyme had lower polymerization activity
than the truncated version [12]. However, as the experi-

ments were performed on a single primer–template
substrate, we decided to test the enzymatic activity of
both proteins on different substrates, each containing a
different base pair at the primer–template junction.
AtPOLK and AtPOLKDC proteins were assayed for
DNA polymerase activity measuring extension of four
5¢-end-labelled 21-mer primers annealed to their corres-
ponding 40-mer template (Fig. 1B). Both AtPOLK
and AtPOLKDC synthesized DNA, extending the pri-
mer to a size comparable to the full-length product
generated by the Klenow fragment of E. coli DNA
polymerase I (Fig. 1B). As reported for mammalian
pol j [13], neither wild-type nor truncated AtPOLK
efficiently copy to the end of the template, finishing
one or two nucleotides before the terminus (Fig. 1B,
and data not shown). We found that deletion of 193
residues at the C-terminus had a major effect on the
enzymatic activity of the protein on all substrates. The
polymerization activity of full-length AtPOLK was
significantly lower than that of AtPOLKDC, requiring
a fivefold higher enzyme concentration than the
truncated polymerase to replicate fully a 40-nucleotide
template. In addition, in all four substrates a range of
incomplete extension products consistent with distribu-
tive synthesis was seen in reaction mixtures containing
the wild-type enzyme, represented by a stepladder
pattern on the gel beginning with primer +1 dNMP.
In contrast, AtPOLKDC showed longer products than
the full-length enzyme at all concentrations tested.
These data are in agreement with our previous finding

of a higher processivity of AtPOLK DC relative to the
wild-type enzyme [12]. Thus, truncation of the 193
C-terminal amino acids stimulates the DNA polym-
erase activity and processivity of AtPOLK on different
DNA substrates.
The C-terminally truncated AtPOLK DNA
polymerase is not affected in DNA binding
We next examined whether the differences in polymer-
ization activity between AtPOLK and AtPOLKDC
could arise from differences in their relative binding
affinities for the primer–template DNA substrate.
Wild-type and truncated AtPOLK were both assayed
for their capacity to bind various DNA substrates
using a gel electrophoretic mobility shift method
(Fig. 2). First, we tested the ability of wild-type and
truncated AtPOLK to bind single-stranded, double-
stranded and template–primer DNA structures. When
various labelled DNAs were incubated with either
AtPOLK or AtPOLKDC, the formation of stable
protein–DNA complexes could be detected as shifted
bands after nondenaturing gel electrophoresis. As
shown in Fig. 2A, both AtPOLK and AtPOLKDC
preferentially bind template–primer DNA substrates or
single-stranded DNA, and bind double-stranded DNA
poorly. Binding to the primer–template structure
results in a single retardation band (Fig. 2A,B), which
might represent a protein–DNA interaction providing
a stable polymerization-competent conformation of the
primer terminus at the enzyme active site, such as that
observed with other DNA polymerases [14]. As shown

in Fig. 2B, binding of the primer–template structure to
the truncated AtPOLK was essentially identical with
that of the full-length protein. We found analogous
results with other combinations of primer–template
DNA (data not shown). Therefore, truncation of the
193 C-terminal amino acids of AtPOLK does not
significantly affect its binding affinity to DNA in vitro.
These results suggest that the C-terminal domain of
AtPOLK is dispensable for DNA binding, although its
presence negatively affects its DNA polymerization
activity.
Fidelity analysis of AtPOLK and AtPOLK
n
C
We next analysed the catalytic efficiency and fidelity of
AtPOLK and AtPOLKDC during DNA synthesis. To
determine the fidelity of both full-length and truncated
AtPOLK, we analysed the steady state kinetics by
measuring the incorporation of correct and incorrect
Role of C-terminus in AtPOLK DNA polymerase M. V. Garcı
´
a-Ortiz et al.
3342 FEBS Journal 274 (2007) 3340–3350 ª 2007 The Authors Journal compilation ª 2007 FEBS
deoxynucleotides opposite each of the four template
bases. A DNA polymerase extension assay was used to
measure the rate of deoxynucleotide incorporation, cal-
culated by dividing the amount of product formed by
the reaction time. The observed rate was plotted as a
function of the dNTP concentration and the data were
fitted to the Michaelis–Menten equation. The apparent

K
m
and V
max
steady state kinetic parameters for the
incorporation of both correct and incorrect deoxy-
nucleotides were obtained from each fitted curve and
used to calculate the catalytic efficiency (k
cat
⁄ K
m
)
(Table 1) and the frequency of deoxynucleotide mis-
incorporation [f
inc
¼ (k
cat
⁄ K
m
)
incorrect
⁄ (k
cat
⁄ K
m
)
correct
]
opposite each of the four template bases (Fig. 3).
AtPOLK and AtPOLKDC misincorporated dCTP

opposite template A, C, and T, dTTP opposite tem-
plate G and C, and dATP opposite template C. The
fidelity of full-length AtPOLK, measured as the
frequency of deoxynucleotide misincorporation (f
inc
),
ranged from 2.3 · 10
)3
(for the misincorporation of
Table 1. Steady-state kinetic parameters for AtPOLK and AtPOLKDC. Data are mean ± SE from at least two independent experiments. Only
those combinations of template base and incoming nucleotide for which incorporation was detected are shown.
dNTP
k
cat
(min
)1
) K
m
(lM) k
cat
⁄ K
m
(lM
)1
Æmin
)1
)
AtPOLK AtPOLKDC AtPOLK AtPOLKDC AtPOLK AtPOLKDC
Template C
dATP 0.024 ± 0.001 0.059 ± 0.010 52.60 ± 64.40 390.46 ± 272.15 4.6 · 10

-4
1.5 · 10
-4
dGTP 0.125 ± 0.010 0.111 ± 0.005 12.51 ± 3.59 0.41 ± 0.08 1.0 · 10
-2
0.27
dTTP 0.057 ± 0.001 0.119 ± 0.006 83.30 ± 8.34 28.25 ± 8.85 6.8 · 10
-4
4.2 · 10
-3
dCTP 0.0038 ± 0.0004 0.081 ± 0.001 156.80 ± 85.80 171.68 ± 45.03 2.4 · 10
-5
4.7 · 10
-4
Template T
dATP 0.157 ± 0.014 0.152 ± 0.005 2.61 ± 0.78 0.13 ± 0.02 6.0 · 10
-2
1.17
dCTP 0.038 ± 0.001 0.131 ± 0.003 62.29 ± 24.38 72.94 ± 9.23 6.1 · 10
-4
1.8 · 10
-3
Template A
dTTP 0.207 ± 0.010 0.145 ± 0.003 4.70 ± 0.94 0.69 ± 0.05 4.4 · 10
-2
2.1 · 10
-1
dCTP 0.045 ± 0.003 0.063 ± 0.003 100.41 ± 46.48 233.38 ± 36.95 4.5 · 10
-4
2.7 · 10

-4
Template G
dTTP 0.022 ± 0.001 0.131 ± 0.007 29.16 ± 7.30 10.07 ± 4.10 7.6 · 10
-4
1.3 · 10
-2
dCTP 0.061 ± 0.004 0.116 ± 0.006 7.76 ± 1.60 0.61 ± 0.11 7.9 · 10
-3
1.9 · 10
-1
AB
Fig. 2. DNA-binding capacity of AtPOLK and AtPOLKDC. Enzymes were incubated with various labelled DNA molecules as described in
Experimental procedures. After nondenaturing gel electrophoresis, enzyme–DNA complexes were identified by their retarded mobility com-
pared with that of free DNA, as indicated. (A) DNA-binding affinity for different DNA substrates. Two concentrations (1.0 and 1.5 l
M)of
AtPOLK (wt) or AtPOLKDC(DC) were incubated with 0.5 l
M each of labelled single-stranded, double-stranded or primer–template DNA sub-
strates. (B) DNA-binding affinity for a primer–template DNA structure. Increasing amounts of AtPOLK or AtPOLKDC (0.5, 1.0 and 1.5 l
M)
were incubated with a labelled primer–template DNA substrate (0.5 l
M).
M. V. Garcı
´
a-Ortiz et al. Role of C-terminus in AtPOLK DNA polymerase
FEBS Journal 274 (2007) 3340–3350 ª 2007 The Authors Journal compilation ª 2007 FEBS 3343
dCTP opposite template C) to 9.6 · 10
)2
(for the mis-
incorporation of dTTP opposite template G) (Fig. 3).
We conclude that AtPOLK is a low-fidelity DNA

polymerase, with error rates in the range of those of
other Y-family DNA polymerases [6,15–19].
The frequencies of deoxynucleotide misincorporation
(f
inc
) opposite four templates are higher for the full-
length enzyme than for AtPOLKDC (Fig. 3). These
differences in fidelity represent different ratios of
catalytic efficiencies for correct and incorrect
nucleotide insertion. As shown in Table 1, AtPOLKDC
inserted every correct nucleotide and most of the
incorrect nucleotides more efficiently than did
AtPOLK. Thus, the higher fidelity exhibited by the
truncated version of AtPOLK is not due to a decrease
in the incorporation of wrong nucleotides but to a
greater ability to insert the correct nucleotide.
Interestingly, the higher catalytic efficiency of
truncated AtPOLK for the correct insertion opposite
each of the four template bases primarily results from
a significant reduction in the apparent K
m
for the
incoming nucleotide, whereas only minor differences
were observed in k
cat
values. For example, the  30-
fold increase in the efficiency of G insertion opposite
the C template nucleotide was accompanied by a  30-
fold decrease in the K
m

for G, whereas the k
cat
did not
change significantly (Table 1).
These results collectively suggest that the C-terminal
domain of AtPOLK negatively affects its catalytic effi-
ciency for correct insertion, decreasing the fidelity of
the enzyme.
The C terminus affects the relative contributions
of direct extension and misalignment during
mismatch extension
AtPOLK is able to extend primer-terminal mispairs
[12]. Therefore, we examined the different abilities of
AtPOLK and AtPOLKDC to extend mismatched
primer–template termini on undamaged DNA. It has
been previously reported that human pol j is able to
extend a mispaired primer terminus by incorporating
the next correct nucleotide (direct extension) or by
misalignment of the template and primer nucleotides
[20]. To explore the capacity of wild-type and trun-
cated AtPOLK to extend mispaired primer termini
by direct extension or via misalignment, we analysed
the steady state kinetics to determine the catalytic
efficiency of nucleotide incorporation following a
mismatched template–primer terminus. Two suitable
DNA substrates were used to discriminate between
the two modes of mismatch extension (Table 2).
Substrate I contains a mispaired G:T primer–template
terminus followed by an A and a G in the two
consecutive downstream template positions. In this

substrate the G:T mispair can only be extended by the
direct incorporation of a T opposite the next A in the
template. Substrate II is identical with substrate I
except for the presence of a C in the first downstream
Table 2. Steady-state kinetic parameters for G:T mispair extension by AtPOLK and AtPOLKDC. Data are mean ± SE from at least two inde-
pendent experiments. ND, not detected.
Substrate Sequence dNTP
k
cat
(min
)1
) K
m
(lM) k
cat
⁄ K
m
(lM
)1
Æmin
)1
)
AtPOLK AtPOLKDC AtPOLK AtPOLKDC AtPOLK AtPOLKDC
Substrate I 5¢ GAG dTTP 0.029 ± 0.002 0.123 ± 0.010 36.95 ± 10.93 49.00 ± 18.90 7.8 · 10
)4
2.5 · 10
)3
3¢ CTTAGA- dATP, dGTP
or dCTP
ND ND ND ND ND ND

Substrate II 5¢ GAG dGTP 0.048 ± 0.003 0.088 ± 0.010 61.65 ± 36.69 80.41 ± 33.18 7.8 · 10
)4
1.1 · 10
)3
3¢ CTTCGA- dCTP 0.073 ± 0.010 0.130 ± 0.004 181.95 ± 35.36 12.98 ± 1.82 4.0 · 10
)4
1.0 · 10
)2
dATP
or dTTP
ND ND ND ND ND ND
Fig. 3. Misincorporation frequencies for AtPOLK and AtPOLKDC.
Steady-state assays were performed as described in Experimental
procedures, and the k
cat
and K
m
parameters (Table 1) were used to
calculate the frequency of deoxynucleotide incorporation (f
inc
)by
applying the equation f
inc
¼ (k
cat
⁄ K
m
)
incorrect
⁄ (k

cat
⁄ K
m
)
correct
.
Role of C-terminus in AtPOLK DNA polymerase M. V. Garcı
´
a-Ortiz et al.
3344 FEBS Journal 274 (2007) 3340–3350 ª 2007 The Authors Journal compilation ª 2007 FEBS
template position. In this substrate the G:T mispair
can be extended either by direct incorporation of a G
opposite the next C in the template or by misalignment
of the primer-terminal G with the next C followed by
the incorporation of a C.
We incubated either AtPOLK or AtPOLKDC in the
presence of DNA substrates I or II and different con-
centrations of dATP, dGTP, dCTP or dTTP. The
measured rate of nucleotide incorporation was plotted
as a function of dNTP concentration, and k
cat
and K
m
values were determined as described in the previous
section and in the Experimental procedures. As shown
in Table 2, both wild-type and truncated AtPOLK
extended the primer terminal G in DNA substrate I,
but only by incorporation of T, which is the next cor-
rect nucleotide. In contrast, both enzymes incorporated
either G or C when incubated with substrate II, which

can realign the mismatched primer terminus using the
neighbouring complementary templating base C. Thus,
both AtPOLK and AtPOLKDC are able to extend a
mispaired primer terminus, by either direct extension
or misalignment of the template and primer nucleo-
tides.
Interestingly, however, both proteins use these two
modes of mispair extension to different degrees. Exten-
sion through misalignment is performed by AtPOLK
with a similar efficiency to extension by direct
incorporation (efficiencies of 4 · 10
)4
and 7.8 · 10
)4
,
respectively), whereas AtPOLKDC carried out
misalignment 10 times more efficiently than direct
extension (efficiencies of 1.2 · 10
)2
and 1.1 · 10
)3
,
respectively). Although the two enzymes perform direct
extension with comparable efficiencies, AtPOLKDC
carries out extension through misalignment 25 times
more efficiently than does AtPOLK. Again, this
increase largely results from a significant reduction in
the apparent K
m
for the incoming nucleotide, with

minor differences in k
cat
values (Table 2). These results
suggest that differences in catalytic efficiency for inser-
tion of correct nucleotides may determine the relative
contributions of direct extension and misalignment
during mismatch extension carried out by Y-DNA
polymerases.
Error-prone and error-free lesion bypass
by wild-type and truncated AtPOLK
To examine the relative aptitudes of wild-type and
truncated AtPOLK to bypass DNA lesions, we ana-
lysed their ability to replicate from templates contain-
ing a single 7,8-dihydro-8-oxoGuanine (8-oxoG), a
single 1,N
6
-ethenoadenine (edA), or an abasic site
(Fig. 4A). We found that AtPOLK is unable to bypass
the abasic site or the edA adduct, but is able to insert
nucleotides opposite the 8-oxodG lesion and moder-
ately extend from the resulting primer end (Fig. 4A,
AB
Fig. 4. DNA synthesis by AtPOLK and AtPOLKDC on templates containing 8-oxoG, edA, or an abasic site. (A) A 5¢-end-labelled 21-nucleotide
primer was annealed to a 40-nucleotide oligonucleotide template containing G (lanes 1, 2 and 6), 8-oxoG (lanes 3 and 7), edA (lanes 4 and 8),
or an abasic site (lanes 5 and 9) at the position indicated by X. AtPOLK (wt, 500 n
M) or AtPOLKDC(DC,100 nM) was incubated with the
DNA substrate (100 n
M) in the presence of each of four dNTPs. The reaction mixture in lane 1 contained no enzyme. (B) Identification of
nucleotides incorporated opposite 8-oxoG by AtPOLK and AtPOLKDC. Reactions were carried out in the presence of each dNTP individually
(A, T, G, C) or all four dNTPs (N

4
). Reaction mixtures in lanes 7 and 14 contained Klenow enzyme (250 nM) with all four dNTPs. The reaction
mixture in lane 1 contained no enzyme.
M. V. Garcı
´
a-Ortiz et al. Role of C-terminus in AtPOLK DNA polymerase
FEBS Journal 274 (2007) 3340–3350 ª 2007 The Authors Journal compilation ª 2007 FEBS 3345
lane 3). AtPOLKDC showed the same bypass specifici-
ty as AtPOLK, being blocked by the abasic site and
the edA adduct, but not by the 8-oxoG lesion. How-
ever, after insertion opposite 8-oxodG, it performed
extension from the 3¢-primer terminus with signifi-
cantly higher efficiency than the wild-type enzyme
(Fig. 4A, lane 7).
The miscoding potential of 8-oxoG arises from its
ability to form stable base pairs with either a C or an
A residue [21]. To identify the nucleotide incorporated
opposite 8-oxoG, we performed DNA synthesis assays
with dATP, dCTP, dGTP or dTTP individually. As
shown in Fig. 4B, both AtPOLK and AtPOLKDC
inserted either A or C opposite 8-oxoG. Primer exten-
sion was not detected with dTTP and dGTP. Klenow
was used as a control, and, as previously reported
[21,22], it was strongly inhibited at chain extension at
template positions 5¢- to the modified base (Fig. 4B,
lanes 7 and 13).
To measure the ratio of dATP:dCTP incorporation
during the bypass of 8-oxoG, we measured the steady-
state kinetic parameters of nucleotide insertion oppos-
ite the lesion. The kinetics of insertion of dATP or

dCTP opposite 8-oxoG were determined as a function
of deoxynucleotide concentration under steady-state
conditions (Table 3). The apparent steady-state k
cat
and K
m
values for each nucleotide incorporation were
determined as described in Experimental procedures,
and the relative incorporation efficiency was calculated
as the ratio of the efficiency (k
cat
⁄ K
m
) of incorrect
nucleotide incorporated to the efficiency (k
cat
⁄ K
m
)of
correct nucleotide incorporated (Table 3).
As indicated by the k
cat
⁄ K
m
values in Table 3,
AtPOLK incorporated either A or C opposite 8-oxoG
lesions less efficiently than did the truncated protein.
The higher catalytic efficiency of AtPOLKDC resulted
primarily from a decrease in the apparent K
m

for the
incoming dNTP, as previously observed during DNA
synthesis in undamaged DNA. The relative incorpor-
ation efficiency to the incorporation of a C opposite
undamaged G ranged from 0.17, for insertion of C
opposite 8-oxodG by AtPOLKDC, to 0.57, for inser-
tion of A opposite 8-oxoG by ATPOLK.
Interestingly, the ratio of the dATP:dCTP insertion
was different in the two proteins. AtPOLK showed
twice the relative incorporation efficiency for A oppos-
ite 8-oxodG than for C (0.57 versus 0.27). However,
AtPOLKDC inserted both A and C with similar effi-
ciency (0.19 and 0.17, respectively). This discrepancy in
the ratio of the dATP:dCTP insertion between the two
proteins mainly arises from differences in the apparent
K
m
values for dATP or dCTP. Whereas AtPOLK
shows twice the K
m
for dCTP as for dATP, similar
values were observed for both deoxynucleotides with
AtPOLKDC (Table 3).
Therefore, although both wild-type and truncated
AtPOLK are able to perform either error-free or error-
prone bypass of 8-oxoG, the full-length protein shows
a preference for the latter given its proclivity towards
A insertion. On the other hand, truncation of the
C-terminal domain increases the bypass efficiency and
decreases the efficiency of A insertion, thus reducing

mutagenic translesion synthesis. Taken together these
results suggest that the presence of the C terminus
affects the relative contributions of error-free and
error-prone bypass activity of AtPOLK.
Discussion
The mutagenic potential of Y-family DNA poly-
merases obliges cells to regulate their access to DNA,
specifically recruiting them when and where they are
needed. The ‘DNA polymerase switch model’ postu-
lates a transient replacement of the replicative DNA
polymerase in the vicinity of the lesion by one or
several error-prone polymerases before resumption of
Table 3. Steady-state kinetic parameters of nucleotide insertion reactions opposite 8-oxoG template residues by AtPOLK and AtPOLKDC.
Data are mean ± SE from at least two independent experiments.
DNA substrate
Incoming
nucleotide
k
cat
(min
)1
) K
m
(lM) k
cat
⁄ K
m
(lM
1
Æmin

)1
)
Relative incorporation
efficiency
AtPOLK AtPOLKDC AtPOLK AtPOLKDC AtPOLK AtPOLKDC AtPOLK AtPOLKDC
Insertion opposite G
5’ GTAGAG
3’ CATCTCGGA-
dCTP 0.061 ± 0.04 0.116 ± 0.006 7.76 ± 1.60 0.61 ± 0.11 7.9 · 10
)3
1.9 · 10
)1
11
Insertion opposite 8-oxoG
5’ GTAGAG
3’ CATCTCGGA-
dCTP 0.105 ± 0.006 0.137 ± 0.004 49.79 ± 8.06 4.27 ± 0.69 2.1 · 10
)3
3.2 · 10
)2
0.27 0.17
8’-oxo dATP 0.110 ± 0.01 0.138 ± 0.04 24.40 ± 8.42 3.72 ± 0.29 4.5 · 10
)3
3.7 · 10
)2
0.57 0.19
Role of C-terminus in AtPOLK DNA polymerase M. V. Garcı
´
a-Ortiz et al.
3346 FEBS Journal 274 (2007) 3340–3350 ª 2007 The Authors Journal compilation ª 2007 FEBS

high-fidelity replication [23]. Current understanding of
this process favours the idea that switching is modu-
lated via interaction of the C-terminal domain of
Y-family DNA polymerases with the replication proc-
essivity clamp PCNA. In this model, PCNA might
function as a ‘tool-belt’, enabling efficient access to
the blocking lesion by different polymerases [24].
Despite the well-documented function of the C-ter-
minus in polymerase targeting and recruitment, our
understanding of its role in Y-family DNA poly-
merase catalytic activity is still limited. Efforts to
relate the enzymatic properties of Y-family DNA poly-
merases to their structural characteristics have focused
on the catalytic core, neglecting the poorly conserved
C-terminal region. To our knowledge, no detailed kin-
etic analysis comparing full-length and C-terminally
truncated Y-DNA polymerases has been previously
reported. However, there have been conflicting reports
about the effect of C-terminal truncation in eukaryotic
DinB orthologues on enzymatic activity. A protein
from amino acids 19–526, which lacks the last 344
amino acids of human pol j, has been reported to
possess a DNA polymerase activity equivalent to that
of the wild-type enzyme [25]. In contrast, another
truncated human pol j containing residues 1–562
shows similar activity but reduced processivity com-
pared with the full-length protein [26]. We have previ-
ously reported that the DNA polymerase activity and
processivity of AtPOLK is markedly enhanced upon
deletion of 193 amino acids from its C-terminus [12].

Differences between AtPOLK and human pol K C-ter-
minal domains may be responsible for the discrepancy
observed between mammalian and plant proteins.
Human pol j, for example, contains two UBZs of the
C
2
HC type (UBZ4) at their C-terminus [10], whereas
AtPOLK shows a single UBZ domain of the C
2
H
2
type (UBZ3) (Fig. S1).
Here, we have made a detailed comparison of the
catalytic properties of both the full-length AtPOLK
and its C-terminally truncated counterpart. We found
that truncation of the AtPOLK C-terminus produced
a high-efficiency mutant protein with increased fidelity.
The DNA-binding capacity of the truncated protein
was not affected, as both AtPOLK and AtPOLKDC
displayed similar affinities for a primer–template struc-
ture. This result argues against the possibility that the
full-length protein does not fold properly, resulting in
a large fraction of inactive protein.
The lower fidelity of wild-type AtPOLK results not
from enhanced catalytic efficiency for misincorporation
relative to the C-terminally truncated enzyme but rather
from a lower catalytic efficiency for Watson–Crick
incorporations than with the C-terminally truncated
enzyme. These results are in agreement with a previous
study reporting that f

inc
of a number of different DNA
polymerases correlates inversely with their catalytic effi-
ciency for correct nucleotide insertion, which thus
implies that fidelity is primarily governed by the ability
to insert the correct nucleotide [27].
The higher catalytic efficiency of truncated AtPOLK
for correct insertion opposite each of the four template
bases is achieved primarily by a reduction in the
apparent K
m
for the nucleotide. It is possible that dele-
tion of the C-terminal 193 amino acids of AtPOLK
modifies the conformation of the active site, causing
higher affinity of the enzyme for the nucleotide. How-
ever, it is important to recall that K
m
cannot be corre-
lated to initial dNTP binding with certainty, and
perhaps the effect of the C-terminal truncation instead
affects rate-limiting conformational changes that
immediately follow dNTP binding [28]. Interestingly,
it has been reported that PCNA binding to the
C-terminal region of Y-family polymerases stimulates
their efficiency to incorporate nucleotides correctly,
primarily through a reduction in the apparent K
m
of
the reaction [29,30]. It has been proposed that this
reduction is due to a PCNA-induced conformational

change in the polymerase active site [2]. The results
reported here are consistent with a possible role of the
C-terminus in modulating the conformational state of
the enzyme active site, raising the possibility that
protein–protein interactions affecting this region may
kinetically control the activity of Y polymerases.
Deletion of the C-terminus also has important conse-
quences during mismatch extension, causing a propen-
sity for the truncated enzyme to extend mispaired
primer termini through misalignment rather than by
direct extension. It is important to note that the confor-
mation of the primer–template junction must be quite
different in both circumstances. The enzyme must
accommodate a mispaired primer–template terminus
during direct extension, whereas a paired terminus is
available after misalignment. In fact, and as we previ-
ously observed with incorporation of correct nucleotides
after properly paired prime-template termini, the higher
efficiency of AtPOLKDC at extending by misalignment
essentially reflects an increase in the catalytic efficiency
for insertion of a ‘correct’ C opposite the second con-
secutive templating base. Therefore, the kinetic control
of DNA polymerase activity, perhaps achieved through
protein–protein interactions via the C-terminus, may
determine the relative contributions of direct incorpor-
ation and misalignment during mismatch extension
carried out by Y-family DNA polymerases.
We found that the ability of AtPOLK to bypass
8-oxoG is also influenced by its C-terminus. Most
M. V. Garcı

´
a-Ortiz et al. Role of C-terminus in AtPOLK DNA polymerase
FEBS Journal 274 (2007) 3340–3350 ª 2007 The Authors Journal compilation ª 2007 FEBS 3347
replicative DNA polymerases preferentially insert A
opposite 8-oxodG [21], causing G:C fi T:A transver-
sions [31]. Insertion preferences among Y-family DNA
polymerases vary considerably. Yeast and human
pol g [32,33] and archaean Dpo4 [34,35] preferentially
insert C opposite the lesion. In contrast, human pol j
inserts A more efficiently than C opposite 8-oxodG
[36–38], and human pol i is significantly blocked by
the lesion [39,40]. We found that AtPOLK shows a rel-
ative incorporation efficiency for A opposite 8-oxodG
that is twice that of C. However, AtPOLKDC inserted
both A and C with similar efficiency. On the other
hand, the truncated protein showed  10-fold higher
catalytic efficiency than the wild-type enzyme for
nucleotide insertion opposite 8-oxoG. Thus, deletion
of the C-terminus increases the bypass efficiency of
the protein but decreases its potential for mutagenic
translesion synthesis.
Taken together, the results reported here suggest
that the 193 C-terminal amino acids of AtPOLK may
modulate the catalytic activity of the protein, affecting
its catalytic efficiency and fidelity during synthesis on
undamaged DNA templates, its capacity to extend
mismatches through misalignment, and its bypass effi-
ciency through error-prone and error-free bypass.
Given the requirement for kinetically controlling error-
prone DNA polymerases when they are in the replica-

tion fork, the possibility exists that, in addition to
function in DNA polymerase targeting and recruit-
ment, the C-terminus of Y-family DNA polymerases
also plays a role in modulating their enzymatic activity
through protein–protein interactions. Elucidation of
the precise role of the C-terminal domain in Y-family
DNA polymerases will need more experimental work
and additional structural data on full-length enzymes.
Experimental procedures
Proteins and DNA substrates
His-tagged AtPOLK and AtPOLKDC proteins were
expressed in E. coli and purified as described previously
[12]. Oligonucleotides used (Supplementary material,
Table S1) were synthesized by Operon and were purified by
PAGE before use. Double-stranded DNA substrates were
prepared by mixing a 1.5-lm solution of a 5¢-fluorescein-
labelled 21-mer oligonucleotide primer (upper-strand
oligonucleotide) with a 1.0-lm solution of an unlabelled
40-nucleotide oligomer template (lower-strand oligonucleo-
tide), heating to 95 °C for 5 min and slowly cooling to
room temperature. Ultrapure dNTPs and Klenow enzyme
were obtained from Roche (Basel, Switzerland).
DNA polymerase assays
The standard DNA polymerase reaction mixture (10 lL)
contained 20 mm potassium phosphate buffer (pH 7.0),
4mm MgCl
2
, 0.4 mgÆmL
)1
BSA, 8% glycerol, 12.5 mm

dithiothreitol, and 100 lm each deoxynucleotide (dGTP,
dATP, dTTP, dCTP), except where noted. Substrate and
enzyme concentrations are specified in the figure legends
and text. Reactions were carried out at 30 °C for 30 min
unless indicated otherwise and terminated by addition of
10 lL formamide gel loading buffer (90% formamide,
1 · Tris ⁄ borate ⁄ EDTA buffer). After denaturation at
95 °C for 10 min, products were resolved by electrophor-
esis on a denaturing 15% polyacrylamide gel (acryl-
amide ⁄ bisacrylamide ¼ 19 : 1, 1 · Tris ⁄ borate ⁄ EDTA, 7 m
urea) (Owl Electrophoresis System, Portsmouth, NH) pre-
run for 30 min at 450 V. Fluorescein-labelled DNA was
visualized using the blue fluorescence mode of the FLA-
5100 imager and analysed using multigauge software
(Fujifilm, Tokyo, Japan).
Analysis of steady-state kinetics
AtPOLK or AtPOLKnC (50–100 nm ) was incubated with
100–200 nm primer–template substrate in the presence of
increasing concentrations of a single deoxynucleotide. After
incubation for 15 min at 30 °C under standard DNA
polymerase assay conditions, reactions were stopped and run
on a 15% polyacrylamide gel, containing 7 m urea, to
separate the unextended and extended DNA primers. Integ-
rated gel band intensities were measured using a FLA-5100
imager and multigauge software. The observed rate of
nucleotide incorporation (extended primer) was plotted as a
function of dNTP concentration. A Michaelis–Menten curve,
where v ¼ (V
max
[dNTP]) ⁄ (K

m
+ [dNTP]), was fitted to the
data by nonlinear regression (sigmaplot; Systat Software,
San Jose, CA). The k
cat
(V
max
⁄ [enzyme]) and K
m
steady-state
parameters defining the fitted curve were used to calculate
the frequency of deoxynucleotide incorporation (f
inc
)by
applying the equation f
inc
¼ (k
cat
⁄ K
m
)
incorrect
⁄ (k
cat
⁄ K
m
)
correct
.
Electrophoretic mobility-shift assay

Standard binding reactions were performed in a volume
of 10 lL containing 25 mm potassium phosphate buffer
(pH 7.4), 0.2 mgÆmL
)1
BSA, 5 mm dithiothreitol, 2.5%
glycerol, 15 m m KCl, 20 mm NaCl, 5¢-fluorescein-labelled
DNA (500 nm), 1 ng poly(dI-dC) and the indicated amounts
of protein. Reaction mixtures were incubated on ice for
15 min before being loaded on to 8% nondenaturing
polyacrylamide gels (acrylamide ⁄ bisacrylamide, 37.5 : 1)
and electrophoresed at 200 V at 4 °Cin1· Tris/acetate/
EDTA buffer.
Role of C-terminus in AtPOLK DNA polymerase M. V. Garcı
´
a-Ortiz et al.
3348 FEBS Journal 274 (2007) 3340–3350 ª 2007 The Authors Journal compilation ª 2007 FEBS
Acknowledgements
This research was supported by grant BMC2003-04350
from the Ministerio de Educacio
´
n y Ciencia, Spain, to
RRA. Financial support from the Junta de Andalucı
´
a
is also gratefully acknowledged.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. Structure-based sequence alignment of
AtPOLK, human pol j, S. solfataricus Dpo4, Dbh,
and E. coli DinB.
Table S1. DNA sequence of oligonucleotides used as
substrates.
This material is available as part of the online article
from
Please note: Blackwell Publishing is not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corres-
ponding author for the article.
Role of C-terminus in AtPOLK DNA polymerase M. V. Garcı
´
a-Ortiz et al.
3350 FEBS Journal 274 (2007) 3340–3350 ª 2007 The Authors Journal compilation ª 2007 FEBS