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 t
Trang 1polymerase 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: ge1roarr@uco.es
(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.
Trang 2and 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 n M ) was used as a positive control in lanes 8, 16, 24 and 32.
Trang 3templates, 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 AtPOLKDC 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 AtPOLKnC
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
Trang 4deoxynucleotides 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
Km and Vmax 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 (kcat⁄ Km) (Table 1) and the frequency of deoxynucleotide mis-incorporation [finc ¼ (kcat⁄ Km)incorrect⁄ (kcat⁄ Km)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 temtem-plate C The fidelity of full-length AtPOLK, measured as the frequency of deoxynucleotide misincorporation (finc), 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
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
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
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
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
1.9 · 10 -1
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 ).
Trang 5dCTP 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
(finc) 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 Km for the incoming nucleotide, whereas only minor differences were observed in kcat 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 Kmfor G, whereas the kcatdid 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.
kcat(min)1) Km(l M ) kcat⁄ K m (l M )1Æmin)1)
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
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
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 finc¼ (k cat ⁄ K m )incorrect⁄ (k cat ⁄ K m )correct.
Trang 6template 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 kcatand Km
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 Km for the incoming nucleotide, with minor differences in kcatvalues (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 scontain-ingle 7,8-dihydro-8-oxoGuanine (8-oxoG), a single 1,N6-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,
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 n M ) 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 (N4) Reaction mixtures in lanes 7 and 14 contained Klenow enzyme (250 n M ) with all four dNTPs The reaction mixture in lane 1 contained no enzyme.
Trang 7lane 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 kcat
and Km 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 (kcat⁄ Km) of incorrect
nucleotide incorporated to the efficiency (kcat⁄ Km) of
correct nucleotide incorporated (Table 3)
As indicated by the kcat⁄ Km 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 Km for the
incoming dNTP, as previously observed during DNA
synthesis in undamaged DNA The relative incorpor-ation efficiency to the incorporincorpor-ation 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
Km values for dATP or dCTP Whereas AtPOLK shows twice the Km 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 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
kcat(min)1) Km(l M ) kcat⁄ K m (l M1Æmin)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 1 1 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
Trang 8high-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
C2HC type (UBZ4) at their C-terminus [10], whereas
AtPOLK shows a single UBZ domain of the C2H2
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 finc 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 Kmfor 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 Kmcannot 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 Km 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
Trang 9replicative DNA polymerases preferentially insert A
opposite 8-oxodG [21], causing G:Cfi 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 addireplica-tion 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 95C 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),
4 mm MgCl2, 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 30C 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
95C 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 30C 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 v¼ (Vmax[dNTP])⁄ (Km+ [dNTP]), was fitted to the data by nonlinear regression (sigmaplot; Systat Software, San Jose, CA) The kcat(Vmax⁄ [enzyme]) and Kmsteady-state parameters defining the fitted curve were used to calculate the frequency of deoxynucleotide incorporation (finc) by applying the equation finc¼ (kcat⁄ Km)incorrect⁄ (kcat⁄ Km)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 mm 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 4C in 1 · Tris/acetate/ EDTA buffer
Trang 10This 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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