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 ´ ´ ´ Marıa Victoria Garcıa-Ortiz, Teresa Roldan-Arjona and Rafael R Ariza ´ ´ Departamento de Genetica, Universidad de Cordoba, Spain Keywords Arabidopsis thaliana; base-pair mismatch; DNA damage; DNA replication; translesion DNA synthesis Correspondence ´ R R Ariza, Departamento de Genetica, Edificio Gregor Mendel, Campus de ´ Rabanales s ⁄ n, Universidad de Cordoba, ´ 14071-Cordoba, Spain Fax: +34 957 212 072 Tel: +34 957 218 979 E-mail: ge1roarr@uco.es (Received 10 April 2007, accepted 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 polymerases 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-conserved 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 propensity to extend mispaired primer termini through misalignment and enhanced error-free bypass activity on DNA templates containing 7,8-dihydro-8-oxoGuanine These results suggest that, in addition to facilitating 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 Cells are equipped not only with high-fidelity enzymes that accurately replicate the genome, but also with specialized 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 Saccharomyces cerevisiae pol g (Rad30) and Rev1 [4] UmuClike 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 aminoacid 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] Abbreviations PCNA, proliferating cell nuclear antigen, UBZ, ubiquitin-binding Zn-finger motif; 8-oxoG, 7,8-dihydro-8-oxoGuanine; edA, 1,N6-ethenoadenine 3340 FEBS Journal 274 (2007) 3340–3350 ª 2007 The Authors Journal compilation ª 2007 FEBS ´ M V Garcıa-Ortiz et al 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-terminal region with a low degree of sequence conservation 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 proliferating 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 evolutionarily conserved in all Y-family polymerases and are required for their recruitment to replication factories [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 facilitates their recruitment to stalled replication forks Role of C-terminus in AtPOLK DNA polymerase 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 previously 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 conservation (Fig 1A) In the C-terminal region, AtPOLK contains 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 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 nM) 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 nM) 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 FEBS Journal 274 (2007) 3340–3350 ª 2007 The Authors Journal compilation ª 2007 FEBS 3341 ´ M V Garcıa-Ortiz et al Role of C-terminus in AtPOLK DNA polymerase templates, enhanced its ability to extend mismatches through misalignment, and strongly influenced its translesion 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-terminal 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 experiments 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 corresponding 40-mer template (Fig 1B) Both AtPOLK and AtPOLKDC synthesized DNA, extending the primer 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 distributive 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 3342 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 polymerase 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 polymerization 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, doublestranded 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 FEBS Journal 274 (2007) 3340–3350 ª 2007 The Authors Journal compilation ª 2007 FEBS ´ M V Garcıa-Ortiz et al Role of C-terminus in AtPOLK DNA polymerase A B Fig 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 compared with that of free DNA, as indicated (A) DNA-binding affinity for different DNA substrates Two concentrations (1.0 and 1.5 lM) of AtPOLK (wt) or AtPOLKDC (DC) were incubated with 0.5 lM each of labelled single-stranded, double-stranded or primer–template DNA substrates (B) DNA-binding affinity for a primer–template DNA structure Increasing amounts of AtPOLK or AtPOLKDC (0.5, 1.0 and 1.5 lM) were incubated with a labelled primer–template DNA substrate (0.5 lM) used to calculate the catalytic efficiency (kcat ⁄ Km) (Table 1) and the frequency of deoxynucleotide misincorporation [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 template G and C, and dATP opposite template 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 deoxynucleotides opposite each of the four template bases A DNA polymerase extension assay was used to measure the rate of deoxynucleotide incorporation, calculated 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 deoxynucleotides were obtained from each fitted curve and Table 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 kcat (min)1) AtPOLK dNTP Template dATP dGTP dTTP dCTP Template dATP dCTP Template dTTP dCTP Template dTTP dCTP kcat ⁄ Km (lM)1Ỉmin)1) Km (lM) AtPOLKDC AtPOLK 0.059 0.111 0.119 0.081 52.60 12.51 83.30 156.80 AtPOLKDC AtPOLK 390.46 0.41 28.25 171.68 4.6 1.0 6.8 2.4 AtPOLKDC C 0.024 0.125 0.057 0.0038 ± ± ± ± 0.001 0.010 0.001 0.0004 ± ± ± ± 0.010 0.005 0.006 0.001 ± ± ± ± 64.40 3.59 8.34 85.80 ± ± ± ± 272.15 0.08 8.85 45.03 · · · · 10-4 10-2 10-4 10-5 1.5 · 10-4 0.27 4.2 · 10-3 4.7 · 10-4 T 0.157 ± 0.014 0.038 ± 0.001 0.152 ± 0.005 0.131 ± 0.003 2.61 ± 0.78 62.29 ± 24.38 0.13 ± 0.02 72.94 ± 9.23 6.0 · 10-2 6.1 · 10-4 1.17 1.8 · 10-3 0.207 ± 0.010 0.045 ± 0.003 0.145 ± 0.003 0.063 ± 0.003 4.70 ± 0.94 100.41 ± 46.48 0.69 ± 0.05 233.38 ± 36.95 4.4 · 10-2 4.5 · 10-4 2.1 · 10-1 2.7 · 10-4 0.022 ± 0.001 0.061 ± 0.004 0.131 ± 0.007 0.116 ± 0.006 29.16 ± 7.30 7.76 ± 1.60 10.07 ± 4.10 0.61 ± 0.11 7.6 · 10-4 7.9 · 10-3 1.3 · 10-2 1.9 · 10-1 A G FEBS Journal 274 (2007) 3340–3350 ª 2007 The Authors Journal compilation ª 2007 FEBS 3343 ´ M V Garcıa-Ortiz et al Role of C-terminus in AtPOLK DNA polymerase a significant reduction in the apparent Km for the incoming nucleotide, whereas only minor differences were observed in kcat values For example, the  30fold increase in the efficiency of G insertion opposite the C template nucleotide was accompanied by a  30fold decrease in the Km for G, whereas the kcat did not change significantly (Table 1) These results collectively suggest that the C-terminal domain of AtPOLK negatively affects its catalytic efficiency for correct insertion, decreasing the fidelity of the enzyme Fig Misincorporation frequencies for AtPOLK and AtPOLKDC Steady-state assays were performed as described in Experimental procedures, and the kcat and Km parameters (Table 1) were used to calculate the frequency of deoxynucleotide incorporation (finc) by applying the equation finc ¼ (kcat ⁄ Km)incorrect ⁄ (kcat ⁄ Km)correct dCTP opposite template C) to 9.6 · 10)2 (for the misincorporation 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 fulllength 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 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 truncated 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 Steady-state kinetic parameters for G:T mispair extension by AtPOLK and AtPOLKDC Data are mean ± SE from at least two independent experiments ND, not detected kcat (min)1) kcat ⁄ Km (lM)1Ỉmin)1) Km (lM) Substrate Sequence dNTP AtPOLK AtPOLKDC AtPOLK AtPOLKDC AtPOLK AtPOLKDC Substrate I 5¢ GAG 3¢ CTTAGA- 0.029 ± 0.002 ND 0.123 ± 0.010 ND 36.95 ± 10.93 ND 49.00 ± 18.90 ND 7.8 · 10)4 ND 2.5 · 10)3 ND Substrate II 5¢ GAG 3¢ CTTCGA- dTTP dATP, dGTP or dCTP dGTP dCTP dATP or dTTP 0.048 ± 0.003 0.073 ± 0.010 ND 0.088 ± 0.010 0.130 ± 0.004 ND 61.65 ± 36.69 181.95 ± 35.36 ND 80.41 ± 33.18 12.98 ± 1.82 ND 7.8 · 10)4 4.0 · 10)4 ND 1.1 · 10)3 1.0 · 10)2 ND 3344 FEBS Journal 274 (2007) 3340–3350 ª 2007 The Authors Journal compilation ª 2007 FEBS ´ M V Garcıa-Ortiz et al Role of C-terminus in AtPOLK DNA polymerase 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 concentrations of dATP, dGTP, dCTP or dTTP The measured rate of nucleotide incorporation was plotted as a function of dNTP concentration, and kcat and 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 correct 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 nucleotides Interestingly, however, both proteins use these two modes of mispair extension to different degrees Extension through misalignment is performed by AtPOLK with a similar efficiency to extension by direct incorporation (efficiencies of · 10)4 and 7.8 · 10)4, A 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 kcat values (Table 2) These results suggest that differences in catalytic efficiency for insertion 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 analysed their ability to replicate from templates containing a single 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 moderately extend from the resulting primer end (Fig 4A, B Fig 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, and 6), 8-oxoG (lanes and 7), edA (lanes and 8), or an abasic site (lanes and 9) at the position indicated by X AtPOLK (wt, 500 nM) or AtPOLKDC (DC,100 nM) was incubated with the DNA substrate (100 nM) in the presence of each of four dNTPs The reaction mixture in lane 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 and 14 contained Klenow enzyme (250 nM) with all four dNTPs The reaction mixture in lane contained no enzyme FEBS Journal 274 (2007) 3340–3350 ª 2007 The Authors Journal compilation ª 2007 FEBS 3345 ´ M V Garcıa-Ortiz et al Role of C-terminus in AtPOLK DNA polymerase Table 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 kcat (min)1) DNA substrate Incoming nucleotide AtPOLK Insertion opposite G 5’ -GTAGAG dCTP 3’ -CATCTCGGAInsertion opposite 8-oxoG 5’ -GTAGAG dCTP 3’ -CATCTCGGA8’-oxo dATP 0.061 ± 0.04 AtPOLKDC 0.116 ± 0.006 AtPOLK AtPOLKDC AtPOLK Relative incorporation efficiency AtPOLKDC AtPOLK 7.76 ± 1.60 0.61 ± 0.11 7.9 · 10)3 1.9 · 10)1 AtPOLKDC 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 24.40 ± 8.42 3.72 ± 0.29 4.5 · 10)3 3.7 · 10)2 0.57 0.19 0.110 ± 0.01 0.138 ± 0.04 lane 3) AtPOLKDC showed the same bypass specificity as AtPOLK, being blocked by the abasic site and the edA adduct, but not by the 8-oxoG lesion However, after insertion opposite 8-oxodG, it performed extension from the 3¢-primer terminus with significantly 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 extension 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 and 13) To measure the ratio of dATP:dCTP incorporation during the bypass of 8-oxoG, we measured the steadystate kinetic parameters of nucleotide insertion opposite 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 3346 kcat ⁄ Km (lM1Ỉmin)1) Km (lM) synthesis in undamaged DNA The relative incorporation 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 insertion 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 opposite 8-oxodG than for C (0.57 versus 0.27) However, AtPOLKDC inserted both A and C with similar efficiency (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 error-free or errorprone 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 polymerases obliges cells to regulate their access to DNA, specifically recruiting them when and where they are needed The ‘DNA polymerase switch model’ postulates a transient replacement of the replicative DNA polymerase in the vicinity of the lesion by one or several error-prone polymerases before resumption of FEBS Journal 274 (2007) 3340–3350 ª 2007 The Authors Journal compilation ª 2007 FEBS ´ M V Garcıa-Ortiz et al high-fidelity replication [23] Current understanding of this process favours the idea that switching is modulated via interaction of the C-terminal domain of Y-family DNA polymerases with the replication processivity 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-terminus in polymerase targeting and recruitment, our understanding of its role in Y-family DNA polymerase catalytic activity is still limited Efforts to relate the enzymatic properties of Y-family DNA polymerases to their structural characteristics have focused on the catalytic core, neglecting the poorly conserved C-terminal region To our knowledge, no detailed kinetic 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 compared with the full-length protein [26] We have previously 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-terminal 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 structure 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 Role of C-terminus in AtPOLK DNA polymerase 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 efficiency 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 Km for the nucleotide It is possible that deletion 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 However, it is important to recall that Km cannot be correlated 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 consequences during mismatch extension, causing a propensity for the truncated enzyme to extend mispaired primer termini through misalignment rather than by direct extension It is important to note that the conformation 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 previously 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 consecutive 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 incorporation 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 FEBS Journal 274 (2007) 3340–3350 ª 2007 The Authors Journal compilation ª 2007 FEBS 3347 ´ M V Garcıa-Ortiz et al Role of C-terminus in AtPOLK DNA polymerase replicative DNA polymerases preferentially insert A opposite 8-oxodG [21], causing G:C fi T:A transversions [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 relative 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 efficiency through error-prone and error-free bypass Given the requirement for kinetically controlling errorprone DNA polymerases when they are in the replication fork, the possibility exists that, in addition to function in DNA polymerase targeting and recruitment, 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 DNA polymerase assays The standard DNA polymerase reaction mixture (10 lL) contained 20 mm potassium phosphate buffer (pH 7.0), mm MgCl2, 0.4 mgỈmL)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 unless indicated otherwise and terminated by addition of 10 lL formamide gel loading buffer (90% formamide, · Tris ⁄ borate ⁄ EDTA buffer) After denaturation at 95 °C for 10 min, products were resolved by electrophoresis on a denaturing 15% polyacrylamide gel (acrylamide ⁄ bisacrylamide ¼ 19 : 1, · Tris ⁄ borate ⁄ EDTA, m urea) (Owl Electrophoresis System, Portsmouth, NH) prerun for 30 at 450 V Fluorescein-labelled DNA was visualized using the blue fluorescence mode of the FLA5100 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 at 30 °C under standard DNA polymerase assay conditions, reactions were stopped and run on a 15% polyacrylamide gel, containing m urea, to separate the unextended and extended DNA primers Integrated 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 (Vmax[dNTP]) ⁄ (Km + [dNTP]), was fitted to the data by nonlinear regression (sigmaplot; Systat Software, San Jose, CA) The kcat (Vmax ⁄ [enzyme]) and Km steady-state parameters defining the fitted curve were used to calculate the frequency of deoxynucleotide incorporation (finc) by applying the equation finc ¼ (kcat ⁄ Km)incorrect ⁄ (kcat ⁄ Km)correct 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¢-fluoresceinlabelled 21-mer oligonucleotide primer (upper-strand oligonucleotide) with a 1.0-lm solution of an unlabelled 40-nucleotide oligomer template (lower-strand oligonucleotide), heating to 95 °C for and slowly cooling to room temperature Ultrapure dNTPs and Klenow enzyme were obtained from Roche (Basel, Switzerland) 3348 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 mgỈmL)1 BSA, mm dithiothreitol, 2.5% glycerol, 15 mm KCl, 20 mm NaCl, 5¢-fluorescein-labelled DNA (500 nm), ng poly(dI-dC) and the indicated amounts of protein Reaction mixtures were incubated on ice for 15 before being loaded on to 8% nondenaturing polyacrylamide gels (acrylamide ⁄ bisacrylamide, 37.5 : 1) and electrophoresed at 200 V at °C in · Tris/acetate/ EDTA buffer FEBS Journal 274 (2007) 3340–3350 ª 2007 The Authors Journal compilation ª 2007 FEBS ´ M V Garcıa-Ortiz et al Role of C-terminus in AtPOLK DNA polymerase 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 15 16 References Friedberg EC (2005) Suffering in silence: the tolerance of DNA damage Nat Rev Mol Cell Biol 6, 943–953 Prakash S, Johnson RE & Prakash L (2005) Eukaryotic translesion synthesis DNA 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PIP motif and the ubiquitin-binding domains facilitates their recruitment to stalled replication forks Role of C-terminus in AtPOLK DNA polymerase and displacement of the replicative DNA polymerase