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Brief report
Human DNA polymerase lambda is a proficient extender of primer ends paired to 7,8-dihydro-8-oxoguanine Angel J. Picher, Luis Blanco ∗ ´ Centro de Biolog´ıa Molecular “Severo Ochoa” (CSIC-UAM), Universidad Autonoma, 28049 Madrid, Spain
a r t i c l e
i n f o
a b s t r a c t
Article history:
Pol ! is a DNA repair enzyme with a high affinity for dNTPs, an intrinsic dRP lyase activity, a
Received 26 March 2007
BRCT domain involved in interactions with NHEJ factors, and also capable to interact with
Received in revised form
the PCNA processivity factor. Based on this potential, Pol ! could play a role in BER, V(D)J
20 June 2007
recombination, NHEJ and TLS.
Accepted 21 June 2007
Here we show that human Pol ! uses a templating 7,8-dihydro-8-oxoguanine (8oxoG) base, a common mutagenic form of oxidative damage, as efficiently as an undamaged dG, but giving rise to the alternative insertion of either dAMP or dCMP. However, Pol !
Keywords:
strongly discriminated against the extension of the mutagenic 8oxoG:dAMP pair. Conversely,
DNA polymerase
Pol ! readily extended the non-mutagenic 8oxoG:dCMP pair with an efficiency that was
Translesion synthesis
even higher than that displayed on undamaged dG:dCMP pair. A similar capacity for non-
Non-homologous end joining
mutagenic extension was also shown to occur in the case of O6 -methylguanine (m6G), a mutagenic and cytotoxic DNA adduct. A comparison of these novel properties of human Pol ! with those of other DNA polymerases involved in TLS will be discussed. Interestingly, when double-strand breaks are associated to base damage, modifications as 8oxoG could be eventually part of the synapsis required to join ends, and therefore, the capacity of Pol ! either to insert opposite 8oxoG or to extend correct base pairs containing such a damage could be beneficial for its role in NHEJ. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
The human genome encodes 16 DNA polymerases [1], classified into four discrete families (A, B, X and Y) based on differences in the primary structure of their catalytic subunits [2]. Family X DNA polymerases are small enzymes present in different organisms ranging from viruses to higher eukaryotes [3–6]. Members of this family vary considerably in their biochemical features and associated functions, taking part in different processes like base excision repair (BER),
non-homologous end joining (NHEJ), V(D)J recombination and lesion bypass [1]. DNA polymerase ! (Pol !) is a family X member that is widespread among higher eukaryotes, both in animals and plants. The catalytic domain of human Pol ! is similar in sequence to human Pol " (32% amino-acid identity), and both enzymes share many of their biochemical properties, including a dRP lyase activity, required to complement the DNA synthesis step associated with BER, as shown both in vitro and in vivo [7,8]. Compared to Pol ", Pol ! has a higher affin-
Corresponding author. Tel.: +34 91 4978493; fax: +34 91 4974799. E-mail address:
[email protected] (L. Blanco). 1568-7864/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2007.06.007 ∗
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ity for dNTPs, leading to the suggestion that it would be the DNA repair enzyme of choice when intracellular dNTP concentrations are low [9]. Moreover, structural analysis of Pol ! has revealed a high overall structural similarity with Pol ", but also different conformational changes associated with catalysis [10,11]. Although the exact biological functions of Pol ! are not yet certain, the observation that Pol ! has an extraordinary ability to generate frame-shift errors [12] and an efficient mismatch extension capacity [13], suggests an ability to use the intermediates generated during NHEJ, one of the mechanisms to repair double-strand breaks (DSBs). In fact, filling short gaps during XRCC4-LigaseIV-dependent re-joining of DSBs requires the presence of Pol ! in HeLa cell extracts, suggesting that Pol ! contributes to NHEJ in human cells [14]. More recently, it has been shown that Pol ! is capable of performing junctional additions during NHEJ in vitro, based on its BRCT-mediated efficient interaction with the NHEJ factors Ku, XRCC4 and Ligase IV [15–17]. Additionally, Pol ! has been implicated in the immunoglobulin heavy chain recombination, during a specialized form of NHEJ, V(D)J recombination [18]. DNA lesions often block the progression of the replication fork, but replication through such lesions (bypass) can be achieved by the action of specialized DNA polymerases (i.e. family Y), in a process denominated translesion DNA synthesis (TLS). Error-free lesion bypass predominantly incorporates the correct nucleotide opposite the damage, whereas error-prone lesion bypass leads to the preferential incorporation of an incorrect nucleotide. Discrimination at the elongation steps immediately following the insertion opposite the damage can also contribute as a mutation-avoiding mechanism during TLS [19]. 7,8-Dihydro-8-oxoguanine (8oxoG) is a common form of oxidative damage, with steady-state levels of about 103 to 104 lesions per mammalian cell [20,21], which are specifically recognized by the OGG1 and MYH glycosilases to initiate BER [22]. If left unrepaired, this lesion would be highly mutagenic giving rise to dG·dC to dT·dA transversions [23]. This is because 8oxoG pairs with both dCMP and dAMP. In the 8oxoG·dCMP base pair, the glycosidic bonds of both residues are in the orthodox anti configuration, and these bases form normal Watson–Crick hydrogen bonds [24,25]. In the 8oxoG·dAMP base pair, the glycosidic bond of the 8oxoG residue is in the unusual syn configuration base pairing along its Hoogsteen edge with dAMP by forming two hydrogen bonds [26,27]. O6 -Methylguanine (m6G) is a mutagenic and cytotoxic DNA adduct that can be formed in vivo by such diverse agents as tobacco smoke, methylnitrosourea, and other alkylating agents [28]. This lesion gives rise to dG to dA transition mutations, because m6G base pairs both with dCMP and dTMP. DNA polymerases incorporate dTMP opposite m6G more often than dCMP, because the m6G·dTMP mispair retains the Watson–Crick geometry more closely than the m6G·dCMP base pair [29–32]. In addition to DNA polymerases Pol #, Pol $, Pol % and Pol &, Pol ! has also been implicated in TLS at abasic sites [33,34]. As in the case of Pol ! [13], these enzymes are relatively promiscuous in extending mismatched primer termini [35–38]. For these similarities, we determine the ability of Pol ! to replicate through 8oxoG and m6G, both at the insertion and extension
step. The data obtained are considered in comparison to other polymerases implicated in replication and several repair processes like BER, NHEJ and TLS.
2.
Materials and methods
2.1.
Materials
Synthetic oligonucleotides purified by PAGE were obtained from Invitrogen or Eurogentec. Template-primer molecules used for insertion assays were generated by annealing P1 primer (5" CTGCAGCTGATGCGC 3" ) to three T4 templates (5" GTACCCGGGGATCCGTACNGCGCATCAGCTGCAG 3" where N is G, 8oxoG or m6G). Template-primer molecules used for extension assays were generated by annealing P2 primers (5" CTGCAGCTGATGCGCN 3" , where N is A, C or T) to three T4 templates. All primers were labeled at its 5" -end with ['-32 P] ATP (3000 Ci/mmol, Amersham) and T4 polynucleotide kinase (New England Biolabs). The primers were then hybridized to template oligonucleotides to generate all different molecules in the presence of 50 mM Tris–HCl pH 7.5, and 0.3 M NaCl, and heating to 80 ◦ C for 10 min before slowly cooling to room temperature overnight. Human Pol ! core fragment (39 kDa) was expressed and purified as previously described [10]. Human Pol " was a generous gift of Dr. S.H. Wilson (NIEHS, Research Triangle Park, NC).
2.2.
Primer extension assays
The incubation mixture (20 (l) contained 50 mM Tris–HCl pH 7.5, 1 mM DTT, 4% glycerol, 0.1 mg/ml BSA, 5 nM labeled primer strand, 100 nM of each DNA polymerase (human Pol " or human Pol !), the indicated concentration of MgCl2 or MnCl2 , and the indicated concentration of each dNTP. Reactions were started by adding the indicated concentration of dNTP and incubated at 37 ◦ C for 20 min. After incubation, reactions were stopped by adding loading buffer (10 mM EDTA, 95% formamide, 0.03% bromophenol blue, and 0.3% cyanol blue). Extension of the labeled primer strand was analyzed by 8 M urea and 20% PAGE, and autoradiography.
2.3.
Steady-state kinetics assays
The incubation mixture (20 (l) contained 50 mM Tris–HCl pH 7.5, 2.5 mM MgCl2 , 1 mM DTT, 4% glycerol, 0.1 mg/ml BSA, 0.25 (M DNA, and 10 nM of human Pol !. dNTP (dCTP or dATP) concentrations used for insertion assays opposite dG or 8oxoG ranged from 0.02 to 2 (M. dGTP concentrations used for extension assays from dG·dCMP control base pair ranged from 0.01 to 5 (M. dGTP concentrations used for extension assays from 8oxoG containing base pairs ranged from 0.01 to 200 (M. dGTP concentrations used for extension assays from base pairs with m6G as template base ranged from 0.01 to 500 (M. Reactions were started by adding the indicated concentration of dNTP and incubated at 37 ◦ C for 20 min. After incubation, reactions were stopped by adding loading buffer. Samples were then run on 20% polyacrilamide sequencing gels containing 8 M urea, to separate the unextended and
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extended DNA primers. Gel band intensities were quantified using a BAS reader 1500 (Fujifilm). The observed rate of nucleotide incorporation (extended primer) was then plotted as a function of nucleotide concentration, and the apparent Km and Vmax parameters were obtained from the best fit to the Michaelis-Menten equation {V = Vmax × [dNTP]/(Km + [dNTP])} using nonlinear regression (Kaleidagraph, Synergy Software, www.synergy.com). In all cases the quality of the fit of the data was acceptable. The relative insertion/extension ◦ ◦ ), was calculated using the followefficiency (fins or fext ◦ ing equation: f = (kcat /Km )damaged /(kcat /Km )undamaged , where kcat = Vmax /[enzyme].
3.
Results
3.1.
Insertion opposite 8oxoG and m6G by human Pol !
In humans, all DNA polymerases from family X (Pol ", Pol !, Pol ( and TdT) lack a proofreading 3" → 5" exonuclease, but they differ in their interaction with DNA and, consequently, in their biological function during DNA repair [1]. As templatedependent enzymes, Pol " and Pol ! preferentially incorporate the correct nucleotide (dCMP) into a matched primer terminus that has dG as the templating base (Fig. 1A, left panels).
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On the other hand, when 8oxoG is the templating base, the behavior significantly differed (Fig. 1A, middle panels). Pol " preferentially inserted the “correct” nucleotide (dCMP) opposite the lesion, although at a lower rate compared to insertion of dCMP opposite undamaged dG. Pol " also inserted dAMP opposite 8oxoG, although at even lower level, accordingly with previous findings [39]. Unlike Pol ", Pol ! readily inserted both dCMP and dAMP opposite 8oxoG, paralleling dCMP insertion opposite undamaged dG. Insertion opposite m6G, which required activating Mn+2 ions, showed also significant differences between these two enzymes (Fig. 1A, right panels). Pol " performed the mutagenic incorporation of dTMP opposite m6G as efficiently as the correct dCMP insertion opposite undamaged dG, whereas the “correct” insertion (dCMP) opposite m6G occurred at a very low rate, accordingly with previous findings [40]. Contrary to Pol ", Pol ! poorly inserted both the mutagenic (dTMP) and “correct” (dCMP) nucleotides, at a similar rate than dGMP (probably inserted by dislocation of the m6G templating base). Therefore, these two family X members (Pol " and Pol !) would mainly promote error-prone bypass if they encountered a m6G lesion, via mutagenic insertion of dTMP opposite m6G or via dislocation of the lesion situated in the template strand.
Fig. 1 – TLS of 8oxoG and m6G lesions by human Pol ! and Pol !. (A) Insertion reactions opposite dG, 8oxoG or m6G by human Pol ! and human Pol !. The scheme shows the sequence of the DNA used in the assay. Reactions were carried out as described under Section 2, using 10 "M of each indicated dNTP. MgCl2 (2.5 mM) was used as activating ion in all cases, except for the insertion opposite m6G (MnCl2 , 1 mM). Extension of the 5" end labeled primer (*) was examined by PAGE. (B) Extension of primers paired to 8oxoG and m6G by human Pol ! and Pol !. The scheme shows the sequence of the DNA used in the assay. Extension reaction from the possible pairs that have dG, 8oxoG or m6G as template base. Reactions were carried out as described under Section 2, using MgCl2 (2.5 mM) as activating ion. dGTP concentrations are indicated in the figure. Extension of the 5" end labeled primer (*) was examined by PAGE. Please cite this article in press as: A.J. Picher, L. Blanco, Human DNA polymerase lambda is a proficient extender of primer ends paired to
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Table 1 – Steady-state kinetic parameters of insertion opposite dG and 8oxoG by human Pol ! Template
dNTP
Vmax ((M min−1 )
Km ((M)
kcat (min−1 )
kcat /Km (min−1 (M−1 )
◦ fins
dG 8oxoG 8oxoG
dCTP dCTP dATP
(4.04 ± 0.79) × 10−3 (3.27 ± 0.43) × 10−3 (3.52 ± 0.40) × 10−3
1.11 ± 0.10 1.32 ± 0.28 0.84 ± 0.02
4.04 × 10−1 3.27 × 10−1 3.52 × 10−1
3.64 × 10−1 2.48 × 10−1 4.19 × 10−1
6.81 × 10−1 1.15
Data are means (±standard error) from at least three independent experiments.
3.2. Steady-state kinetic analysis of nucleotide insertion opposite 8oxoG by human Pol ! Human Pol ! was used to examine the steady-state kinetics of insertion opposite either dG or 8oxoG as template bases. Primer extension was assessed for each dNTP representing a preferential insertion (dCMP opposite a dG template, and dAMP or dCMP opposite a 8oxoG template). The rate of nucleotide incorporation, obtained as described in Section 2, was plotted as a function of nucleotide concentration and Vmax and Km apparent values were then determined. From ◦ ), which is these values, the relative insertion efficiency (fins the ratio between the apparent kcat /Km of insertion of dCMP opposite undamaged dG and the apparent kcat /Km of insertion of dCMP or dAMP opposite 8oxoG, was then calculated. As shown in Table 1, human Pol ! inserts dAMP or dCMP ◦ opposite 8oxoG as efficiently as opposite undamaged dG (fins values close to 1), although Pol ! slightly prefers (1.7-fold) to insert dAMP opposite 8oxoG, favouring error-prone bypass of 8oxoG at the insertion step.
3.3. Pol ! preferentially extends a primer terminus correctly paired to 8oxoG and m6G template lesions Next, we compared the ability of Pol " and Pol ! to extend from the possible 3" primer termini paired or mispaired to 8oxoG or m6G lesions (Fig. 1B). As expected, the two DNA polymerases extended the undamaged dG·dCMP base pair by insertion of the next complementary nucleotide (dGMP) (Fig. 1B, left panels), Pol ! displaying a higher extension rate due to its higher affinity for dNTPs [9]. Then, we studied the extension of the two most frequent base pairs containing 8oxoG: 8oxoG·dCMP and 8oxoG·dAMP (Fig. 1B, middle panels). Pol " and Pol ! extended 8oxoG·dCMP much better than 8oxoG·dAMP. However, when compared to the extension of an undamaged dG·dCMP base pair, Pol " extended 8oxoG·dCMP similarly, whereas Pol ! showed a significant increase in the extension of 8oxoG·dCMP compared to the extension of the
undamaged dG·dCMP base pair (compare the extension reaction at 1 (M dGTP in Fig. 1B, left and middle panels), which suggests a specific predisposition of Pol ! for extending the “correct” 8oxoG·dCMP base pair. A similar study, analyzing the extension of the possible base pairs generated opposite m6G, was carried out and revealed different polymerization patterns by these two template-dependent enzymes from family X (Fig. 1B, right panels). Pol " poorly extended the m6G·dCMP base pair, and it was unable to extend the mutagenic m6G·dTMP base pair. Pol ! significantly extended the error-free pair m6G·dCMP, better than the mutagenic m6G·dTMP base pair, but worse than an undamaged dG·dCMP base pair. In summary, these two template-dependent DNA polymerases from family X prefer to extend (discriminate in favour of) the error-free base pair m6G·dCMP, but their relative extension efficiency (compared to undamaged DNA) appears to be different in each case.
3.4. Steady-state kinetic analysis of extension beyond pairs containing 8oxoG and m6G lesions by human Pol ! To further characterize the spectrum and efficiency of extension of a primer paired to 8oxoG and m6G lesions by Pol !, we measured the steady-state kinetics parameters of these reactions. As shown in Table 2, Pol ! extends the “correct” 8oxoG·dCMP base pair much more efficiently (23-fold) than the mutagenic pair 8oxoG·dAMP, even showing a catalytic efficiency higher (5-fold) than that shown for extension of undamaged dG·dCMP base pair. Therefore, Pol ! combines discrimination and high efficiency at the extension step of 8oxoG bypass, potentially contributing to error-free TLS. Moreover, Pol ! extends the “correct” m6G·dCMP base pair more efficiently than the mutagenic m6G·dTMP base pair (3.4-fold), again indicating that Pol ! could provide error-free discrimination at extension during TLS. In this case, the catalytic efficiency for extending the “correct” m6G·dCMP base pair is lower (4.4-fold) than that displayed on undamaged dG·dCMP base pair.
Table 2 – Steady-state kinetic parameters of extension from nucleotides opposite dG, 8oxoG or m6G by human Pol ! Template·primer
Vmax ((M min−1 )
Km ((M)
kcat (min−1 )
kcat /Km (min−1 (M−1 )
◦ fext
dG·dCMP 8oxoG·dCMP 8oxoG·dAMP m6G·dCMP m6G·dTMP
(3.38 ± 0.42) × 10−3 (5.87 ± 0.82) × 10−3 (2.55 ± 0.26) × 10−4 (4.87 ± 0.79) × 10−3 (1.86 ± 0.34) × 10−3
8.66 ± 0.96 3.06 ± 0.22 3.05 ± 0.58 5.50 ± 0.94 7.17 ± 0.93
3.38 × 10−1 5.87 × 10−1 2.55 × 10−2 4.87 × 10−2 1.86 × 10−2
3.90 × 10−2 1.92 × 10−1 8.36 × 10−3 8.86 × 10−3 2.59 × 10−3
4.92 2.14 × 10−1 2.27 × 10−1 6.64 × 10−2
Primer extension was measured in the presence of dGTP, the next correct nucleotide. Data are means (±standard error) from at least three independent experiments.
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Overall, the steady-state kinetic analysis indicates that Pol ! has a remarkably high efficiency for extending the correct nucleotide (dCMP) paired to a 8oxoG lesion, and also a significant efficiency for extending the correct nucleotide (dCMP) paired to a m6G lesion. In both cases, Pol ! shows discrimination, promoting the error-free bypass of both lesions.
4.
Discussion
Steady-state kinetics studies have been carried out with a wide range of DNA polymerases to quantify: (1) the efficiency and accuracy of nucleotide insertion opposite 8oxoG and m6G containing templates; (2) the efficiency and discrimination during extension from the possible pairs formed opposite those lesions. Most eukaryotic DNA polymerases involved in replication incorporate nucleotides opposite 8oxoG with both a low relative efficiency (in comparison with incorporation opposite an undamaged dG) and low accuracy (wrong dAMP insertion predominates over right dCMP insertion) [41–43]. On the other hand, most family Y DNA polymerases incorporate nucleotides opposite 8oxoG with high accuracy but the relative efficiency values vary from one to another, Pol $ from Saccharomyces cerevisiae being the only one that combines both high accuracy and high relative efficiency [44–47]. The only family X DNA polymerase evaluated to date, Pol ", has low accuracy and also a low relative efficiency in using dam-
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aged versus undamaged bases [39]. As shown here, as derived from steady-state kinetic analysis, human Pol ! possesses a high relative efficiency for inserting nucleotides opposite 8oxoG, but a low accuracy. Very similar data has been recently obtained by employing single-turnover kinetic methods [48]. So, in humans, none of the DNA polymerases studied to date using in vitro assays and in the absence of ancillary proteins, guarantees the error-free insertion opposite 8oxoG by combining high accuracy and high efficiency. Regarding the extension step of 8oxoG lesion bypass, most eukaryotic DNA polymerases studied combine low discrimination (Fig. 2A) and low relative efficiency (Fig. 2B) [41,43–46]. However, in this report we demonstrate the great potential of Pol ! to promote the error-free bypass of 8oxoG at the extension step, due to its high discrimination combined with such a high relative efficiency that allows extending the “correct” 8oxoG·dCMP base pair even more efficiently than the undamaged dG·dCMP base pair (Table 2). As shown in Fig. 2, Pol ! is the DNA polymerase that shows the best discrimination and relative efficiency values at the extension step of 8oxoG lesion bypass. Therefore, Pol ! would be the best candidate to guarantee an error-free extension step during bypass of the 8oxoG lesion. Regarding the extension step of m6G bypass, most eukaryotic DNA polymerases combine relative high discrimination and low relative efficiency [40,43,44,49]. As reported here, Pol ! presents a similar behavior, having a relatively high discrim-
Fig. 2 – Extension from pairs having 8oxoG as a template base by different eukaryotic DNA polymerases. (A) Discrimination is expressed as the ratio between the extension efficiency of the “correct” 8oxoG·dCMP base pair and the extension efficiency of the mutagenic 8oxoG·dAMP base pair. Data represented corresponds to calf thymus Pol # [41], Saccharomyces cerevisiae Pol # [43], S. cerevisiae Pol $ [45], human Pol $ [46], human Pol % [44] and human Pol ! (Table 2). (B) Relative efficiency of extension from pairs having 8oxoG as a template base by different eukaryotic DNA polymerases. Relative efficiency of extension is expressed as the ratio between the catalytic efficiency of extension from the possible pairs formed opposite 8oxoG (8oxoG·dAMP or 8oxoG·dCMP) versus that from undamaged dG·dCMP base pair. Data represented corresponds to calf thymus Pol # [41], S. cerevisiae Pol # [43], S. cerevisiae Pol $ [45], human Pol $ [46], human Pol % [44] and human Pol ! (Table 2). Family B members are represented in light gray, family Y members are represented in gray and the family X member is represented in black. Please cite this article in press as: A.J. Picher, L. Blanco, Human DNA polymerase lambda is a proficient extender of primer ends paired to
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ination and moderate relative extension efficiency, lower than that shown on undamaged substrate. Therefore, none of the DNA polymerases studied so far using in vitro assays and in the absence of ancillary proteins, would ensure the error-free bypass of m6G lesion at the extension step. In summary, Pol ! appears to be adapted to extend primers correctly paired to 8oxoG lesions (error-free pathway), although it is capable to extend 8oxoG-containing mismatches at a lower but significant extent. Moreover, Pol ! is the only DNA polymerase with a higher catalytic efficiency to extend a damaged base pair (8oxoG·dCMP) versus the undamaged one (dG·dCMP). Overall, and considering the interaction of Pol ! with PCNA [33], these data support a role of Pol ! as a TLS error-free extender during the bypass of 8oxoG (and perhaps of m6G) lesions. Interestingly, a very recent report has shown that the presence of PCNA and RPA modulates the accuracy of Pol ! insertion opposite 8oxoG [50], strongly favouring the correct insertion of dCMP. Therefore, Pol ! would be fully suited for both insertion and extension steps during in vivo TLS at 8oxoG containing templates. These new properties of Pol ! could have also important implications in its role in gap-filling synthesis during the NHEJ repair process. Exogenous and endogenous agents that provoke DSBs could also induce chemical modifications at the nitrogen bases of nucleotides located close to the site of the DSB. Therefore, those modified nucleotides would be at the heart of the re-joining reaction, either as templates during gap-filling or as part of the synapsed portion connecting the two ends and serving as primer for eventual gap-filling and ligation. In this context, Pol ! would be well suited to accept end-connections containing lesions, improving the efficiency and discrimination when extending pairs formed by modified nitrogen bases at the template strand. Moreover, in the absence of PCNA and RPA, the low accuracy of Pol ! when inserting opposite 8oxoG would not be a handicap during end joining. As fidelity is not a main issue during NHEJ, taking the maximal advantage from the dual templating potential of the 8oxoG lesion could be instrumental to bridge damaged DNA ends. Interestingly, a similar capacity to bypass 8oxoG templating bases has been recently reported with the polymerization domain of LigD [51], one of the two sole components of the NHEJ system operating in mycobacteria. Why is Pol ! so efficient when using 8oxoG containing templates? Compared to Pol ", Pol ! makes a considerable lower number of contacts with the DNA at the primer terminus [10,11], which is probably related to its great potential for extending mismatched base pairs [13]. Moreover, Pol ! has a much higher affinity for the incoming nucleotides [9], a factor that could be important to stabilize the oxygen at the C8 position of 8oxoG to form a catalytically active ternary complex, as it has been suggested [52]. A resolution of the crystal structure of Pol ! complexed with different 8oxoG containing substrates would provide invaluable information to explain its ability to negotiate these lesions during TLS or NHEJ processes.
Acknowledgements We thank Dr. Samuel Wilson for providing human Pol ", and Dr. Thomas A. Kunkel and Dr. Lawrence F. Povirk for help-
ful discussions and critical reading of the manuscript. This work was supported by Ministerio de Ciencia y Tecnolog´ıa Grant BMC 2003–00186 and by an institutional grant to Centro ´ Ramon ´ de Biolog´ıa Molecular “Severo Ochoa” from Fundacion Areces. AJP was recipient of a fellowship from the Ministerio ´ y Ciencia. de Educacion
references
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