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J. Biol. Chem., Vol. 281, Issue 35, 25026-25040, September 1, 2006
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From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021
Received for publication, April 6, 2006 , and in revised form, June 28, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The LigD POL domain catalyzes either nontemplated single nucleotide additions to a blunt-ended duplex DNA or fill-in synthesis at a 5'-tailed duplex DNA; these are the molecular signatures of mutagenic mycobacterial NHEJ in vivo at bluntend and 5'-overhang DSBs, respectively (2, 9, 10). POL activity in vitro is optimal in the presence of manganese (2, 5). rNTPs are preferred over dNTPs as substrates for nontemplated bluntend addition (2,5). During templated synthesis in the presence of dNTPs on a DNA primer-template with an 18-nucleotide 5' tail, the primer is elongated to the end of the template strand and then further extended with a single nontemplated nucleotide (2, 5). LigD POL can also add templated ribonucleotides to a DNA primer, but extension is limited to about 4 cycles of rNMP incorporation because the primer-template is rendered progressively less active as ribonucleotides accumulate at the 3' end (6). These properties suggest that the initial insertions preceding the strand sealing step of NHEJ might involve rNMP incorporation and that the ability of LigD to use rNTPs as substrates might be advantageous for the repair of chromosomal breaks that arise in quiescent cells, insofar as the dNTP pool might be limiting when bacteria are not actively replicating.
The ribonucleotide addition reactions of LigD POL are of particular interest given its structural similarity to the catalytic subunit of archaeal/eukaryal primase (9, 11–14), the enzyme responsible for synthesizing the RNA primers for lagging strand DNA replication in archaeal and eukaryal species. The crystal structures of Pseudomonas LigD POL and Pyrococcus horikoshii primase align with a root mean squared deviation of 3.7 Å at 203 C
positions (9, 12). LigD POL lacks a distinctive helical domain appended to archaeal primase as an insert to the sequence of the catalytic domain; it also lacks the zinc-binding motif located within the primase catalytic domain (9). Thus, LigD POL is a minimized version of an archaeal primase-polymerase. It is the first example of such a protein in the bacterial domain of life, and one with a novel role in DNA repair rather than priming of DNA replication.
Eukaryal DNA primase is optimally active in the presence of manganese (15) and displays an exceptionally high rate of nucleotide misincorporation during templated RNA synthesis (16–19). Archaeal DNA primase, too, is optimally active in the presence of manganese (20–22). Archaeal primase has templated DNA and RNA polymerase activities and nontemplated 3'-terminal transferase activity with dNTP or rNTP substrates (22–25). To our knowledge, studies of the fidelity of archaeal primase-polymerase during templated synthesis have not been reported. The issue of whether LigD POL is a relatively faithful or unfaithful enzyme during templated synthesis is pertinent, given the mutagenic quality of NHEJ in vivo.
Here we examine the fidelity of the POL component of Pseudomonas LigD, focusing on the following questions. How does the rate of incorporation of the correctly base-paired nucleotide compare with the rates of misincorporation of incorrect nucleotides? Is POL fidelity influenced by whether the incoming substrate is a ribonucleotide or a deoxyribonucleotide? How does LigD POL perform in extending a primer containing a preformed 3' base mismatch? Does its capacity to extend a mismatch depend on whether the enzyme is in DNA polymerase or RNA polymerase mode?
We also address the effects of two kinds of DNA lesions on LigD POL activity: abasic sites and polycyclic aromatic hydrocarbon-DNA adducts. Abasic lesions, which can arise by spontaneous hydrolysis of the N-glycosidic bond or base excision catalyzed by DNA glycosylases, comprise a very common form of DNA damage. Abasic sites can elicit different responses from different DNA polymerases, ranging from arrest of the replication fork to bypass of the lesion. Bypass entails two distinct polymerization steps: insertion of a dNMP across from the abasic site (ostensibly without the benefit of instructional base pairing) and subsequent extension from the 3'-dNMP:abasic terminus (26–28). Here we query how LigD POL behaves when it encounters an abasic site on the template strand at positions opposite from or immediately flanking the 3' nucleotide of the primer strand.
Polycyclic aromatic hydrocarbons (PAHs) such as benzo-[a]pyrene (BP) and benzo[c]phenanthrene (BPh) are potent environmental carcinogens. BP and BPh are converted in vivo to bay- or fjord-region diol epoxides, which react with the deoxyadenosine (dA) or deoxyguanosine (dG) bases in DNA to form covalent PAH-DNA adducts that either occupy the minor groove (BPdG) or intercalate between base pairs (BPhdA) (29). Synthetic oligonucleotides containing single PAH adducts have been used to study how these mutagenic lesions affect DNA polymerases (30–32). Here we report the impact of BPdG and BPhdA adducts in the template strand on the translesion insertion and extension reactions of LigD POL.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-D-galactopyranoside-induced Escherichia coli BL21(DE3) by nickel-agarose chromatography as described (5). The protein concentration was determined with the Bio-Rad dye reagent with bovine serum albumin as the standard.
Primer-Templates—Oligonucleotide primer strands were 5' 32P-labeled by reaction with T4 polynucleotide kinase and [
-32P]ATP, then purified by native gel electrophoresis, and annealed to a 4-fold molar excess of an unlabeled complementary DNA strand to form the primer-templates depicted in the figures. Oligonucleotides containing single tetrahydrofuran abasic sites or single covalent PAH-DNA adducts of known chirality were described previously (33–35).
Assay of Templated Primer Extension—Reaction mixtures containing 50 mM Tris-HCl (pH 7.5), 5 mM dithiothreitol, 5 mM MnCl2, 25 nM 32P-labeled primer-template, 1.4 µM LigD POL, and nucleotides as specified were incubated at 37 °C. Aliquots (20 µl) were withdrawn at the times specified and quenched immediately by adding an equal volume of 96% formamide, 20 mM EDTA. The 32P-labeled products were resolved by electrophoresis through a 15-cm 18% polyacrylamide gel containing 7 M urea in TBE (90 mM Tris borate, 2.5 mM EDTA). The products were visualized by autoradiography. Where indicated, primer extension was quantified by scanning the gel with a Fujifilm BAS-2500 imager. The percentage of input primer extended was plotted as a function of reaction time. The data were then normalized to the end point values for extension (defined as 100%) and the apparent rate constant (k) for the initial addition step was calculated by fitting the normalized data to the equation: 100 – % extension(norm) = 100 e–kt.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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A set of four primer-templates was prepared that consisted of a 13-mer 5' 32P-labeled DNA primer strand annealed to a complementary 18-mer DNA strand to form a 13-bp duplex with a five-nucleotide single-stranded 5' tail (Figs. 1, 2, 3, 4). The primertemplates differed only in the identity of the template deoxynucleotide base immediately flanking the 3'-OH primer terminus. We measured the rate of extension of the primertemplate (25 nM) by excess POL (1.4 µM) in the presence of single dNTP or rNTP substrates (10 µM), and thereby probed all possible misincorporation events. A shorter time course was monitored for the reactions containing the correct dNTP and rNTP (e.g. 5 s, 15 s, 30 s, 1 min, and 2 min) than for those containing only mispaired nucleotides (which were sampled at 1, 2, 5, 10, and 20 min in panels A in Figs. 1, 2, 3, 4).
Fig. 1A illustrates the selectivity of nucleotide incorporation at a dT in the template strand. When presented with the correct deoxyribonucleotide dATP, LigD POL sequentially incorporated two dAMP residues in response to the TT sequence in the template strand. Whereas 44% of the input primer was extended by at least one nucleotide in 5 s with dATP, 93% of the input primer was elongated in 5 s with ATP. Extension by two nucleotides was virtually complete within 1 min for both dATP and ATP and there was no further elongation beyond the n+2 position within the time frame of the experiment. By quantifying the fraction of the input primer extended, we obtained the kinetic profiles for the first adenosine nucleotide addition step shown in Fig. 1B. The correct ribonucleotide was more rapidly utilized by LigD POL (apparent rate constant of 
0.6 s–1 for ATP) than was the correctly paired deoxynucleotide (the kobs for dATP being 0.13 s–1). Control experiments showed that the apparent rate constant and the end point of the primer extension reaction in the presence of ATP were unchanged when the concentration of POL was decreased from 1.4 to 0.7 or 0.35 µM, which implied that POL binding to the primer-template was not rate-limiting and that the reaction could be treated as a pseudo-first order process (data not shown). Also, the rate constant for dATP addition did not increase when the POL concentration was doubled to 2.8 µM (data not shown), signifying that enzyme binding to the primer-template was not rate-limiting for the DNA polymerase reaction.
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2% of the rate at which the correct dAMP residue was incorporated. LigD POL also misincorporated single dGMP and dCMP residues opposite the template dT, to the extent that 53 and 35% of the input primer was elongated after 20 min in the presence of dGTP and dCTP, respectively (Fig. 1A).
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12-fold faster than dGMP; and extension with CMP was 5-fold faster than dCMP.
Effects of the Templated Base on POL Fidelity—Fig. 2A shows the kinetics of elongation on a primer-template containing a templated dA nucleotide flanking the 3'-OH of the primer strand. LigD POL rapidly incorporated dTMP when provided with the correct dTTP substrate, such that nearly all of the input primer was extended within 1 min. Note that the POL did not cease after adding the first TMP, but proceeded to add a second dTMP nucleotide opposite the n+2 T on the template strand. This result confirms the finding in Fig. 1A that POL is adept at misincorporating T opposite T. The addition of the correct ribonucleotide UMP was complete in 5 s and the reaction was largely limited to a single step of incorporation. The kinetic profile plotted in Fig. 2B shows that UTP (k 
0.6 s–1) was utilized at least 12-fold faster than dTTP (k = 0.048 s–1) for the first addition step. Here, dGTP was the best of the misincorporated deoxynucleotides (initial rate 1.6% of dTTP), followed by dATP (initial rate 1% of dTTP) and dCTP (0.3% of dTTP). Note that two nucleotides were added in the presence of dATP, which reflects the correct addition of dAMP opposite the n+2 dT template base despite the presence of a dA:dA mispair at the primer terminus. Ribonucleotide misincorporation was fastest with ATP, where we again observed a second cycle of AMP incorporation opposite the n+2 dT (Fig. 2A). By including measurements of dATP primer extension at shorter time points (Fig. 2B), we calculated an apparent rate constant of 0.02 s–1 for AMP misincorporation opposite dA; this value was 30-fold less than the rate of correct UMP incorporation. Nonetheless, the initial rate of AMP incorporation was 50-fold faster than the rate of dAMP addition. GTP (k = 0.008 s–1) was utilized at 1.3% the rate of UTP, but 10-fold faster than dGTP. CTP-dependent extension (k = 0.0017 s–1) was 0.3% of the rate with UTP, but still 10-fold faster than the rate with dCTP.
The salient themes from the above experiments, that LigD POL is unfaithful and is even more unfaithful as an RNA polymerase than a DNA polymerase, are reinforced by the kinetics of nucleotide addition opposite a templated dC (Fig. 3) and a templated dG (Fig. 4). In brief, dGMP and GMP were both added rapidly opposite the templated dC base (Fig. 3A). From the data in Fig. 3B, we calculated rate constants of 0.31 and 0.57 s–1 for correct dGMP and GMP addition, respectively. All of the incorrect dNTPs were comparatively poor substrates for misincorporation opposite dC (with initial rates of extension on the order of 0.1–0.3% of the initial rate with dGTP). However, rNTPs were readily misincorporated opposite dC (Fig. 3A), with ATP being fastest (k = 0.016 s–1), followed by UTP (k = 0.008 s–1) and CTP (k = 0.003 s–1) (Fig. 3B). The initial rates of AMP, UMP, and CMP addition were 30-, 30-, and 10-fold faster than the rates of dAMP, dTMP, and dCMP, respectively.
dCMP and CMP were incorporated rapidly opposite the templated dG base (Fig. 4A), with rate constants of 0.25 and 0.6 s–1, respectively (Fig. 4B). dTTP was the best deoxynucleotide substrate for misincorporation, at 0.5% the rate of dCTP utilization. All rNTPs were readily misincorporated opposite dG, with UTP being fastest (k = 0.04 s–1), followed by ATP (k = 0.017 s–1) and GTP (k = 0.006 s–1).
Elongation of Primer-Templates with a 3' Base Mispair— Having established that LigD POL can generate a 3' mispair by adding an incorrect nucleotide, it was of interest to test how POL behaved when confronted with such mispaired primer termini in the presence of the full complement of dNTP or rNTP substrates. Thus, we prepared four sets of primer-templates (with four primer-templates per set) composed of a 5' 32P-labeled 13-mer primer DNA strand annealed to an 18-mer DNA oligonucleotide to form 13-bp duplexes with five-nucleotide single-stranded 5' tails. Within each of the four sets, the 3'-nucleoside of the primer was identical (there being a 3'-dT set, a 3'-dA set, etc.) and the four template strands in each set differed only in the identity of the deoxynucleoside base opposite the 3'-nucleotide of the primer strand. In this way, we probed rates of dNTP and rNTP-dependent primer extension at all possible 3' base mispairs.
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The remarkable finding was that LigD POL was much more adept at extending mispaired ends in the presence of ribonucleotides (50 µM) than it was with dNTPs (Fig. 5B). rNMP addition at the correctly paired 3'-dC:dG terminus was rapid and efficient, with 97% of the input primer being extended by 1–3 nucleotides after 1 min. As noted previously (6), POL incorporation of rNMPs petered out after 3 or 4 cycles, prior to complete fill-in of the available 5' tail. Consequently, there is no opportunity here for POL to add a nontemplated rNMP in a reaction analogous to the nontemplated dNMP addition to a filled-in blunt end seen in Fig. 5A. rNMP addition to the 3'-dC: dT, 3'-dC:dA, and 3'-dC:dC mispair termini was complete in 20 min, at which time the products consisted predominantly of the n+1 species and a minor fraction of n+2 strand (Fig. 5B). We estimated that POL was 7–8-fold faster at adding rNMPs than dNMPs at each of the mispaired ends analyzed in Fig. 5.
Fig. 6 shows the kinetics of extension at paired and mispaired 3'-dA primer termini. The correctly paired dA:dT primer was extended quickly in the presence of dNTPs (86% of the input primer was extended by at least 1 nucleotide at 1 min) or rNTPs (95% of the primer elongated at 1 min). The DNA polymerase reaction end point occurred after complete fill-in synthesis and one cycle of nontemplated dNMP addition to the blunt duplex end (Fig. 6A); the RNA polymerase reaction was limited to 3–4 cycles of templated rNMP incorporation (Fig. 6B). The dA:dA mispair was extended with dNMPs at 9% the rate of the correctly paired primer end; the dA:dC and dA:dG termini were extended at 4–5% the rate of the correctly paired control (Fig. 6A). Ribonucleotides were added 9-fold more rapidly than deoxys at the respective mispaired dA ends, with the dA:dA mispair being the most effective configuration for the RNA polymerase (93% primer utilization in 5 min), followed by dA:dG and dA:dC (Fig. 6B).
Fig. 7 shows similar outcomes for POL activity at paired and mispaired 3'-dT primer termini. The correctly paired dT:dA primer was extended by 5 or 6 dNMPs at the reaction end point, but RNA synthesis was limited to 3 cycles of rNMP addition. The mispairs were extended with dNMPs at 2 (dT:dT), 7 (dT:dG), and 9% (dT:dC) the rate of the correctly paired end (Fig. 7A). dT:dG and dT:dC were the most effective of the mispair configurations for rNMP addition (complete primer utilization within 5 min), followed by dT:dT (Fig. 7B). Ribonucleotides were added 8-, 10-, and 20-fold more rapidly than deoxys at the mispaired dT:dG, dT:dC, and dT:dT ends, respectively.
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3 nucleotides shorter on the abasic template than the products formed on the unmodified template (Fig. 9B). We surmise that the complete bypass of the abasic site leading to fill-in synthesis entailed a slippage mechanism, whereby the TT dinucleotide at the end of the primer strand slips forward to pair with the AA dinucleotide downstream of the abasic lesion, as illustrated in Fig. 9B. POL extension from this conformation of the primer would yield the 3-nucleotide shorter fill-in product that we observed.
Preferences for Nucleotide Addition Opposite an Abasic Template Lesion—Extension of the 12-mer primer on the unmodified and abasic template strands was analyzed in reaction mixtures containing only a single dNTP or rNTP substrate (10 µM). The unmodified template containing a dT flanking the primer 3'-OH directed rapid incorporation of the correctly paired dAMP, followed by slow misincorporation of a second dAMP opposite the n+2 dA of the template strand (Fig. 10A). POL catalyzed near quantitative addition of dAMP opposite the abasic lesion (by 20 min), albeit at about one-tenth the rate of the templated dAMP addition reaction (Fig. 9A). Misincorporation of dGMP opposite dT was slower than dAMP addition by 40-fold and only a single n+1 product was detected at 20 min (Fig. 10D). Yet, the rate and extent of dGMP insertion opposite the abasic site was about the same as its addition opposite the dT nucleotide (Fig. 10D). We estimate that dATP was about 4-fold better than dGTP as a substrate for addition at an abasic template lesion.
As expected, we found that ATP and GTP were better substrates than dATP and dGTP for addition opposite dT on the unmodified 12-mer/30-mer primer template. Yet, ATP and GTP were also far superior to dATP/dGTP (by at least a factor of 20) as substrates for incorporation by POL opposite an abasic site (Fig. 10, A and D). Thus, POL prefers a ribonucleotide whether or not there is an instructive base on the template strand.
The patterns of primer extension on the unmodified 12-mer/30-mer DNA in the presence of dTTP or dCTP revealed further evidence of template slippage. For example, the dCTP reaction resulted in extension by two nucleotides, with scant accumulation of the n+1 species as an intermediate (Fig. 10C). The likely explanation is that the primer-template adopts the slipped-forward configuration depicted in Fig. 9B, which directs templated incorporation of two consecutive dCMP nucleotides. The same pattern is observed when CTP is the substrate, expect that the overall reaction is faster than with dCTP and the n+1 intermediate is more evident early on (at 1 min), which reflects slowing of the second ribo incorporation event at a 3'-ribo primer terminus.
In the presence of dTTP, the primer is consumed slowly, but is extended by up to 4 or 5 nucleotides, which is more than what is expected if POL first misincorporates T opposite T and then correctly incorporates two more Ts opposite the AA dinucleotide at n+2/n+3. Rather, the predicted n+2 and n+3 extension products are underrepresented compared with n+1 and n+4. We infer that n+4 is generated by backward slippage of the T-rich primer terminus on the A-rich segment of the template strand, allowing an extra T to be added. Although dTTP, dCTP, UTP, and CTP were as effective (or more so) for primer extension on the abasic primer-template than they were on the unmodified DNA, we cannot unambiguously ascribe these extensions to incorporation opposite the abasic lesion, given the propensity of POL to utilize the slipped conformation of the primer-template when only C or T are provided.
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The situation was quite different for the extension reaction at a3'-dT:abasic primer terminus depicted in Fig. 11B, in which virtually all of the primer strand was extended to the end of the template and there was no accumulation of a paused n+1 intermediate. The mispaired dT:dT control was extended with a similar kinetic profile (Fig. 11B). The relatively facile extension of these two 3'-dT primer strands annealed to the 30-mer template contrasts with the much feebler reactivity of LigD POL at the mispaired dT:dT primer-template used in Fig. 7A. The key difference is that the template strand of the 13-mer/30-mer primer template allows for slippage of the 3'-TTT trinucleotide over the flanking mispaired dT or abasic site on the template so that the T-rich primer end can pair with the AA dinucleotide at n+1/n+2 on the template strand.
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When the 12-mer primer terminus was located two nucleotides upstream of the BPhdA adduct, POL catalyzed rapid and efficient incorporation of the first templated dNMP to form a 13-mer (Fig. 12A). The next step of dNMP insertion across from BPhdA occurred slowly; the 14-mer extension product accumulated steadily over 20 min and few primers were elongated beyond that position. In contrast, POL extended the control primer by 6 nucleotides to fill in the unmodified template, then added a nontemplated nucleotide to the blunt end.
When the primer end was poised immediately prior to BPhdA, POL slowly added a single dNMP opposite the lesion (Fig. 12B). Few of the primers on the BPhdA templates were extended past the lesion. Adjusting the primer 3' end so that it was directly opposite BPhdA strongly inhibited addition of the next nucleotide (Fig. 12C). Yet, activity was regained when the primer terminus was moved 1 nucleotide past the BPhdA lesion (Fig. 12D).
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Arrest of LigD POL by a Benzo-[a]pyrene-dG Adduct on the Template Strand—The 18-mer template strands used in primer-extension experiments in Fig. 13 contained a C-10 R or S 7,8-diol 9,10-epoxide adduct of BP at a single dG 6 nucleotides from the 5' end. In duplex DNA, the trans opened BPdG adducts fit into the minor groove; the S adduct points toward the 5' end of the modified strand, whereas the R adduct points toward the 3' end (29). We varied the length of the primer strand so that its 3'-terminal nucleotide was either 1 nucleotide upstream of the adduct (Fig. 13A), opposite the adduct (Fig. 13B), or 1 nucleotide past the adduct (Fig. 13C). dNMP insertion across from the BPdG adduct was suppressed (40-fold) compared with the rate of extension of a control primertemplate (Fig. 13A). Whereas little of the n+1 product of translesion insertion on the S BPdG template was extended, a higher fraction of conversion to n+2 was noted on the R BPdG template (Fig. 13A). The suppression of the translesion extension reaction was recapitulated when the substrate was configured to place the 3' primer end opposite the lesion (Fig. 13B), and the S diastereomer again exerted a tighter restraint on extension by more than one step. The striking finding was that phasing the primer terminus 1 nucleotide past the BPdG lesion did not restore POL activity (Fig. 13C). Thus, the "footprint" of BPdG adduct interference with LigD POL extends further than that of the BPhdA adduct. We infer that the POL makes important interactions with the minor groove of the DNA duplex segment at the primer terminus.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The properties of LigD POL in vitro are most consistent with a short-tract synthetic function during DNA repair, given that its rate of templated nucleotide addition is much slower than that of replicative bacterial DNA polymerases. In the present study we see that it takes LigD POL between 1 and 5 min to completely fill-in with dNMPs a 5-nucleotide overhang attached to a 13-bp duplex; POL takes 5 min to fill in an 18-nucleotide 5' overhang to a 12-bp duplex. As noted here and previously (6), the RNA polymerase activity of LigD POL decays as successive rNMPs are incorporated at the 3' primer terminus, which argues that rNTPs would yield an even shorter repair patch than dNTPs. Yet, these properties do not augur badly for the role of LigD in bacterial DSB repair, which is entirely dependent on Ku (2), a protein that has a DNA end-binding function (3), and interacts physically with LigD via the POL domain (2,8). It is assumed that Ku recruits LigD to DSBs where its action is needed. It seems unlikely that LigD POL is called upon to add long tracts of nucleotides in vivo, given that DSB formation in response to DNA damaging agents likely entails closely staggered breaks on the two strands of the duplex. The critical issue with respect to the biological plausibility of a rNMP addition function for LigD POL is whether or not the ligase component of LigD can effectively seal a strand containing one or more ribonucleotides at the reactive 3'-OH terminus. We find that Pseudomonas LigD is adept at this sealing reaction.3
The crystal structure of LigD in complex with Mn-ATP and Mn-dATP substrates provides clues to its preference for rNMP additions (9). The divalent cations and triphosphate moieties are bound identically to the enzyme in the Mn-ATP versus Mn-dATP cocrystals, but the nucleosides adopt different conformations. The adenosine of ATP extends away from the triphosphate group and is engaged to the enzyme via stacking of the adenine base on Phe604 and a network of direct and water-mediated contacts to the O2' and O3' atom of the ribose sugar. In contrast, the deoxyadenosine of dATP is folded back over the triphosphate so that the adenine base stacks on the guanidinium moiety of the Arg778 side chain; in this conformation, there are no apparent protein contacts to the O3' of the deoxyribose sugar. Changing Phe604 to alanine suppressed both rNMP and dNMP incorporation, whereas changing Arg778 to alanine had minimal impact on POL activity (9). These results suggested that, although ribonucleotides and deoxyribonucleotides can assume different conformations in the substrate-binding cleft, the ATP mode is the productive one for templated nucleotide addition. Conceivably, the two binding modes are in equilibrium, in which case rNTPs would more readily adopt the productive mode by virtue of the protein contacts to the ribose O2' that favor the extended conformation with stacking on Phe604.
The POL structure also provides clues to its infidelity, insofar as the 
stack on Phe604 is the only contact between the enzyme and the adenine base of the ATP substrate. Similar stacking of the pyrimidine base on Phe604 was seen in cocrystals of LigD Pol with Mn-CTP and Mn-UTP (9). Given that the nucleotide and metal can clearly bind to the POL domain in the absence of instructions from a primer-template, that the NTP base is exposed on the surface of the enzyme and makes no base-edge contacts to the protein, and that there are no obvious mobile structural elements that could close around the NTP base, it is unlikely that LigD POL could adopt a tight substrate fit mechanism (39) to ensure high fidelity. A simple view would be that the faster incorporation of the correct nucleotide versus the "next best" of the incorrect nucleotides (which is on the order of 15–35-fold for the rNTP substrates) reflects the contributions of Watson-Crick base pairing. This model can ultimately be tested by examining the utilization of apolar base isosteres of the template bases and the rNTP/dNTP substrates (40–42).
The introduction of a base mispair at the 3'-OH terminus of a primer-template can either: (i) impede further elongation by the polymerase that created the mismatch, or (ii) allow further elongation by that polymerase, thereby embedding the error within the newly synthesized polynucleotide. Whereas many DNA polymerases come equipped with a proofreading 3'-exonuclease function that can remove 3' mispaired nucleotides, the POL domain of LigD evidently does not have such an activity, insofar as we detected no transient shortening of any of the 3' mispaired primer strands during the extension reactions studied in Figs. 5, 6, 7, 8. Our experiments show that 3' mispairs can be extended with high yield by LigD POL, albeit at diminished rates compared with 3' paired primer-templates. The magnitude of the rate decrement in subsequent nucleotide incorporation varies according to the nature of the 3' mispair, but it was generally true that the mispaired ends were extended more rapidly with rNTP substrates than with dNTPs, no matter what the mispair configuration. These findings suggest that LigD POL is itself capable of embedding mispairs within repair tracts (particularly RNA repair patches). This contrasts with the division of labor observed in some error-prone lesion bypass polymerase systems, where one polymerase is responsible for incorporating a nucleotide opposite the lesion on the template strand, but cannot then extend the terminus it created. Instead, a second DNA polymerase specializes in extending the aberrant 3' end of the primer-template (26).
The nucleotide incorporation and mismatch extension properties observed here for LigD POL resonate with the prior studies concerning the infidelity of the homologous eukaryal primase, including the capacity of primase to embed incorrectly paired rNMPs (16). The fact that LigD POL can ignore template instructions when provided with the wrong nucleotide is in line with the fact that it is adept at adding nontemplated ribo- or deoxyribonucleotides at a blunt duplex DNA end.
There has been little attention paid to how members of the archaeal/eukaryal primase enzyme family respond to DNA damage in the template strand. This issue is relevant now that LigD POL is clearly identified as a member of this family with a role in DNA repair rather than primer synthesis. The present characterization of LigD POL shows that it is not adept by itself at bypassing abasic sites, which are among the most common types of DNA damage. POL is capable of catalyzing the translesion insertion step with high yield across from a tetrahydrofuran abasic site. Ribonucleotides are again superior to the cognate deoxynucleotides as substrates for this reaction. Yet, POL is impaired in the subsequent extension step. These properties suggest that were LigD confronted with an abasic site in vivo, then successful translesion synthesis (not involving a frameshift) would require the participation of another polymerase that performs the extension step (26). Alternatively, LigD POL might acquire improved translesion synthesis activity under the influence of an accessory factor.
Our experiments highlight an alternative pathway of lesion bypass by LigD POL, whereby the enzyme allows slippage of the primer terminus across an abasic site (or a 3' mismatch) to pair with downstream bases of the template strand. Bypass via this route results in a shorter fill-in product, indicative of a microdeletion of the looped out template segment. One might question the wisdom of a lesion bypass mechanism that generates frameshift mutations. However, this is a minor concern in the setting of bacterial NHEJ, which is already highly mutagenic, because of the frameshifts generated by LigD when it "correctly" fills in 5' overhangs or adds nontemplated nucleotides at broken DNA ends prior to the sealing step (2, 9). The slippage response to an abasic lesion that we invoke for LigD POL has been described previously for the Y-family polymerase Dpo4, especially the capture of crystal structures of Dpo4 bound to a primer-template with the abasic lesion looped out to allow alternative pairing (43–45). Although LigD POL has not been crystallized bound to DNA, the general location of the DNA binding surface is evident from the structure of the POL-ATP complex (9), which suggests that the DNA primer-template will not be tightly enveloped by the POL protein.
We find that POL is incapable by itself of bypassing BPhdA and BPdG adducts. Here again, POL can generate reasonably high yields of the insertion product, albeit slowly, but is impeded to a greater degree at the extension step. The deleterious effects of a BPhdA adduct are ameliorated once the primer is positioned ahead of the lesion, which presumably allows the BPh to intercalate from the major groove into the duplex of the primer-template. In contrast, the BPdG adducts continue to interfere with POL elongation when the primer is advanced to a position 1 nucleotide ahead of the lesion. We surmise that occupancy of the minor groove by BPdG in the duplex segment at the 3' primer terminus is responsible for this effect, as suggested by the crystal structure of Bacillus DNA polymerase on a primer-template containing BPdG paired with the 3' nucleotide of the primer (32).
Our inference from these results is that LigD POL is more acutely reliant on contacts with the minor groove of the DNA terminus than with the major groove. Indeed, we propose that the reason POL activity decays when ribonucleotides are added at the 3' primer terminus is because the resulting RNA-DNA hybrid adopts an A-like secondary structure with altered minor groove dimensions compared with B-form DNA.
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1 American Cancer Society Research Professor. To whom correspondence should be addressed. E-mail: s-shuman{at}ski.mskcc.org.
2 The abbreviations used are: LigD, ligase D; POL, polymerase domain; NHEJ, nonhomologous end joining; DSB, double strand break; PAH, polycyclic aromatic hydrocarbon; BP, benzo[a]pyrene; BPh, benzo[c]phenanthrene.
3 H. Zhu and S. Shuman, unpublished data.
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