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Nucleic Acids Research Pages 4804-4810  

Role of the DNA ligase III zinc finger in polynucleotide binding and ligation
Introduction
Materials And Methods
   Expression and purification of recombinant proteins from E.coli
   Site-directed mutagenesis
   Polynucleotide substrates
   DNA ligase assays
   Cell-free BER assays
   Electrophoretic mobility shift assay (EMSA)
Results
   Role of the zinc finger motif in ligation of simple nicked DNA substrates
   Role of the zinc finger in DNA base excision repair (BER) in vitro
   The zinc finger stimulates DNA ligase III activity on nicked RNA homopolymers
   The zinc finger motif stimulates binding of DNA ligase III to nicked RNA/DNA homopolymers
   Role of the zinc finger in ligation of nicked RNA by XRCC1-DNA ligase III complex
Discussion
Acknowledgements
References

Role of the DNA ligase III zinc finger in polynucleotide binding and ligation

Role of the DNA ligase III zinc finger in polynucleotide binding and ligation

Richard M. Taylor, Jenna Whitehouse, Enrico Cappelli1, Guido Frosina1 and Keith W. Caldecott*

School of Biological Sciences, G.38 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK and 1DNA Repair Unit, CSTA Laboratory-Instituto Nazionale Ricerca Cancro, L.go Rosanna Benzi 10,16132 Genova, Italy

Received August 8, 1998; Revised and Accepted September 11, 1998

ABSTRACT

Mammalian DNA ligase III exists as two distinct isoforms denoted [alpha] and [beta]. Both forms possess a motif that is homologous to the putative zinc finger present in poly(ADP-ribose) polymerase. Here, the role of this motif in the binding and ligation of nicked DNA and RNA substrates in vitro has been examined in both isoforms. Disruption of the putative zinc finger did not affect DNA ligase III activity on nicked DNA duplex, nor did it abolish DNA ligase III-[alpha] activity during DNA base excision repair in a cell-free assay. In contrast, disruption of this motif reduced 3-fold the activity of both DNA ligase III isoforms on nicked RNA present in RNA/DNA homopolymers. Furthermore, whereas disruption of the motif did not prevent binding of DNA ligase III to nicked DNA duplex, binding to nicked RNA homopolymers was reduced ~10-fold. These results suggest that the putative zinc finger does not stimulate DNA ligase III activity on simple nicked DNA substrates, but indicate that this motif can target the binding and activity of DNA ligase III to nicked RNA homopolymer. The implications of these results to the cellular role of the putative zinc finger are discussed.

INTRODUCTION

The ligation of nicks in duplex DNA is required during DNA repair, genetic recombination and DNA replication. Multiple DNA ligase polypeptides have been identified in mammalian cells and are denoted DNA ligases I-V (1-3). DNA ligase III exists as two isoforms that differ at their C-terminus and which are produced by a tissue- and cell type-specific splicing mechanism (4,5). The two isoforms are denoted DNA ligase III-[alpha] (103 kDa) and DNA ligase III-[beta] (96 kDa), with the C-terminal 77 amino acids of the former replaced with 17-18 different residues in the latter. As a consequence of this difference only DNA ligase III-[alpha] interacts with XRCC1 protein in vitro and in vivo (4,5). XRCC1 is required for normal levels of DNA ligase III-[alpha] in Chinese hamster ovary (CHO) cells and XRCC1 mutant EM-C11 extracts lacking normal levels of DNA ligase III-[alpha] are unable to support efficient DNA ligation during DNA base excision repair (BER) in a cell-free assay (6-8). Expression of DNA ligase III-[beta] appears to be restricted to meiotic cells and on this basis has been proposed to be involved in homologous recombination (4).

Both [alpha]- and [beta]-isoforms of DNA ligase III possess a putative zinc finger motif at the N-terminus that is highly homologous (42% identity) to one present in poly(ADP) ribose polymerase (PARP) (2,9-11). It should be noted that the designation of this motif as a zinc finger is putative, since formal demonstration of this requires knowledge of its tertiary structure. A truncated N-terminal fragment of DNA ligase III-[alpha] that contains the putative zinc finger displays weak nick-sensing activity, but the effect of this motif on DNA ligase III activity has not been examined (10). This motif may fulfil a role in increasing the binding affinity of DNA ligase III for nicked DNA or may increase the affinity of the polypeptide for nicked substrates of a specific secondary structure. To examine these possibilities, we have compared the activity of recombinant DNA ligase III-[alpha] and DNA ligase III-[beta] with that of mutant derivatives lacking an intact zinc finger on various synthetic polynucleotide substrates in vitro.

MATERIALS AND METHODS

Expression and purification of recombinant proteins from E.coli

His-DNA ligase III polypeptides were expressed in Escherichia coli from the vector pET16B (Novagen). Constructs encoding His-DNA ligase III-[alpha] and His-DNA ligase III-[alpha]d50-63 have been described previously, with the latter previously denoted His-DNA ligase IIIZn- (10). pET16BHL3-[alpha]R31I, encoding His-DNA ligase III-[alpha]R31I, was derived by site-directed mutagenesis as described below. pET16BHL3-[beta], encoding His-DNA ligase III-[beta], was constructed by replacing a MluNI-ClaI fragment from the DNA ligase III-[alpha] open reading frame (ORF) with the corresponding fragment from DNA ligase III-[beta], thus replacing the C-terminal 77 amino acids specific to the former polypeptide with the C-terminal amino acids specific to the latter. Recombinant polypeptides were purified by immobilized metal-chelate affinity chromatography as outlined previously (7). For some experiments (e.g. those presented in Fig. 6), recombinant DNA ligase III-[alpha] was further purified by affinity chromatography using a column comprised of MBP-X573-592; a maltose binding protein fused to the DNA ligase III-[alpha] binding site of XRCC1 (7,12). Affinity purified DNA ligase III-[alpha] polypeptide possessing a C-terminal FLAG tag were used to confirm results obtained with nicked RNA homopolymer (results not shown). All proteins were purified to apparent homogeneity as measured by SDS-PAGE and staining with Coomassie blue. It should be noted that DNA ligase activity was not detected in preparations of an unrelated polypeptide (XRCC1) which was purified by the same procedure in parallel with DNA ligase III.

Site-directed mutagenesis

pET16BHL3-[alpha]R31I encodes His-DNA ligase III-[alpha]R31I, a derivative of His-DNA ligase III-[alpha] that harbours a site-specific point mutation. This mutation was generated using synthetic oligonucleotides and a Quickchange Site-Directed Mutagenesis kit (Stratagene), as described by the manufacturer. Theprimers used were 5[prime]-TGAAGGGCGTATGCATTATTGGCAAAGTGG-3[prime] (forward) and 5[prime]-CCACTTTGCCAATAATGCATACGCCCTTCA-3[prime] (reverse). To prevent unwanted PCR-generated mutations outside the target region, an open reading frame encoding only a small region of DNA ligase III-[alpha] (residues 1-353) was present during mutagenesis. The presence of the site-directed mutation and the absence of additional mutations was confirmed by dideoxy sequencing prior to reconstituting the complete His-DNA ligase III-[alpha] ORF by subcloning. A version of His-DNA ligase III-[beta] harbouring the site-specific mutation was generated by the transfer of a CelII fragment, containing the Arg31->Ile point mutation, from pET16BHL3-[alpha]R31I into pET16BHL3-[beta] to create pET16BHL3-[beta]R31I.

Polynucleotide substrates

Oligonucleotides (dT)16 and (rA)12-18 and polynucleotides poly(dA) and poly(rA) were purchased from Pharmacia LKB Biotechnology. Oligonucleotide (dT)70 was synthesized by Zeneca Pharmaceuticals and purified by FPLC. Dephosphorylated oligonucleotides (5 µg) were labelled with [[gamma]-32P]ATP (50 µCi, 4500 Ci/mmol; ICN) using T4 polynucleotide kinase (Boehringer Mannheim) with cold ATP present at 2 µM. Unincorporated nucleotides were removed using Nuc-Trap columns (Stratagene). The labelled oligonucleotide was mixed with an equimolar amount of the appropriate polynucleotide, incubated at 85°C for 10 min and slowly cooled to room temperature to allow annealing. NaCl was added to 50 mM and the duplexes stored at -20°C. Specific oligonucleotides used to generate a 70 bp duplex DNA with a single nick were synthesized by Zeneca Pharmaceuticals and purified by FPLC. A 20 bp RNA oligonucleotide of heterologous sequence was synthesized by MWG-Biotech UK Ltd (Milton Keynes, UK). The radiolabelled oligonucleotides were prepared as described above and mixed with appropriate unlabelled oligonucleotides in an equimolar ratio, heated to 70°C for 10 min and cooled slowly to room temperature to allow annealing. Oligo(dT)16·poly(dA) and oligo(dT)16·poly(rA) are comprised of 16 bp dTMP oligomers annealed to dAMP or AMP polymers of ~300 or 465-660 bp, respectively. Oligo(rA)12-18·(dT)70 is comprised of 12-18 bp AMP oligomers annealed to 70 bp dTMP oligomers.

DNA ligase assays

DNA ligase III polypeptides and radiolabelled substrate were incubated at 30°C for the times indicated in 10-40 µl of ligation buffer (60 mM Tris-HCl, pH 8.0, 10 mM MgCl, 50 µg/ml BSA, 5 mM DTT, 1 mM ATP). Reactions were stopped by the addition of 4 vol of sample buffer (90% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol FF in 1× TBE), heated to 85°C for 10 min, cooled rapidly on ice and polynucleotides resolved on 10% polyacrylamide-8 M urea gels. Dried gels were analysed by autoradiography and densitometry.


Figure 1. DNA ligase III and PARP possess homologous zinc finger motifs. Schematic comparing the putative zinc finger motif of DNA ligase III with the homologous motif in PARP. The cysteine and histidine amino acids putatively involved in zinc coordination are shown. Residues underlined and in upper case are conserved between the two polypeptides. Arrows and boxes indicate those residues deleted or altered in His-DNA ligase III-[alpha]d50-63 (L3[delta]50-63) and His-DNA ligase III-[alpha]/[beta]R31I (L3R31I), respectively.

Cell-free BER assays

Reactions were conducted as described previously (8). Briefly, pGEM X or pGEM T (0.3 µg) plasmid was incubated for 30 min at 30°C in reaction buffer containing dNTPs, [[alpha]-32P]dTTP and cell extract from parental CHO-9 or mutant EM-C11 cells (20 µg protein). Where indicated, recombinant His-DNA ligase III-[alpha] (1 µg) was pre-incubated at 30°C for 20 min prior to initiating the repair reaction. Following incubation, plasmid DNA was recovered and digested with SmaI and HindIII to release a radiolabelled 33 bp fragment if BER repair was complete or a radiolabelled 16 bp fragment if repair synthesis occurred in the absence of DNA ligation (8; Fig. 3A). Fragments were separated by denaturing PAGE and 16-33 bp fragments in which repair replication had occurred were detected by autoradiography.

Electrophoretic mobility shift assay (EMSA)

Radiolabelled duplex substrates were incubated with a 50-fold excess (by weight) of supercoiled competitor plasmid (DNA) and 6.4 pmol (0.5 µg) of recombinant DNA ligase III-[alpha] or DNA ligase III-[beta] on ice for 20 min in binding buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, 0.1 mg/ml BSA). One fifth volume of loading buffer (30% glycerol and 0.25% bromophenol blue in 1× TBE) was added and samples subjected to non-denaturing polyacrylamide gel electrophoresis at 15 mA in pre-chilled1× TBE (5% gels; BioRad Mini Protean II apparatus). Gels were fixed, dried and subjected to autoradiography.

RESULTS

Role of the zinc finger motif in ligation of simple nicked DNA substrates

The zinc finger motif was disrupted in His-DNA ligase III-[alpha] and His-DNA ligase III-[beta] either by deletion (His-DNA ligase III-[alpha]d50-63; 10; Fig. 1) or by substitution of Arg31 with Ile (His-DNA ligase III-[alpha]R31I and His-DNA ligase III-[beta]R31I; Fig. 1). Arg31 was changed to Ile because the corresponding mutation in PARP (R138I) abrogates the nick-sensing activity of this polypeptide, as measured by south-western blotting (11). The deletion and point mutants behaved similarly in all of these experiments and were thus used interchangeably. A comparison of the activity of His-DNA ligase III-[alpha]R31I and His-DNA ligase III-[beta]R31I with their respective parental forms on a 70mer oligonucleotide duplex possessing a single nick (Fig. 2A) revealed that the mutant polypeptides possessed full activity on this substrate under the conditions employed (Fig. 2B and C). Increased binding affinity conferred by the zinc finger may only manifest at low concentrations of DNA strand breakage and, therefore, His-DNA ligase III-[alpha] and His-DNA ligase III-[alpha]d50-63 were compared in the presence of titrated amounts of nicked duplex. However, mutant His-DNA ligase III-[alpha] was fully active in these experiments (Fig. 2D). The activity of His-DNA ligase III-[alpha]d50-63 was also examined on nicked duplex in the presence of excess un-nicked competitor (Fig. 2E), which better reflects the environment in which strand breaks are present in vivo. In addition DNA ligase III-[alpha]d50-63 activity was also examined on oligo(dT)16·poly(dA) (Fig. 2F) and oligo (dA)16·poly(dT) homopolymers, to examine whether the zinc finger targets DNA ligase III to regions of extensive DNA strand breakage. However, mutant DNA ligase III-[alpha] polypeptide also displayed full activity in these experiments (data not shown).


Figure 2. The zinc finger motif does not stimulate ligation of nicked DNA oligonucleotides or oligo(dT)16·poly(dA) homopolymer. (A) Schematic showing the nicked 70mer oligonucleotide duplex. The single-strand break (arrow) and radiolabelled 5[prime]-phosphate (asterisk) are indicated. DNA ligase III-[alpha] (B) or DNA ligase III-[beta] (C) polypeptides were incubated with nicked 70mer (12 or 15 nM, respectively) for 30 min at 30°C, at the concentrations indicated. (D) DNA ligase III-[alpha] polypeptides (5 nM) were incubated with nicked 70mer duplex for 60 min at 30°C, at the concentrations indicated. (E) DNA ligase III-[alpha] polypeptides were incubated with nicked 70mer (7.6 nM) and competitor DNA (50-fold excess by weight of SalI linker; New England Biolabs) for 30 min at 30°C, at the concentrations indicated. (F) DNA ligase III-[alpha] polypeptides were incubated with oligo(dT)16·poly(dA) (2.2 nM) for 30 min at 30°C, at the concentrations indicated. The radiolabelled phosphate on oligo(dT)16 is indicated (asterisk). Reaction products were separated by denaturing PAGE and dried gels (10%) subjected to autoradiography.


Figure 3. The zinc finger motif is not required for DNA ligation during BER in vitro. (A) The SmaI-HindIII fragments of pGEM X and pGEM T are shown, as are the sizes of BER intermediates prior to repair synthesis (15mer), after repair synthesis (16mer) and after DNA ligation (33mer). The radiolabelled dTMP incorporated during BER is boxed. (B) BER reactions containing [[alpha]-32P]dTTP, 20 µg cell extract protein from CHO-9 (CH9) or EM-C11 (E11) and 300 ng of either pGEM X (lanes 1-4) or pGEM T (lanes 5 and 6) were incubated at 30°C for 20 min. Where indicated, 1 µg of either His-DNA ligase III-[alpha] (L3, lane 3) or DNA ligase III-[alpha]d50-63 (L3[delta]Zn, lane 4) was preincubated with EM-C11 cell extract prior to the reaction. Purified reaction products were incubated with SmaI and HindIII and subsequently separated by denaturing PAGE and the dried gels subjected to autoradiography. The fraction of BER patches that remained unligated was calculated from the percentage of radiolabelled SmaI-HindIII fragment that was present as 16mer [16mer/(16mer + 33mer)].

Role of the zinc finger in DNA base excision repair (BER) in vitro

Cell extracts derived from the XRCC1 mutant CHO cell line EM-C11 lack normal levels of DNA ligase III-[alpha] and consequently exhibit a reduced ability to ligate BER patches in a cell-free assay (8). Here, the cell-free assay was used to examine whether the zinc finger motif is required for the role of DNA ligase III-[alpha] in BER. Wild-type CHO-9 or EM-C11 cell extract was incubated with pGEM X plasmid harbouring a single abasic site or control pGEM T plasmid containing a thymine base at the corresponding position (Fig. 3A; 8). In this assay, completed excision repair of the abasic site is indicated by specific incorporation of [32P]dTMP into the 33 bp SmaI-HindIII fragment of pGEM X (Fig. 3A). Excision repair patches that are not ligated manifest as reduced amounts of radiolabelled 33mer and increased amounts of radiolabelled 16mer (Fig. 3A). As observed previously (8), EM-C11 extract was less able to support DNA ligation during BER than was CHO-9 extract, with 37 and 11% of BER patches remaining unligated after 20 min, respectively (Fig. 3B, lanes 1 and 2). Addition of 1 µg of either His-DNA ligase III-[alpha] or His-DNA ligase III-[alpha]d50-63 reduced the number of unligated BER patches to levels present in CHO-9 reactions (Fig. 3B, lanes 3 and 4), indicating that the zinc finger motif is not essential for the role of DNA ligase III-[alpha] in BER.

The zinc finger stimulates DNA ligase III activity on nicked RNA homopolymers

We next examined whether disruption of the zinc finger motif affected DNA ligase III activity on nicked RNA/DNA homo-polymeric duplex. Although not physiological substrates, these are useful for identifying differences in substrate specificity between different DNA ligases (1,13). The zinc finger mutations in His-DNA ligase III-[alpha]d50-63 and His-DNA ligase III-[alpha]R31I reduced activity ~3-fold on oligo(rA)·(dT)70 under conditions of limiting enzyme concentration and over a wide range of substrate concentrations (Fig. 4A-C). Moreover, His-DNA ligase-[beta]R31I exhibited 3-fold less activity than His-DNA ligase-[beta] on oligo(rA)·(dT)70 (Fig. 4D), indicating that both isoforms of DNA ligase III require the zinc finger for full activity on this substrate. Identical results were observed with a second nicked RNA/DNA homopolymer, oligo (rA)·poly (dT), which possesses a homo-polymeric TMP backbone of undefined length (data not shown). In contrast, mutation of the zinc finger did not affect the activity of DNA ligase III on the nicked DNA/RNA substrate oligo(dT)16·poly(rA), indicating that the stimulatory effect of this motif was specific to RNA/DNA hybrids that possessed nicked RNA (Fig. 5A). Furthermore, stimulation was specific to RNA homopolymer, because mutant DNA ligase III was fully active on substrate in which oligo(rA) was replaced with a 20mer oligoribonucleotide of heterogeneous sequence (Fig. 5B).


Figure 4. The zinc finger motif stimulates ligation of nicked RNA/DNA homopolymers. (A) DNA ligase III-[alpha] polypeptides were incubated with oligo(rA)12-18·(dT)70 (27.5 nM) for 15 min at 30°C, at the concentrations indicated. (B) DNA ligase III-[alpha] polypeptides (60 nM) were incubated with oligo(rA)12-18·(dT)70 (30 nM) at 30°C for the times indicated. (C) DNA ligase III-[alpha] polypeptides (125 nM) were incubated at 30°C for 15 min with oligo(rA)12-18·(dT)70 present at the concentrations indicated. (D) DNA ligase III-[beta] polypeptides were incubated with oligo(rA)12-18·(dT)70 (40 nM) at 30°C for 30 min, at the concentrations indicated. Ligated products were analysed as in Figure 2. Where indicated, ligation activity was quantified by densitometry and expressed graphically as the percentage or total amount of substrate ligated.


Figure 5. The ability of the zinc finger motif to stimulate ligation is specific to nicked RNA homopolymer in vitro. (A) DNA ligase III-[alpha] polypeptides were incubated at the concentrations indicated with labelled oligo(dT)16·poly(rA) (4 nM) for 60 min at 30°C. (B) DNA ligase III-[beta] polypeptides were incubated with 63 nM non-homopolymeric nicked RNA/DNA hybrid (sequence indicated) for 30 min at 30°C at the concentrations indicated. [[alpha]-32P]phosphate labels are indicated by asterisks. Reaction products were analysed as in Figure 2.


Figure 6. The zinc finger motif stimulates binding to nicked RNA/DNA homopolymers. Reactions contained competitor DNA (50-fold excess by weight), 60 fmol labelled oligo(rA)12-18·(dT)70 (A), 100 fmol labelled 70mer duplex containing a single-strand nick (B) or 100 fmol labelled un-nicked 70mer duplex (C) and either 6.4 pmol His-DNA ligase III-[alpha] (lanes 1), 6.4 pmol His-DNA ligase III-[alpha]R31I (lanes 2), 4 pmol His-DNA ligase III-[beta] (lanes 4), 4 pmol His-DNA ligase III-[beta]R31I (lanes 5) or no recombinant protein (lanes 3 and 6). (D) The zinc finger motif present in XRCC1-DNA ligase III-[alpha] complex stimulates ligation of nicked RNA homopolymer. [[alpha]-32P]oligo(rA)12-18·(dT)70 (87.5 nM) was incubated for 30 min at 30°C with His-DNA ligase III-[alpha] (top) or His-DNA ligase III-[alpha]R31I (bottom), present at the concentrations indicated. Prior to use, DNA ligase III-[alpha] (3.2 pmol) was incubated on ice with BSA (10 pmol, lanes 1-5) or XRCC1-His (10 pmol, lanes 6-10) for 10 min in a total volume of 10 µl to allow formation of protein complexes. Reaction products were analysed as in Figure 2.

The zinc finger motif stimulates binding of DNA ligase III to nicked RNA/DNA homopolymers

Wild-type and mutant DNA ligase III-[alpha] and -[beta] were compared in EMSA assays to determine whether differences in ligation activity were reflected in substrate binding. Indeed, binding of His-DNA ligase III-[alpha]R31I and His-DNA ligase III-[beta]R31I to oligo(rA)12-18·(dT)70 was reduced ~10-fold compared with His-DNA ligase III-[alpha] or His-DNA ligase III-[beta], respectively, in this assay (Fig. 6A). In contrast, the major complex formed between DNA ligase III-[alpha] or DNA ligase III-[beta] with nicked 70mer duplex did not require the zinc finger motif (Fig. 6B), consistent with the zinc finger being dispensable for ligation of this substrate. These results were observed under a variety of protein and substrate concentrations.

A small amount of wild-type DNA ligase III complex was formed with nicked 70mer that migrated faster than the major complex formed with this substrate (Fig. 6B, lanes 1 and 4). This complex reflects the binding of an N-terminal fragment of DNA ligase-III of ~20-30 kDa, which contains the zinc finger motif but which lacks the C-terminal three-quarters of the DNA ligase, including those putative DNA binding sites common to other DNA ligases (unpublished observations). Consequently, the formation of this complex was entirely zinc finger dependent (Fig. 6B, lanes 2 and 5).

Role of the zinc finger in ligation of nicked RNA by XRCC1-DNA ligase III complex

DNA ligase III-[alpha] interacts with XRCC1 polypeptide in vitro and in vivo (5-8,12). It was therefore pertinent to examine whether the zinc finger stimulates activity of XRCC1-DNA ligase III-[alpha] heterodimer. Indeed, when complexes formed by preincubation of DNA ligase III-[alpha] with XRCC1 were examined for ability to ligate oligo(rA)·(dT)70, mutant DNA ligase III-[alpha] again displayed a reduced activity on the nicked RNA substrate (Fig. 6D). Interestingly, XRCC1 also weakly stimulated DNA ligase III-[alpha] activity on this substrate (~2-fold), irrespective of zinc finger status (Fig. 6D, compare lanes 1-5 with 6-10). Thus, the zinc finger motif and XRCC1 binding together stimulated DNA ligase III-[alpha] activity, 6-fold.

DISCUSSION

DNA ligases are ubiquitous enzymes and are fundamental to the maintenance of genetic stability. Mammalian DNA ligase III differs from all other known DNA ligases in that it possesses a zinc finger motif. This motif is highly homologous to a `nick-sensing' zinc finger present in PARP (2) and a truncated fragment of DNA ligase III possessing this domain exhibits weak nick-sensing activity in gel retardation assays (10). Here, we have investigated in detail the role of this motif in the binding and activity of DNA ligase III isoforms on a variety of polynucleotide substrates in vitro.

DNA strand breaks continuously arise at low levels in living cells during the excision repair of spontaneous DNA base damage, and a role for DNA ligase III-[alpha] in the rejoining of such breaks is suggested by a number of studies (6,8,10,14). It was thus considered possible that the zinc finger motif increases the affinity of DNA ligase III for DNA strand breaks present at low levels. However, disruption of the zinc finger motif did not affect ligation activity on a nicked 70mer duplex, either when this substrate was titrated to concentrations as low as technically possible for the ligation assay or in the presence of supercoiled competitor DNA (50-fold excess, by weight). Indeed, we failed to detect any effect of the zinc finger motif on the activity of DNA ligase III-[alpha] or DNA ligase III-[beta] on nicked DNA substrates under any condition examined. Consistent with this, both isoforms efficiently bound nicked 70mer in the absence of an intact zinc finger, though the small differences observed in some experiments (Fig. 6B; 10) may reflect a weak contribution by this motif. The ability to bind nicked 70mer exhibited by His-DNA ligase III-[alpha]R31I and His-DNA ligase III-[beta]R31I did not reflect `leaky' zinc finger activity, because complete deletion of the zinc finger did not further reduce binding (R.Taylor and K.Caldecott, unpublished data). Taken together, these data indicate that the zinc finger motif is not required for binding of DNA ligase III to simple nicked DNA substrates and does not stimulate ligation of such substrates. Furthermore, that mutant DNA ligase III-[alpha] was able to complement the ligation defect exhibited by EM-C11 cell extract suggests that this motif is similarly not required for rejoining DNA strand breaks within the context of BER in vitro.

In contrast to nicked DNA substrates, the zinc finger motif did stimulate (~3-fold) DNA ligase III-[alpha] and DNA ligase III-[beta] activity on nicked RNA/DNA hybrids. Reduced DNA ligase III activity on oligo(rA)·(dT)70 was observed at all enzyme and substrate concentrations examined, including when complexed with XRCC1. Moreover, the zinc finger increased binding of DNA ligase III-[alpha] and DNA ligase III-[beta] to oligo(rA)·(dT)70 ~10-fold, as measured by gel retardation assays. The stimulation of DNA ligase III-[alpha] activity by the zinc finger motif was specific to substrates containing nicked RNA homopolymer. Such substrates are useful for defining enzyme substrate specificity (1,13), but are unlikely to reflect a target of DNA ligase III in vivo. Rather, it is possible that the zinc finger is required to stabilize DNA ligase III binding to certain intermediates of DNA metabolism and that nicked RNA/DNA homopolymers fortuitously possess secondary structures similar to such intermediates. For example, annealing of oligo(rA) 16-18mers to poly(dT) can generate a variety of structures in addition to nicks, such as single-strand flaps, single/multiple base pair gaps and single-strand loops. Similar structures can arise in cellular DNA as intermediates of DNA replication, DNA excision repair or DNA recombination, all of which require DNA ligation. We suggest that the zinc finger motif facilitates this step by targeting DNA ligase III to these intermediates, a possibility currently under investigation.

ACKNOWLEDGEMENTS

We are indebted to Dr Kevin Hudson and Zeneca Pharmaceuticals for providing oligonucleotides. This work was funded by the Medical Research Council (KC and RT, grant no. G9603130), Zeneca Pharmaceuticals (KC) and the Italian Association for Cancer Research (EC and GF).

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*To whom correspondence should be addressed. Tel: +44 161 275 5311; Fax: +44 161 275 5600; Email: keith.caldecott@man.ac.uk

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