What are the key determinants for nick recognition by DNA ligases? A DNA nick typically consists of 5′-phosphorylated and 3′-OH termini on opposites sides of a break in the phosphodiester backbone. Shuman and colleagues have shown that the 5′-phosphate is absolutely required for nick recognition by ATP-dependent ligases but the 3′-OH is dispensable for DNA binding (51
). Formation of nick-specific complexes is reduced significantly if the nicks lack a 5′-phosphate (53
). Ligases also discriminate between nicks and gaps. The introduction of a ≥1 nt gap in a nick site effectively abolishes DNA binding (51
). The DNA nick binding sites of the minimal sized ATP-dependent ligases from T7 and Chlorella
virus have been physically mapped using different DNA footprinting approaches (54
). These footprinting studies revealed that the enzymes bind asymmetrically to nicks, extending 7–12 nt on the 5′-phosphate side of the nick and 3–8 nt on the 3′-OH side.
There are currently no structures of a DNA ligase in complex with nicked DNA in the protein database. Molecular modelling studies (55
), using the crystal structure of T7 ligase, suggest that dsDNA binds predominantly in the positively charged interdomain cleft (Fig. ) with domain 2 acting as a movable ‘thumb’ which can open, rotate or close in response to ligand association/dissociation (Figs and ). DNA binding experiments with recombinant domains 1 and 2 of T7 ligase (17
) showed that the larger N-terminal domain 1 has a much higher affinity for DNA than domain 2. DNA can be docked closely into the positively charged cleft region, which extends between the two domains of the protein (Fig. ). This charged groove is lined by a number of conserved motifs and residues which have a strong positive potential. The most favourable DNA-binding site is asymmetrical with respect to the nick site, consistent with the DNA footprinting studies. This model is supported by a number of key observations. Odell and Shuman (54
) have shown that the interdomain linker region (motif V) of PBCV-1 ligase is less accessible to proteolysis when the enzyme is bound to nicked DNA. Photocrosslinking and mutagenesis studies (55
) have implicated two conserved lysine residues of motif V in DNA nick recognition. Doherty and Wigley (17
) reported that domains 1 and 2 of T7 ligase can independently and non-specifically bind to DNA and they suggested that nick sensing is a product of these distinct DNA-binding activities. This suggests that domain 1 together with domain 2 make up the minimal unit for all the ATP-dependent ligases and, probably, NAD+
-dependent bacterial DNA ligases. It is likely that these two domains are responsible for the inherent nick sensing and ligation activities of these enzymes. The additional domains, found in the larger ligases, may enhance DNA binding or target the enzymes to regions of DNA damage or replication, but they are not essential for ligase activity. This is supported by complementation experiments which showed that the smaller PBCV-1 ligase can function in lieu of the more complex ligases I and IV in yeast (39
Figure 5 A view of the model T7 ligase–DNA complex. The DNA–protein complex was achieved by placing the DNA nick site adjacent to the active site followed by energy minimisation and molecular dynamics protocols as detailed by Doherty and Dafforn (more ...)
Nick recognition requires ligases to be adenylated at the active site lysine (51
). A structural basis for this requirement can now be proposed based on the T7 and Tfi
ligase structures. As discussed above, the OB fold (domain 2) has been shown to bind to dsDNA. In the non-adenylated structure of the T7 ligase–ATP complex the proposed DNA-binding face of the OB domain is rotated away from the active site cleft (Fig. B). In contrast, in the covalently bound AMP structure of the NAD+
ligase the equivalent OB domain is facing towards the active site cleft (Fig. B). Why is this domain found in such different conformations in the two structures? In the non-adenylated T7 ligase structure it is likely that the OB domain is orientated to prevent DNA binding in the active site cleft until the conserved active site lysine has become adenylated. In contrast, the OB domain in the adenylated Tfi
ligase structure is rotated around, positioning the DNA-binding surface towards the active site awaiting nick binding. The OB fold appears to play a dual role in the ligation mechanism. Residues on one face of this domain position the β and γ phosphate tail of ATP away from the incoming nucleophile, enhancing the adenylation reaction. Adenylation of the active site lysine acts as a conformational switch, facilitating rotation of the DNA-binding surface (β-barrel face) of the OB fold towards the active site cleft (Fig. ). In this way only adenylated ligase can bind to nicked DNA. This mechanism prevents the formation of non-productive ligase–DNA complexes.
The larger ligases appear to have additional DNA-binding motifs which contribute to the DNA-binding surface. This is most obvious in the Tfi
ligase structure, which has two additional C-terminal domains. Domain 3 consists of a zinc finger subdomain (3a) and four HhH motifs (3b). Lee et al.
) proposed that this domain forms a second DNA-binding site, named the ‘non-catalytic’ DNA-binding site as it is distant from the active site. This proposal is supported by the result of a limited proteolysis study on the homologous Bst
). Timson and Wigley (56
) have shown that the DNA-binding activity of a C-terminal fragment of Bst
ligase is comparable to full-length enzyme. Mackey et al
) identified two functionally distinct regions within mammalian ligase III that interact with nicked DNA. Currently no structural information is available on the mammalian ligases (I–IV) but it is obvious at the sequence level that these enzymes, in common with Tfi
ligase, have a catalytic core (domains 1 and 2) with additional domains, such as BRCT and zinc finger domains, forming distinct N- and C-terminal extensions.
A conserved catalytic mechanism for DNA ligases and nucleotidyl transferases
As we discussed above, DNA ligases are members of the nucleotidyl transferase family of enzymes and proceed through a covalent AMP–enzyme intermediate in which the AMP is attached to the enzyme via a lysine residue (1
). This lysine residue is part of the conserved motif I (5
), one of six co-linear sequence motifs, also found in capping enzymes, RNA ligases and tRNA ligases, with a similar spacing between them (Fig. A) (4
). Shuman and Schwer (4
) proposed that all of these enzymes share a common nucleotidyl transfer mechanism and are likely to have a similar structure. This has since been confirmed by biochemical analysis of mutant enzymes (7
) and, more recently, by the structures of T7 DNA ligase (13
virus RNA capping enzyme (14
), the N-terminal domain 1 of Bst
) and Tfi
). Examination of the positions of the conserved sequence motifs (Fig. A) within these DNA ligase and RNA capping structures (13
) reveals that they are clustered around the NTP-binding site and they form the sides of the groove between domains 1 and 2 (Fig. B). The crystal structures of the T7 and Tfi
ligases (in bold) reveal a conserved role for many of the residues in these motifs. Motif 1 contains the active site lysine (K34, K116
) which forms the covalent AMP adduct. Motif III contains a glutamate residue (E93, E114
) which forms hydrogen bonds with the ribose of the ATP, while the tyrosine (Y149, Y221
) in motif IIIa is stacked against the adenine ring and the essential lysine in motif V (K238, K312
) contacts the α-phosphate group. Examination of the conservation of sequence between the DNA ligases and the capping enzymes, coupled with the crystal structures, provides clues about how nucleotide specificity is achieved. There are two important interactions between the 6-amino group of the adenine ring and ligases, one via the main chain carbonyl of I33/H115
and the other via the side chain of E32/E114
. This latter residue is usually a Glu, though occasionally an Asp or Gln, in ATP-dependent ligases (6
). In contrast, this residue is highly variable in the capping enzymes but is never a Glu, Asp or Gln (4
). In T7 ligase Lys222 and Glu32 form an ion pair at the base of the ATP-binding pocket. A similar ion pair is also present in the Tfi
NAD-dependent DNA ligase (Lys288 and Glu114) (13
). Currently there is no structural information on the binding specificity for the base at the nicotinamide position of NAD+
-dependent ligases. A complete structural understanding of the nucleotide specificity of the two classes of ligases will be essential for the design and development of specific inhibitors of bacterial ligases as potential antibacterial compounds.
Lee et al
) have proposed that the final step of the ligation reaction, deadenylation of the adenylated DNA intermediate and phosphodiester bond formation, is analogous to the polymerising step catalysed by DNA polymerases. The adenylated DNA intermediate in ligation corresponds to deoxyribonucleoside 5′-triphosphate in polymerisation. They argue that DNA ligases are also likely to utilise a similar two divalent metal ion mechanism, proposed previously for DNA polymerases (60
). They have identified a putative metal ion-binding site Asp118, Glu281 and Asp283 in Tfi
ligase (Fig. ) which matches the two cobalt ion-binding site in E.coli
methionine aminopeptidase. These three residues form a highly negatively charged pocket near the active site lysine residue (Lys116) (Fig. ). The strictly conserved Arg196 is likely to interact with the 5′-phosphate end of the nicked strand. A schematic model proposed for the ligase active site is shown in Figure . It is very likely that a similar active site architecture exists in all DNA ligases and related nucleotidyl transferases.
Schematic model of the Tfi ligase active site. Residues that are likely to participate in binding divalent metal ions and the 5′-phosphate end of the nick are indicated.
The biochemical properties of a chimeric polypeptide ACE (A
nzyme), consisting of the N-terminal domain 1 of T7 DNA ligase fused to the C-terminal OB domain 2 of the PBCV-1 capping enzyme, have recently been reported (61
). The ACE protein can become adenylated at the active site lysine in common with DNA ligases. However, ACE is deficient in DNA ligase activity, but remarkably this chimeric enzyme can ‘cap’ RNA molecules by transferring AMP specifically to diphosphate terminated 5′-ends of RNA. The OB domain of ACE confers a novel binding specificity for the 5′-end of mRNA rather than for nicked RNA. The OB fold appears to determine the polynucleotide specificity of these enzymes. This report supports the idea that nucleotidyl transferases have a conserved catalytic mechanism.