Kinetics and thermodynamics of nick sealing by T4 DNA ligase

Cherepanov, Alexey V.; Vries, Simon de

doi:10.1046/j.1432-1033.2003.03824.x

Cited by 62 publications

(72 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Because our preparations of the ligase IV/XRCC4 complex consist predominantly of the pre-adenylylated species (E-AMP), the concentration of the uncharged enzyme that is accessible to the reaction E + ATP f E-AMP + PPi is extremely low, and this has made accurate rate measurements difficult even when using coupled assays; efforts to monitor ligase adenylylation after a pre-incubation with nicked DNA (to effect decharging) are being pursued. 6 Note that we (3) and others (33) have been able to effect burst behavior during DNA ligation but only by using substrates or reaction conditions that are unnatural. 7 DNA ligases exist in ViVo as a mixture of the adenylylated and non-adenylylated species.…”

Section: Fidelity Of Human Dna Ligaseiv/xrcc4mentioning

confidence: 96%

Human DNA Ligase IV and the Ligase IV/XRCC4 Complex: Analysis of Nick Ligation Fidelity

2007

View full text Add to dashboard Cite

In addition to linking nicked/fragmented DNA molecules back into a contiguous duplex, DNA ligases also have the capacity to influence the accuracy of DNA repair pathways via their tolerance/ intolerance of nicks containing mismatched base pairs. Although human DNA ligase I (Okazaki fragment processing) and the human DNA ligase III/XRCC1 complex (general DNA repair) have been shown to be relatively intolerant of nicks containing mismatched base pairs, the human DNA ligase IV/XRCC4 complex has not been studied in this regard. Ligase IV/XRCC4 is the sole DNA ligase involved in the repair of double strand breaks (DSBs) via the non-homologous end joining (NHEJ) pathway. During the repair of DSBs generated by chemical/physical damage as well as the repair of the programmed DSB intermediates of V(D)J recombination, there are scenarios where, at least conceptually, a capacity for ligating nicks containing mismatched base pairs would appear to be advantageous. Herein we examine whether ligase IV/XRCC4 can contribute a mismatched nick ligation activity to NHEJ. Toward this end, we (i) describe an E. coli-based coexpression system that provides relatively high yields of the ligase IV/XRCC4 complex, (ii) describe a unique rate-limiting step, which has bearing on how the complex is assayed, (iii) specifically analyze how XRCC4 influences ligase IV catalysis and substrate specificity, and (iv) probe the mismatch tolerance/intolerance of DNA ligase IV/XRCC4 via quantitative in Vitro kinetic analyses. Analogous to most other DNA ligases, ligase IV/XRCC4 is shown to be fairly intolerant of nicks containing mismatched base pairs. These results are discussed in light of the biological roles of NHEJ.In addition to sealing broken DNA back into a contiguous duplex, DNA ligases also have the capacity to influence the accuracy of the repair pathways they are involved in. This is well illustrated by the example of mammalian base excision repair (BER 1 ), where nicks containing 3′-OH mismatched base pairs, resulting from aberrant gap-filling by DNA polymerase (Pol ), are sealed inefficiently by the DNA ligase III/XRCC1 complex (1). This delay in mismatch ligation is expected to provide a greater window of opportunity for nick editing by the 3′ f 5′ exonuclease activity of APE1 (1, 2), the predominant mammalian AP endonuclease (APE).Though a large body of qualitative and quantitative data suggests that DNA ligases from a broad spectrum of organisms are intolerant of non-Watson-Crick base pairing schemes at the 3′-OH terminus of a nick (3 and references therein), our recent analysis of the African swine fever virus (ASFV)-encoded DNA ligase demonstrated that analogous to the recently discovered error-prone DNA polymerases, there are exceptions to this rule (3). The ASFV DNA ligase is not only tolerant of numerous 3′ mismatched base pairing schemes but also actually displays higher catalytic efficiency for sealing a 3′ C:T 2 mismatch than it does for sealing nicks containing Watson-Crick base pairs. Whether the errortolerance of t...

show abstract

Section: Fidelity Of Human Dna Ligaseiv/xrcc4mentioning

confidence: 96%

Human DNA Ligase IV and the Ligase IV/XRCC4 Complex: Analysis of Nick Ligation Fidelity

2007

View full text Add to dashboard Cite

show abstract

“…Nicked DNA-adenylates are not observed during nick sealing by vaccinia ligase or CVLig, yet, DNA-adenylate does accumulate in reactions of vaccinia and CVLig when the position of the reactive 3Ј-OH moiety is perturbed, either by a single base-mismatch involving the 3Ј-OH nucleotide or by a 1-nt gap between the 3Ј-OH and 5Ј-PO 4 termini (12,25). Similarly, T4 DNA ligase accumulates DNA-adenylate during sealing of nicks that have multiple mismatched bases on the 5Ј-PO 4 side of the nick (26). These findings are highly relevant to our single-molecule study of reverse step 3, insofar as the observed occasional dissociation of ligase is likely triggered by the displacement of the 3Ј-OH that occurs during DNA swiveling.…”

Section: Monitoringmentioning

confidence: 99%

Dynamics of phosphodiester synthesis by DNA ligase

Crut

Nair

Köster

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Ligases are essential actors in DNA replication, recombination, and repair by virtue of their ability to seal breaks in the phosphodiester backbone. Ligation proceeds through a nicked DNA-adenylate intermediate (AppDNA), which must be sealed quickly to avoid creating a potentially toxic lesion. Here, we take advantage of ligase-catalyzed AMP-dependent incision of a single supercoiled DNA molecule to observe the step of phosphodiester synthesis in real time. An exponentially distributed number of supercoils was relaxed per successful incision-resealing event, from which we deduce the torque-dependent ligation probability per DNA swivel. Premature dissociation of ligase from nicked DNA-adenylate accounted for Ϸ10% of the observed events. The ability of ligase to form a C-shaped protein clamp around DNA is a key determinant of ligation probability per turn and the stability of the ligaseAppDNA intermediate. The estimated rate of phosphodiester synthesis by DNA ligase (400 s ؊1 ) is similar to the high rates of phosphodiester synthesis by replicative DNA polymerases.DNA ligation ͉ DNA relaxation ͉ magnetic tweezers T he DNA ligases are essential guardians of genome integrity. They seal 3Ј-OH/5Ј-PO 4 DNA nicks via three chemical steps ( Fig. 1a): (i) ligase reacts with ATP (or NAD ϩ ) to form a covalent ligase-adenylate intermediate, in which AMP is linked via a phosphoamide (P-N) bond to N of a lysine on the enzyme; (ii) AMP is transferred from the ligase to the 5Ј-PO 4 strand at a nick to form a DNA-adenylate intermediate (AppDNA); and (iii) ligase catalyzes attack by the 3Ј-OH of the nick on AppDNA to form a phosphodiester bond and release AMP (1). Recent biochemical and crystallographic studies have illuminated the mechanism of nucleotidyl transfer, how ligases recognize nicks as ''damaged,'' and how protein domain movements and active-site remodeling are used to choreograph the sequential steps of the ligation pathway (2, 3). In particular, the crystal structures of nick-bound ligases have revealed a conserved theme whereby ligases envelope the DNA duplex in a C-shaped protein clamp and elicit changes in DNA conformation, including bending at the nick and the adoption of A-form helical structure on the 3Ј-OH side of the nick (4-6).Chlorella virus ligase (CVLig) is a minimized (298 aa) pluripotent exemplar of the ATP-dependent DNA ligase clade. It consists of an N-terminal nucleotidyltransferase domain and a C-terminal OBfold domain. Although lacking the accessory domains found in cellular ligases, it has an intrinsic nick-sensing function and can sustain mitotic growth, excision repair, and nonhomologous end joining in budding yeast when it is the only ligase present in the cell (7-10). Accordingly, CVLig has proven to be an instructive model system for mechanistic and structural studies (11-15). For example, the atomic structure of the CVLig-AMP intermediate bound to duplex DNA with a 3Ј-OH/5Ј-PO 4 nick highlighted the key role of a ␤-hairpin ''latch'' module emanating from the OB domain in forming the C-shaped...

show abstract

“…[21] The difference in enzyme efficiency (k cat /K M ) decreased by more than five orders of magnitude. When the azobenzene was isomerized to the cis form, the k cat value recovered to 0.94 min…”

mentioning

confidence: 99%

Nick Sealing by T4 DNA Ligase on a Modified DNA Template: Tethering a Functional Molecule on D‐Threoninol

Liang

Fujioka

Asanuma

2011

Chemistry A European J

View full text Add to dashboard Cite

Efficient DNA nick sealing catalyzed by T4 DNA ligase was carried out on a modified DNA template in which an intercalator such as azobenzene had been introduced. The intercalator was attached to a D-threoninol linker inserted into the DNA backbone. Although the structure of the template at the point of ligation was completely different from that of native DNA, two ODNs could be connected with yields higher than 90% in most cases. A systematic study of sequence dependence demonstrated that the ligation efficiency varied greatly with the base pairs adjacent to the azobenzene moiety. Interestingly, when the introduced azobenzene was photoisomerized to the cis form on subjection to UV light (320-380 nm), the rates of ligation were greatly accelerated for all sequences investigated. These unexpected ligations might provide a new approach for the introduction of functional molecules into long DNA strands in cases in which direct PCR cannot be used because of blockage of DNA synthesis by the introduced functional molecule. The biological significance of this unexpected enzymatic action is also discussed on the basis of kinetic analysis.

show abstract

Kinetics and thermodynamics of nick sealing by T4 DNA ligase

Cited by 62 publications

References 37 publications

Human DNA Ligase IV and the Ligase IV/XRCC4 Complex: Analysis of Nick Ligation Fidelity

Human DNA Ligase IV and the Ligase IV/XRCC4 Complex: Analysis of Nick Ligation Fidelity

Dynamics of phosphodiester synthesis by DNA ligase

Nick Sealing by T4 DNA Ligase on a Modified DNA Template: Tethering a Functional Molecule on D‐Threoninol

Contact Info

Product

Resources

About