Kinetic Characterization of Single Strand Break Ligation in Duplex DNA by T4 DNA Ligase

Abstract

Background: T4 DNA ligase catalyzes the formation of phosphodiester bonds in dsDNA.Results: Single turnover rates of all chemical reaction steps exceed steady state turnover rates by 10-fold.Conclusion: Product release or a postligation conformational change is rate-limiting during turnover.Significance: This study represents the first detailed analysis of the kinetic mechanism of nick ligation by T4 DNA ligase.

Coevolution of RtcB and Archease created a multiple-turnover RNA ligase

“…Classical nucleic acid ligases that join 5 ′ -p and 3 ′ -OH termini are multiple-turnover enzymes (Lohman et al 2011), highlighting a peculiar aspect of catalysis by RtcB. Studies on human RtcB and Archease suggest that Archease facilitates the guanylylation of RtcBq after a single turnover, enabling another round of catalysis .…”

Kinetic Analysis of DNA Strand Joining by Chlorella Virus DNA Ligase and the Role of Nucleotidyltransferase Motif VI in Ligase Adenylylation

“…Thus, the attack of the nick 3Ј-OH on AppDNA is an order of magnitude faster than the formation of AppDNA. Similar single-turnover kinetic studies have been performed for human DNA ligase I (HuLig1) (15) and bacteriophage T4 DNA ligase (16), yielding rate constants as follows: HuLig1 (k step2 2.6 s Ϫ1 ; k step3 12 s Ϫ1 ) and bacteriophage T4 DNA ligase (k step2 5.3 s Ϫ1 ; k step3 38 s Ϫ1 ). It would appear that diverse ATP-dependent DNA ligases have similar kinetic properties with respect to steps 2 and 3.…”

“…A recent innovation to the functional analysis of DNA ligation is the application of rapid mix-quench methods to study the transient state kinetics of nick sealing by preformed ligaseadenylate (13)(14)(15)(16). As implemented for ChVLig (13,14), the observed rate profiles fit well to a unidirectional scheme of DNA adenylylation (step 2, Lig-AMP⅐pDNA 3 Lig⅐AppDNA) and subsequent phosphodiester synthesis (step 3, Lig⅐AppDNA 3 Lig⅐AMP⅐DNApDNA), with allowance for reversible branching of Lig⅐AppDNA to an "out-of-pathway" state (e.g.…”

Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3′-OH base mispairs and damaged base lesions

“…) (Lohman et al 2011). It would appear that diverse ATP-dependent DNA ligases have similar kinetic properties with respect to steps 2 and 3, whereby step 2 is rate limiting.…”

“…We and others have applied rapid mix-quench methods to study the transient state kinetics of DNA nick sealing by ATP-dependent DNA ligases (Lohman et al 2011;Shuman 2011a,b, 2012;Taylor et al 2011). Here we extended this approach to T4 Rnl2.…”