Kinetic Mechanism of Human DNA Ligase I Reveals Magnesium-dependent Changes in the Rate-limiting Step That Compromise Ligation Efficiency

Abstract

DNA ligase I (LIG1) catalyzes the ligation of single-strand breaks to complete DNA replication and repair. The energy of ATP is used to form a new phosphodiester bond in DNA via a reaction mechanism that involves three distinct chemical steps: enzyme adenylylation, adenylyl transfer to DNA, and nick sealing. We used steady state and pre-steady state kinetics to characterize the minimal mechanism for DNA ligation catalyzed by human LIG1. The ATP dependence of the reaction indicates that LIG1 requires multiple Mg 2؉ ions for catalysis and that an essential Mg 2؉ ion binds more tightly to ATP than to the enzyme. Further dissection of the magnesium ion dependence of individual reaction steps revealed that the affinity for Mg 2؉ changes along the reaction coordinate. At saturating concentrations of ATP and Mg 2؉ ions, the three chemical steps occur at similar rates, and the efficiency of ligation is high. However, under conditions of limiting Mg 2؉ , the nick-sealing step becomes rate-limiting, and the adenylylated DNA intermediate is prematurely released into solution. Subsequent adenylylation of enzyme prevents rebinding to the adenylylated DNA intermediate comprising an Achilles' heel of LIG1. These ligase-generated 5-adenylylated nicks constitute persistent breaks that are a threat to genomic stability if they are not repaired. The kinetic and thermodynamic framework that we have determined for LIG1 provides a starting point for understanding the mechanism and specificity of mammalian DNA ligases.Breaks in DNA result from spontaneous hydrolysis of the phosphodiester backbone and are formed as transient intermediates during DNA replication and repair pathways. Mammals have three genes encoding DNA ligases that seal these breaks and restore the continuous nature of chromosomes. DNA ligase I (LIG1) 2 is essential for ligation of single-strand breaks in the nucleus, including the ligation of Okazaki fragments during discontinuous DNA replication (1). DNA ligase III (LIG3) is required for mitochondrial DNA replication and repair. Although LIG3 has been assumed to play essential roles in nuclear DNA repair, it was recently shown to be dispensable for nuclear genomic maintenance (2, 3). DNA ligase IV (LIG4) is specialized for repair of nuclear double-strand breaks and is required for nonhomologous end joining and V(D)J recombination (4, 5).The overall reaction catalyzed by DNA ligases involves the formation of a phosphodiester bond between an adjacent 3Ј-hydroxyl and a 5Ј-phosphate in DNA. The reaction proceeds via a universally conserved pathway ( Fig. 1) (6 -8). First, the apoenzyme catalyzes transfer of the AMP group from a nucleotide cofactor to an active site lysine, forming an adenylylated enzyme intermediate. Eukaryotic ligases use ATP as the adenylyl group donor, whereas bacterial ligases utilize either ATP or NAD ϩ . After binding a nicked DNA substrate, the adenylylated enzyme catalyzes transfer of the adenylyl group to the 5Ј-phosphate present at the nick, forming an adenylylated DNA intermediate. In the fina...

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

“…Both data sets were fit well by nonlinear regression to a simple hyperbolic binding equation (Fig. 3, A and B (15). This low-Mg 2ϩ "Achilles' heel" of the HuLig1 mechanism is apparently not shared by ChVLig.…”

“…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.…”

“…Mg 2ϩ in this position would be well poised to stabilize the transition state of the AMP phosphate during step 2 DNA adenylylation and to activate the 3Ј-OH nucleophile during step 3 phosphodiester synthesis. We were especially interested in the effects of Mg 2ϩ concentration in light of the report that limiting Mg 2ϩ changes the rate-limiting step for nick sealing by HuLig1 in such a way as to potentially compromise genome stability (15). It has been appreciated for more than 40 years that acidic pH conditions favor the detection of otherwise fleeting 5Ј-adenylylated polynucleotide species in reactions of DNA and RNA ligases at 5Ј-phosphate ends (20 -22).…”

“…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

“…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.…”