Kinetic mechanism and fidelity of nick sealing by Escherichia coli NAD+-dependent DNA ligase (LigA)

Abstract

Escherichia coli DNA ligase (EcoLigA) repairs 3'-OH/5'-PO4 nicks in duplex DNA via reaction of LigA with NAD(+) to form a covalent LigA-(lysyl-Nζ)-AMP intermediate (step 1); transfer of AMP to the nick 5'-PO4 to form an AppDNA intermediate (step 2); and attack of the nick 3'-OH on AppDNA to form a 3'-5' phosphodiester (step 3). A distinctive feature of EcoLigA is its stimulation by ammonium ion. Here we used rapid mix-quench methods to analyze the kinetic mechanism of single-turnover nick sealing by EcoLigA-AMP. For substrates with correctly base-paired 3'-OH/5'-PO4 nicks, kstep2 was fast (6.8-27 s(-1)) and similar to kstep3 (8.3-42 s(-1)). Absent ammonium, kstep2 and kstep3 were 48-fold and 16-fold slower, respectively. EcoLigA was exquisitely sensitive to 3'-OH base mispairs and 3' N:abasic lesions, which elicited 1000- to >20000-fold decrements in kstep2. The exception was the non-canonical 3' A:oxoG configuration, which EcoLigA accepted as correctly paired for rapid sealing. These results underscore: (i) how EcoLigA requires proper positioning of the nick 3' nucleoside for catalysis of 5' adenylylation; and (ii) EcoLigA's potential to embed mutations during the repair of oxidative damage. EcoLigA was relatively tolerant of 5'-phosphate base mispairs and 5' N:abasic lesions.

Figure 1.

Kinetics of single-turnover nick sealing by EcoLigA at correctly paired 5′-phosphate ends. (A) The singly nicked duplex substrate is shown with the 5′ ³²P-label at the nick denoted by •. The 5′ N:X base pair at the nick (highlighted in the shaded box) is variable. The distributions of radiolabeled AppDNA intermediate (black circles) and sealed 36-mer DNA product (gray squares) during the reaction of EcoLigA–AMP at the indicated 5′ N:X nicks are plotted as a function of time. The curve fits to the kinetic scheme are shown. (B) The step 2 and step 3 rate constants for sealing nicks with the indicated 5′ N:X pairs are shown.

Figure 2.

Kinetics of nick sealing by EcoLigA at correctly paired 3′-OH ends. (A) The singly nicked duplex substrate is shown with the 5′ ³²P-label at the nick denoted by •. The 3′ N:X base pair at the nick (highlighted in the shaded box) is variable. The distributions of radiolabeled AppDNA intermediate (black circles) and sealed 36-mer DNA product (gray squares) during the reaction of EcoLigA–AMP at the indicated 3′ N:X nicks are plotted as a function of time. The curve fits to the kinetic scheme are shown. (B) The step 2 and step 3 rate constants for sealing nicks with the indicated 3′ N:X pairs are shown.

Figure 3.

Effect of ammonium ion on the kinetics of EcoLigA nick sealing. The singly nicked duplex substrate is shown with the 5′ ³²P-label at the nick denoted by •. (A) The distributions of radiolabeled AppDNA intermediate (black circles) and sealed 36-mer DNA product (gray squares) during the reaction of EcoLigA–AMP with the nicked DNA in the absence of ammonium sulfate are plotted as a function of time. The curve fits to the kinetic scheme are shown. (B and C) Single-turnover nick sealing was assayed in the presence of the indicated concentrations of ammonium sulfate. The apparent step 2 and step 3 rate constants (± standard error) are plotted as a function of ammonium sulfate concentration in panels B and C, respectively.

Figure 4.

Effects of 5′ N:X mispairs on the kinetics of nick sealing. The singly nicked duplex substrate is shown at the top with the 5′ ³²P-label at the nick denoted by •. The 5′ N:X site at the nick (highlighted in the shaded box) is variable. (A and B) The distributions of radiolabeled AppDNA intermediate (black circles) and ligated 36-mer DNA product (gray squares) during the reaction of EcoLigA–AMP at the 5′ C:A nick (panel A) and the 5′ G:G nick (panel B) are plotted as a function of time. The curve fits to the kinetic scheme are shown. (C) The step 2 and step 3 rate constants for sealing nicks with the indicated 5′ N:X configurations are shown.

Figure 5.

Effects of 3′ N:X mispairs on the kinetics of nick sealing. The singly nicked duplex substrate is shown at the bottom with the 5′ ³²P-label at the nick denoted by •. The 3′ N:X site at the nick (highlighted in the shaded box) is variable. (A–D) The distributions (±SEM) of radiolabeled AppDNA intermediate (black circles), ligated 36-mer DNA product (gray squares) and the sum of AppDNA plus ligated DNA (open circles) during the reaction of EcoLigA–AMP at the indicated 3′ N:X mispaired nicks are plotted as a function of time. The thick black curves though the open circles show the nonlinear regression fits of the sum of AppDNA plus ligated DNA to a single exponential, from which we derived k_step2. (E) The step 2 rate constants for sealing nicks with the indicated 3′ N:X configurations are shown. The 3′ A:G and 3′ G:A mispaired nicks were deemed inactive as substrates, by the criterion than there was no detectable formation of AppDNA or ligated DNA during a 40 min reaction with EcoLigA–AMP.

Figure 6.

Sealing of nicks with 5′ N:abasic lesions. (A and B) The distributions of radiolabeled AppDNA intermediate (black circles) and ligated 36-mer DNA product (gray squares) during the reaction of EcoLigA–AMP at the 5′ A:abasic nick (panel A) and the 5′ C:abasic nick (panel B) are plotted as a function of time. The curve fits to the kinetic scheme are shown. (C) The step 2 and step 3 rate constants for sealing nicks with the indicated 5′ N:abasic configurations are shown.

Figure 7.

Sealing of nicks with 3′ N:abasic lesions. (A) The distributions (±SEM) of radiolabeled AppDNA intermediate (black circles), ligated 36-mer DNA product (gray squares) and the sum of AppDNA plus ligated DNA (open circles) during the reaction of EcoLigA–AMP at the 3′ G:abasic nick are plotted as a function of time. The thick black curve though the open circles shows the nonlinear regression fit of the sum of AppDNA plus ligated DNA to a single exponential, from which we derived k_step2. (B) The step 2 rate constants for sealing nicks with the indicated 3′ N:abasic configurations are shown. The 3′ C:abasic nick was deemed inactive by the criterion than there was no detectable formation of AppDNA or ligated DNA during a 40 min reaction with EcoLigA–AMP.

Figure 8.

Sealing of nicks with 3′ N:oxoguanine lesions. (A and B) The distributions of radiolabeled AppDNA intermediate (black circles) and ligated 36-mer DNA product (gray squares) during the reaction of EcoLigA–AMP at the 3′ C:oxoG nick (panel A) and the 3′ A:oxoG nick (panel B) are plotted as a function of time. The curve fits to the kinetic scheme are shown. The chemical structure of the A:oxoG pair with oxoG in the syn conformation is shown as an inset in panel B; hydrogen-bonding contacts are indicated by dashed lines. (C) The step 2 and step 3 rate constants for sealing nicks with the indicated 3′-OH N:oxoG configurations are shown.