Mycobacterium tuberculosis RecA intein, a LAGLIDADG homing endonuclease, displays Mn(2+) and DNA-dependent ATPase activity

Abstract

Mycobacterium tuberculosis RecA intein (PI-MtuI), a LAGLIDADG homing endonuclease, displays dual target specificity in response to alternative cofactors. While both ATP and Mn(2+) were required for optimal cleavage of an inteinless recA allele (hereafter referred to as cognate DNA), Mg(2+) alone was sufficient for cleavage of ectopic DNA sites. In this study, we have explored the ability of PI-MtuI to catalyze ATP hydrolysis in the presence of alternative metal ion cofactors and DNA substrates. Our results indicate that PI-MtuI displays maximum ATPase activity in the presence of cognate but not ectopic DNA. Kinetic analysis revealed that Mn(2+) was able to stimulate PI-MtuI catalyzed ATP hydrolysis, whereas Mg(2+) failed to do so. Using UV crosslinking, limited proteolysis and amino acid sequence analysis, we show that (32)P-labeled ATP was bound to a 14 kDa peptide containing the putative Walker A motif. Furthermore, the limited proteolysis approach disclosed that cognate DNA was able to induce structural changes in PI-MtuI. Mutation of the presumptive metal ion-binding ligands (Asp122 and Asp222) in the LAGLIDADG motifs of PI-MtuI impaired its affinity for ATP, thus resulting in a reduction in or loss of its endonuclease activity. Together, these results suggest that PI-MtuI is a (cognate) DNA- and Mn(2+)-dependent ATPase, unique from the LAGLIDADG family of homing endonucleases, and implies a possible role for ATP hydrolysis in the recognition and/or cleavage of homing site DNA sequence.

Figure 1

ATP binding to PI-MtuI as monitored by UV catalyzed crosslinking. Reactions were performed as described in Materials and Methods. The autoradiogram shows the SDS–PAGE analysis of photolabeled proteins. Lane 1, M.tuberculosis RecA protein (16 µM) in the presence of 2 mM MgCl₂ and 1.6 pmol [γ-³²P]ATP; lane 2, M.tuberculosis single-stranded DNA-binding protein (16 µM), 2 mM MgCl₂ and 1.6 pmol [γ-³²P]ATP; lane 3, PI-MtuI (16 µM) with 1.6 pmol [γ-³²P]ATP in the absence of added metal ion (–Me²⁺). The remaining lanes contained PI-MtuI (16 µM) and the indicated cofactor(s): 2.5 mM MgCl₂ (lane 4), 5 mM MgCl₂ (lane 5), 2.5 mM MnCl₂ (lane 6); 5 mM MnCl₂ (lane 7). An asterisk denotes the truncated form of PI-MtuI.

Figure 2

Binding of ³²P-labeled ATP to PI-MtuI as a function of ATP concentration. Reactions were performed with increasing concentrations of ATP as indicated above each lane. Reactions mixtures were analyzed as described in Materials and Methods. (A) Autoradiogram showing crosslinking of ATP to PI-MtuI. (B) Quantification of the extent of binding of labeled ATP. The intensity of bands was quantified after correcting the background using UVI-BandMap software ver. 99 and plotted against concentration of ATP using Graphpad Prism ver. 2.0. The graph shows the average of two independent experiments.

Figure 3

ATP hydrolysis catalyzed by PI-MtuI. Reactions were performed in the absence or presence of cofactors and/or DNA as described in Materials and Methods. An autoradiogram of a typical TLC analysis showing ADP generated during the reaction in the absence (lanes 2 and 9) or presence (lanes 1, 3–8 and 10–13) of different cofactors as indicated above each lane. The positions of ATP and ADP are shown on the left.

Figure 4

Effect of a metal ion on ATP hydrolysis catalyzed by PI-MtuI. ATPase assays were performed with increasing concentrations of Mg²⁺ or Mn²⁺. Reactions were terminated and analyzed by TLC as described in Materials and Methods. The bands were quantified and plotted as a function of Mg²⁺ or Mn²⁺ concentration. ATP hydrolysis is expressed as a percentage of ADP formed from [α-³²P]ATP with respect to the value obtained in the absence of metal ions. The graph shows the average of two independent experiments.

Figure 5

PI-MtuI is a Mn²⁺- and (cognate) DNA-dependent ATPase. ATP hydrolysis was performed in an assay buffer containing PI-MtuI (3 µM) and cognate or ectopic DNA (16 µM) in the presence of either 3 mM Mn²⁺ or Mg²⁺ as described in Materials and Methods. The bands corresponding to ADP in the autoradiogram were quantified and plotted against time of incubation using Graphpad Prism ver. 2.0. The graph shows the mean of three independent experiments.

Figure 6

ATP hydrolysis catalyzed by PI-MtuI follows Michaelis–Menten kinetics. Reactions were performed at different concentrations of the substrate with a fixed concentration of PI-MtuI in the presence of 3 mM Mn²⁺. Reaction products were separated by TLC and visualized by autoradiography. Quantification was performed as described in Materials and Methods. The rate of the reaction was calculated from the slopes of such plots. The figure shows 1/v versus 1/[S] in the form of a Lineweaver–Burk plot. The inset shows the summary of kinetic parameters for wild-type PI-MtuI. The graph shows the mean ± SD of three independent experiments.

Figure 7

Mutation of conserved amino acid residues in the LAGLIDADG motifs of PI-MtuI affect binding of ATP and its hydrolysis. (A) Autoradiogram showing the binding of ATP by wild-type and variants of PI-MtuI. (B) Quantification of the extent of binding of ATP by wild-type and variants of PI-MtuI shown in (A). (C) Kinetics of ATPase activity of wild-type and variants of PI-MtuI. The ATPase assay was performed as described in Materials and Methods. The graph shows the mean ± SD of three independent experiments.

Figure 8

Kinetics of cognate DNA cleavage by wild-type and variants of PI-MtuI. Reactions containing wild-type (A) or variants (B and C) of PI-MtuI were performed as described in Materials and Methods. (D) Quantification of the extent of cognate DNA cleavage by wild-type and variants of PI-MtuI shown in (A–C). The graph shows the mean of two independent experiments. Form I, negatively supercoiled DNA; Form II, nicked circular DNA; Form III, linear double-stranded DNA.

Figure 9

Isolation of 14 kDa peptide. PI-MtuI was crosslinked to [γ-³²P]ATP and subjected to limited proteolysis with chymotrypsin as described in Materials and Methods. (A) Coomassie blue stained SDS–PAGE gel showing the partial chymotryptic digest. (B) Autoradiogram of the gel shown in (A).

Figure 10

The effect of cofactors and DNA on limited proteolysis of PI-MtuI. Products of the proteolysis reaction in the absence of cofactors (A) or in the presence of 3 mM Mn²⁺ and 1.5 mM ATP (B) or cognate DNA (16 µM), 3 mM Mn²⁺ and 1.5 mM ATP (C). In each lane, ∼7 µg of PI-MtuI incubated with 1 µl of 0.1 mg/ml bovine chymotrypsin for the indicated time interval was loaded as described in Materials and Methods. The time of digestion (min) is indicated at the top of each panel. The reaction was terminated by the addition of 5 µl of 5× SDS loading buffer. Samples were separated by 10% SDS–PAGE and visualized by staining with Coomassie blue. Lane M, molecular mass markers (Combithek; Boehringer Mannheim Biochemica), with size in kDa indicated on the left. Asterisks represent the major proteolysis fragments of PI-MtuI.