Characterization of the mycobacterial AdnAB DNA motor provides insights into the evolution of bacterial motor-nuclease machines

Abstract

Mycobacterial AdnAB exemplifies a family of heterodimeric motor-nucleases involved in processing DNA double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal RecB-like nuclease module. Here we conducted a biochemical characterization of the AdnAB motor, using a nuclease-inactivated heterodimer. AdnAB is a vigorous single strand DNA (ssDNA)-dependent ATPase (k(cat) 415 s(-1)), and the affinity of the motor for the ssDNA cofactor increases 140-fold as DNA length is extended from 12 to 44 nucleotides. Using a streptavidin displacement assay, we demonstrate that AdnAB is a 3' --> 5' translocase on ssDNA. AdnAB binds stably to DSB ends. In the presence of ATP, the motor unwinds the DNA duplex without requiring an ssDNA loading strand. We integrate these findings into a model of DSB unwinding in which the "leading" AdnB and "lagging" AdnA motor domains track in tandem, 3' to 5', along the same DNA single strand. This contrasts with RecBCD, in which the RecB and RecD motors track in parallel along the two separated DNA single strands. The effects of 5' and 3' terminal obstacles on ssDNA cleavage by wild-type AdnAB suggest that the AdnA nuclease receives and processes the displaced 5' strand, while the AdnB nuclease cleaves the displaced 3' strand. We present evidence that the distinctive "molecular ruler" function of the ATP-dependent single strand DNase, whereby AdnAB measures the distance from the 5'-end to the sites of incision, reflects directional pumping of the ssDNA through the AdnAB motor into the AdnB nuclease. These and other findings suggest a scenario for the descent of the RecBCD- and AddAB-type DSB-processing machines from an ancestral AdnAB-like enzyme.

FIGURE 1.

AdnAB binds stably to DSB ends. Reaction mixtures containing 0.1 μm (1 pmol) of 5′ ³²P-labeled ssDNA, 3′-tailed, or 5′-tailed DNA as specified, and 1 pmol (indicated by +) or 2 pmol (indicated by ++) of nuclease-dead AdnAB were incubated for 30 min at 22 °C. AdnAB was omitted from control reactions (− lanes). The products were analyzed by native PAGE. The free ³²P-labeled DNAs and slower migrating protein·[³²P]DNA complexes (annotated on the right) were visualized by autoradiography. The DNAs are shown at the bottom, with the 5′ ³²P label denoted by ●.

FIGURE 2.

AdnAB is a helicase that requires no loading strand. Reaction mixtures (10 μl) containing 20 mm Tris-HCl (pH 8.0), 0.9 mm DTT, 2 mm MgCl₂, 2 mm ATP, 0.1 μm (1 pmol) of 5′ ³²P-labeled blunt duplex 24-mer, 3′ dT₂₀-tailed, or 5′ dT₂₀-tailed duplex as specified, and 0.85 pmol of nuclease-dead AdnAB or AdnAB* (a nuclease-dead AdnAB heterodimer with an ATPase-inactivating D285A mutation in the AdnB subunit) were incubated for 15 min at 37 °C. The products were analyzed by electrophoresis through a 15-cm 18% native polyacrylamide gel in 0.5× TBE (45 mm Tris borate, 1.2 mm EDTA) and visualized by autoradiography. AdnAB and AdnAB* were omitted from control reactions (− lanes). DNA-containing reaction mixtures lacking AdnAB that were heat-denatured prior to PAGE are included in lanes marked with ▵.

FIGURE 3.

Properties of the AdnAB helicase. A, reaction mixtures (10 μl) containing 20 mm Tris-HCl (pH 8.0), 0.9 mm DTT, 0.1 μm (1 pmol) 5′ ³²P-labeled 24-bp blunt duplex DNA, 0.85 pmol of nuclease-dead AdnAB, and 2 mm MgCl₂, 2 mm ATP, or 2 mm AMPPNP (where specified by +) were incubated for 15 min at 37 °C. B, ATP titration. Reaction mixtures (10 μl) containing 20 mm Tris-HCl (pH 8.0), 2 mm MgCl₂, 0.9 mm DTT, 0.1 μm (1 pmol) 5′ ³²P-labeled 24-bp blunt duplex DNA, 0.85 pmol of nuclease-dead AdnAB, and increasing amounts of ATP as specified were incubated for 15 min at 37 °C. C, nucleotide specificity. Reaction mixtures (10 μl) containing 20 mm Tris-HCl (pH 8.0), 2 mm MgCl₂, 0.9 mm DTT, 0.1 μm (1 pmol) 5′ ³²P-labeled 24-bp blunt duplex DNA, 0.85 of pmol nuclease-dead AdnAB, and either no nucleotide (− lane) or 1 mm of the indicated NTP or dNTP were incubated for 15 min at 37 °C. Enzyme was omitted from the control reaction in lane -E. D, divalent cation specificity. Reaction mixtures containing 20 mm Tris-HCl (pH 8.0), 2 mm ATP, 0.1 μm (1 pmol) 5′ ³²P-labeled 24-bp blunt duplex DNA, 0.85 pmol of nuclease-dead AdnAB, and 2 mm of the specified divalent cation were incubated for 15 min at 37 °C. The reaction products were analyzed by native PAGE and visualized by autoradiography. DNA-containing reaction mixtures lacking AdnAB that were heat-denatured prior to PAGE are included in lanes marked by ▵.

FIGURE 4.

Directionality of AdnAB translocation on ssDNA. A, schematic representation of directional 5′ streptavidin displacement by the AdnAB motor acting as a cowcatcher to pry apart the otherwise stable SA-biotin interaction. See text for discussion. B, translocation reaction mixtures (10 μl) containing 20 mm Tris-HCl (pH 8.0), 1 mm DTT, 2 mm MgCl₂, 2 mm ATP, 50 nm (0.5 pmol) 5′ ³²P-labeled 5′ or 3′ biotinylated 34-mer ssDNA (shown at the bottom, with B signifying the position of the biotin spacer), 2 μm streptavidin (where indicated by +), 20 μm biotin trap, and 0.25 pmol (+), or 0.5 pmol (++) of nuclease-dead AdnAB were incubated for 15 min at 37 °C. The reactions were quenched with EDTA, and the mixtures were analyzed by native PAGE. The radiolabeled DNAs were visualized by autoradiography. The species corresponding to SA·DNA complex and free DNA are indicated on the right. C, reaction mixtures (10 μl) containing 20 mm Tris-HCl (pH 8.0), 1 mm DTT, 2 mm MgCl₂, 2 mm ATP, 50 nm (0.5 pmol) of 5′ ³²P-labeled 5′ biotinylated 34-mer ssDNA, 2 μm streptavidin (where indicated by +), 20 μm biotin trap, and 0.5 pmol of nuclease-dead AdnAB or AdnAB* (a nuclease-dead AdnAB with an inactive AdnB subunit motor) were incubated for 15 min at 37 °C. The products were resolved by native PAGE. D, reaction mixtures (10 μl) containing 20 mm Tris-HCl (pH 8.0), 1 mm DTT, 50 nm 5′ ³²P-labeled 5′ biotinylated 34-mer ssDNA, 2 μm streptavidin (where indicated by +), 0.5 pmol nuclease-dead AdnAB, and 2 mm ATP, 2 mm MgCl₂ or 20 μm biotin trap, where specified by +, were incubated for 15 min at 37 °C. The products were resolved by native PAGE.

FIGURE 5.

AdnAB domain model based on the structure of DNA-bound RecBC.

FIGURE 6.

Model of the AdnAB nuclease ruler and predictions of experimental testing.

FIGURE 7.

A 5′ biotin-streptavidin adduct is a kinetic obstacle to AdnAB translocation. Reaction mixtures (90 μl) containing 20 mm Tris-HCl (pH 8.0), 1 mm DTT, 2 mm MgCl₂, 2 mm ATP, 2 μm streptavidin, 50 nm (4.5 pmol) 5′ ³²P-labeled 5′ or 3′ biotinylated 34-mer ssDNA, 20 μm biotin, and 4.6 pmol of nuclease-dead AdnAB were incubated at 37 °C. Aliquots (10 μl) were withdrawn at the times specified and quenched with EDTA. The mixtures were analyzed by native PAGE, and the radiolabeled DNA was visualized by autoradiography (A). The distributions of radiolabeled DNA between DNA·SA complex, free DNA, and the well-shift complex were quantified by scanning the gel and are plotted as a function of time in B. The results are consistent with a biphasic process of 5′ SA adduct displacement, depicted in schematic form in A, whereby the AdnAB motor rapidly translocates to abut the 5′ SA complex, followed by a slow step in which SA is pried off the DNA by the AdnAB cowcatcher.

FIGURE 8.

Testing the ATP ruler model: effect of 5′ and 3 streptavidin obstacles on the outcomes of ssDNA cleavage. Reaction mixtures (10 μl) containing 20 mm Tris-HCl (pH 8.0), 1 mm DTT, 2 mm MgCl₂, 50 nm (0.5 pmol) 5′ ³²P-labeled 5′ or 3′ biotinylated 34-mer ssDNA or non-biotinylated 24-mer DNA, and 1 mm ATP, 2 μm streptavidin, or 0.26 pmol of wild-type AdnAB (where indicated by +) were incubated for 15 min at 37 °C. The reactions were quenched with formamide/EDTA, heated for 5 min at 95 °C, and then analyzed by electrophoresis through a 15-cm 18% polyacrylamide gel containing 7 m urea, 45 mm Tris borate, 1.25 mm EDTA. An autoradiograph of the gel is shown. The ATP-induced cleavage products of the biotinylated DNAs are highlighted by ●.