Clutch mechanism of chemomechanical coupling in a DNA resecting motor nuclease

Abstract

Mycobacterial AdnAB is a heterodimeric helicase–nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The N-terminal motor domain of the AdnB subunit hydrolyzes ATP to drive rapid and processive 3′ to 5′ translocation of AdnAB on the tracking DNA strand. ATP hydrolysis is mechanically productive when oscillating protein domain motions synchronized with the ATPase cycle propel the DNA tracking strand forward by a single-nucleotide step, in what is thought to entail a pawl-and-ratchet–like fashion. By gauging the effects of alanine mutations of the 16 amino acids at the AdnB–DNA interface on DNA-dependent ATP hydrolysis, DNA translocation, and DSB resection in ensemble and single-molecule assays, we gained key insights into which DNA contacts couple ATP hydrolysis to motor activity. The results implicate AdnB Trp325, which intercalates into the tracking strand and stacks on a nucleobase, as the singular essential constituent of the ratchet pawl, without which ATP hydrolysis on ssDNA is mechanically futile. Loss of Thr663 and Thr118 contacts with tracking strand phosphates and of His665 with a nucleobase drastically slows the AdnAB motor during DSB resection. Our findings for AdnAB prompt us to analogize its mechanism to that of an automobile clutch.

Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules

“…Due to space constraints, we were limited in the number of investigators we could highlight in this section and apologize to those who have contributed to the development and application of single-molecule assays but were not mentioned herein. Specific techniques that utilize TIRF microscopes to study protein-DNA complexes include DNA or protein tethering [39][40][41][42], DNA combing and curtains [43][44][45][46], DNA tightropes (via oblique angle fluorescence imaging) [47,48], as well as smFRET [40,[49][50][51][52][53] and TIRF/AFM systems [54][55][56]. While the examples mentioned below include data collected on both prism and objective-based TIRF instruments, in most cases, the prismTIRF microscope described above can be used for similarly designed experiments.…”

Structure–activity relationships at a nucleobase-stacking tryptophan required for chemomechanical coupling in the DNA resecting motor-nuclease AdnAB

“…Because the average rate of DSB resection is similar to the apparent k cat of 415 s −1 for ssDNA-dependent ATP hydrolysis, it is surmised that 1 bp is unwound by AdnAB per ATP hydrolyzed. AdnAB is prone to spontaneous pausing at random sites during the resection of long dsDNA substrates; a notable property of AdnAB is that the velocity of DSB resection slows after the enzyme experiences a spontaneous pause ( 10 , 11 ). Outstanding mechanistic issues concern the physical basis for the chemomechanical coupling of ATP hydrolysis to DNA unwinding.…”

“…The cryo-EM structures guided a comprehensive alanine scan of the 16 amino acids at the AdnB-DNA interface aimed at discerning which DNA contacts are necessary to couple ATP hydrolysis to motor activity ( 11 ). Mutant AdnAB enzymes were assayed for DNA-dependent ATP hydrolysis, DNA translocation, and DSB resection in ensemble and single-molecule formats.…”

“…The results pinpointed the AdnB amino acid Trp325 (a constituent of SF1 helicase motif III) as the singular essential constituent of the putative ratchet pawl, insofar as: (i) W325A mutant AdnAB was inert in ATP-dependent DSB resection; (ii) W325A AdnAB was defective for ATP-dependent translocation on ssDNA; (iii) W325A AdnAB had the same specific activity in ssDNA-dependent ATP hydrolysis as wild-type AdnAB; and (iv) W325A and wild-type AdnAB had similar apparent K m values for the ssDNA activator of ATP hydrolysis (236 and 192 nM, respectively), i.e. loss of the tryptophan did not impede ssDNA binding ( 11 ).…”

“…The essential Trp325 makes a π stack on a nucleobase 5′ of a nucleoside that is flipped out between base-stacked segments of the tracking strand (Figure 1A and Supplementary Figure S2 ). Its neighbor Arg326, which makes a cation–π stack on the nucleobase 3′ of the flipped-out nucleoside (Figure 1A and Supplementary Figure S2 ), is not essential for ATP hydrolysis or DNA resection ( 11 ). Leu142 and Phe254, which intercalate into the tracking strand further downstream (Figure 1A and Supplementary Figure S2 ), also proved not to be important for AdnAB motor activity ( 11 ).…”