CMG helicase can use ATPγS to unwind DNA: Implications for the rate-limiting step in the reaction mechanism

Abstract

The adenosine triphosphate (ATP) analog ATPγS often greatly slows or prevents enzymatic ATP hydrolysis. The eukaryotic CMG (Cdc45, Mcm2 to 7, GINS) replicative helicase is presumed unable to hydrolyze ATPγS and thus unable to perform DNA unwinding, as documented for certain other helicases. Consequently, ATPγS is often used to "preload" CMG onto forked DNA substrates without unwinding before adding ATP to initiate helicase activity. We find here that CMG does hydrolyze ATPγS and couples it to DNA unwinding. Indeed, the rate of unwinding of a 20- and 30-mer duplex fork of different sequences by CMG is only reduced 1- to 1.5-fold using ATPγS compared with ATP. These findings imply that a conformational change is the rate-limiting step during CMG unwinding, not hydrolysis. Instead of using ATPγS for loading CMG onto DNA, we demonstrate here that nonhydrolyzable adenylyl-imidodiphosphate (AMP-PNP) can be used to preload CMG onto a forked DNA substrate without unwinding.

Keywords: ATPgammaS; CMG helicase; DNA replication; rate-limiting step; staircase model.

Fig. 1.

CMG can use ATPγS to unwind a DNA fork. (A) Unwinding of a 20-bp forked DNA by CMG. The scheme (Left) explains that the forked DNA is preincubated with CMG and 0.1 mM ATPγS for various times, and then 5 mM ATP is added to initiate unwinding (Right). (B) Native 10% polyacrylamide-gel electrophoresis (PAGE) of the helicase assays. Products formed during the preincubation time with ATPγS are shown in the Left three lanes, and reaction times with ATP are shown in the Right six lanes. (C) Quantitation of the gel data in B; error bars show the SEM.

Fig. 2.

CMG unwinds 20- and 30-bp forked DNAs of different sequences at nearly the same rate using ATPγS or ATP. (A) Scheme of the assays. No preloading step was used. Instead, ATP or ATPγS was present and CMG was added directly to assays. (B) Native PAGE of CMG helicase assays using either ATP or ATPγS to unwind a 20-bp forked DNA assembled from Y20 leading and lagging oligos (SI Appendix, Table S1). (B, Bottom) Quantitation of the results; assays were performed in triplicate and error bars show the SEM. (C) Native PAGE of CMG helicase assays using either ATP or ATPγS, to unwind a 30-bp forked DNA having distinct sequences from the 20-bp fork, and assembled from N30 leading and lagging oligos. (C, Bottom) Quantitation of the gels is shown. Oligo sequences are in SI Appendix, Table S1.

Fig. 3.

Mutational evidence that unwinding by CMG using ATPγS is not due to a contaminant of ATP. (A) Reactions were performed using the N30-bp forked DNA and ATP without preincubation using either wt CMG (first seven lanes) or CMG that carries a mutation in the Walker A box (CMG^K-A) (second set of lanes). Use of twice the concentration of the CMG^K-A mutant is shown in the third set of seven lanes, and use of equal amounts of the CMG^K-A mutant plus the wt CMG is shown in the last seven lanes. (B) The reactions are the same as explained in A, except for use of 0.3 M ATPγS in place of 0.3 M ATP.

Fig. 4.

AMP-PNP supports preloading of CMG onto forked DNA. (A) Scheme of the assay. Preincubation for CMG loading was with 0.3 mM ATPγS or 0.3 mM AMP-PNP after which 5 mM ATP was added. (B) PAGE analysis of products during the preincubation with either ATPγS or AMP-PNP, and after the addition of ATP. (C) Quantitation of triplicate assays using ATPγS in which the error bars on the data points represent the SEM.

Fig. 5.

CMG is less able to unwind long products with ATPγS compared with the use of ATP. (A) Unwinding reactions were performed using either ATP or ATPγS. (B) Substrate and products using ATPγS were analyzed by native PAGE (horizontal slices are shown), and then scanned for intensity by a Typhoon phosphorimager (arbitrary units, but the same units are used for both gels in B and C). (C) The same as B except for use of ATP instead of ATPγS. (D) Quantitation shows that as the length of the duplex increases, the ratio of DNA that is unwound using ATPγS compared with the unwound DNA using ATP becomes smaller (i.e., ATP results in greater unwinding than ATPγS). The red dashed lines through B and C point out the unwound DNA peaks. The ratio of the areas of these peaks is presented in D.

Fig. 6.

Staircase model of hexameric helicase action. In the staircase model of DNA unwinding, the helicase encircles one strand as a staircase, and the subunit at the bottom of the spiral staircase (i.e., subunit A is shown in red) hydrolyzes ATP and then moves to the top of the spiral and exchanges ADP for ATP to rebind DNA. This process is proposed to repeat around the ring to move the staircase along the ssDNA. Studies of CMG as a staircase indicate unique properties of each ATP site (10) as do studies of individual ATP sites in reconstitution studies (35, 36).