(A) Helicase activity of wild type (wt) Mcm2-7, the pentameric preparation (), and single DENQ (top) and RA (bottom) mutant Mcm2-7 complexes. Electrophoretic positions of the fork substrate and dissociated single strand are shown at left. The lane marked ‘boiled (+)’ serves as a positive control and shows the position of the radioactive single strand after unwinding of the fork; the lane marked ‘boiled (−)’ shows the position of the radioactive fork in the absence of dissociation. The identity of the mutant subunit is listed below the gel. (B) Quantitation of unwinding activities shown in (A). Previous results with the KA mutant complexes (14) are listed for comparison. (C) Confirmation that DNA unwinding of the DENQ and RA mutant preparations is Mcm2-7-dependent. Pair-wise helicase assays of the indicated Mcm2-7 and Mcm467 preparations in the presence or absence of an antibody that specifically blocks Mcm467 DNA unwinding (14) are shown.

(A) Correlation between various biochemical and genetic defects of individual Mcm ATPase alleles and their location within the Mcm2-7 ring. Walker A (KA, denoted by black or gray rectangle), Walker B (DENQ, denoted by red circle) and arginine finger (RA, denoted by cyan triangle) mutations of each subunit are mapped onto the putative circular organization of Mcm2-7, as indicated. To illustrate the trends in these data in a simple manner, the activity of each mutation on the indicated property is categorized into one of three groups and labeled as follows: approximately wild-type (120–33% the levels of wild type Mcm2-7, no fill), partially defective (33–10%, shaded), or completely defective (<10% the activity of wild type, solid fill). These categories are for illustration only and do not imply, for example, that a mutant placed in the wild-type category for one activity globally functions as a true wild type Mcm2-7 complex. The summary of association rates was derived from Supplementary Table S4, and missing alleles correspond to those that bind ssDNA too poorly to reliably quantify an association rate. Trends in the relative ATPγS preincubation stimulation of association rates were quantified as follows: unfilled symbols correspond to a rate stimulation between 5.4- (wt) and 2.4-fold; shaded fill corresponds to a rate stimulation of <2.4- to 1.4-fold; and solid fill corresponds to a rate stimulation of <1.4-fold. Trends in the circularization activity were plotted as the ratio of cir1st/cir2nd: unfilled corresponds to a ratio of 0.4–0.7 (∼wt); shaded fill 0.7–0.8; and solid fill 0.9–1.0 (completely defective). Data concerning the KA mutations are summarized from (5,14,15), the ATP hydrolysis and viability data of the DENQ and RA alleles are from (12), and the ssDNA association rate and circularization data for the RA mutants are from (14). (B) Model of Mcm2-7 activity. The Mcm5/3 and 6/2 active sites communicate the status (open versus closed) of the Mcm2/5 gate to the Mcm7/4 motor. When the gate is open (left), helicase activity is inhibited; when the gate is closed (right), helicase activity is stimulated.

DNA binding of mutant Mcm2-7 complexes. (A) Proposed subunit organization of Mcm2-7 (12,13). (B) Silver-stained SDS-PAGE gel of wild-type and indicated mutant Mcm2-7 hexamers. (C) ATPγS-dependent ssDNA binding as a function of (Mcm2-7) for DENQ (top) or RA (bottom) mutant complexes. The number to the right of each curve denotes the mutant subunit; 6x refers to Mcm2-7 complexes in which all six subunits contain the indicated mutation. Boxes around the 150 nM points indicate the data used to create Figure 1D. (D) Summary of Mcm2-7 ssDNA binding by the pentameric preparation () and the single and sextuple (6x) KA (left), DENQ (middle) and RA (right) mutant hexamers (at 150 nM). The data for the Mcm2-7 complexes containing the indicated KA mutants are from ref. 5 and are shown for comparison. Assays for all three sets of mutant complexes were performed under identical conditions. Although the Mcm2-7 complex containing Mcm7KA appears to bind ssDNA, this binding is largely independent of ATP (5), and as such, we consider this mutant to be defective in ssDNA binding.

Effects of ATP preincubation on Mcm2-7 circular ssDNA binding and ssDNA association by the wild-type (wt) and mutant complexes. (A) Circularization assay. This assay measures the ability of a circular ssDNA substrate (i.e. cir) to compete with the standard radiolabeled oligonucleotide substrate for Mcm2-7 binding as a function of ATPγS preincubation. The three vertical bars in the graph for each protein represent (i) normalized relative binding in the absence of circular competitor DNA; (ii) competition experiments in which both the cold circular competitor and radiolabeled oligonucleotide are added to the Mcm complex for 30 min prior to ATPγS addition (cir 1st); and (iii) conditions in which the Mcm preparation was incubated with ATPγS prior to addition of both cold circular ssDNA competitor and radiolabeled oligonucleotide (cir 2nd). Ring closure results for wild type Mcm2-7, Mcm467, the pentameric preparation (), and 2RA complexes have been previously published (14) and are shown here for comparison. (B) Summary of circularization. The ratio of cir1st/cir 2nd for each mutant Mcm2-7 preparation (Supplementary Table 3) is plotted in ranked order. (C) Mcm2-7/ssDNA association as a function of time. The Mcm preparations were either preincubated (open squares) or not (filled diamonds) with ATPγS for 30 min prior to addition of the ssDNA substrate, as described in the ‘Materials and methods’ section. The filter binding method and the radiolabeled ssDNA substrate used are the same as in Figure 1C. Note: the association rate data for the wild-type Mcm2-7, Mcm467, pentamer (), and Mcm2RA have been previously published [(5) and Supplementary Data in ref. 14), and are shown here for comparison. (D) Summary of association rate enhancement of the mutant Mcm2-7 preparations with ssDNA. The ratio of the association rate in the absence of ATPγS preincubation to the association rate in the presence of ATPγS preincubation for each mutant Mcm2-7 preparation (Supplementary Table 4) is plotted in ranked order.