Chi hotspot control of RecBCD helicase-nuclease by long-range intramolecular signaling

Repair of broken DNA by homologous recombination requires coordinated enzymatic reactions to prepare it for interaction with intact DNA. The multiple activities of enterobacterial RecBCD helicase-nuclease are coordinated by Chi recombination hotspots (5′ GCTGGTGG 3′) recognized during DNA unwinding. Chi is recognized in a tunnel in RecC but activates the RecB nuclease, > 25 Ǻ away. How the Chi-dependent signal travels this long distance has been unknown. We found a Chi hotspot-deficient mutant in the RecB helicase domain located > 45 Ǻ from both the Chi-recognition site and the nuclease active site. This unexpected observation led us to find additional mutations that reduced or eliminated Chi hotspot activity in each subunit and widely scattered throughout RecBCD. Each mutation alters the intimate contact between one or another pair of subunits in crystal or cryoEM structures of RecBCD bound to DNA. Collectively, these mutations span a path about 185 Ǻ long from the Chi recognition site to the nuclease active site. We discuss these surprising results in the context of an intramolecular signal transduction accounting for many previous observations.

. Model for homologous recombination and DNA break repair by RecBCD enzyme and its control by Chi hotspots. (A) Pathway of RecBCD-promoted recombination and DNA break repair 1,23 . RecBCD binds a ds DNA end (a) and unwinds the DNA, producing loop-tail structures (b) that are converted into twin-loop structures (c) by annealing of the tails. At Chi, RecBCD nicks the 3′-ended strand (d) and loads RecA (e); later, the RecBCD subunits disassemble (likely at the DNA end with purified components) 20 . The ssDNA-RecA filament invades intact homologous DNA to form a D-loop (f), which can be converted into a Holliday junction and resolved into reciprocal recombinants (g). Alternatively, the 3′-end in the D-loop can prime DNA synthesis and generate a non-reciprocal recombinant (h). See ref. 1 for discussion of alternative models. (B) Atomic structure of RecBCD bound to DNA (PDB 1W36) 14 . RecB is orange, RecC blue, and RecD green. Yellow dashed line indicates the RecC tunnel in which Chi is recognized. Cyan indicates the RecC patch with differential trypsin-sensitivity during the RecBCD reaction cycle 19 . Table 1. Mutants altered in RecC-RecD contact (CD), RecD-RecB contact (DB), and RecB-RecC contact (BC) have little or no Chi hotspot activity. a The indicated mutation was on a derivative of plasmid pSA607 (recBCD + ) in strain V2831 (ΔrecBCD2731). Complete genotypes and the amino acids altered are in Tables S1-S3. b The contact points are composed of the indicated amino acids at the indicated positions. The alleles have mutations deleting the indicated amino acid (△); substitutions (when made) contain the indicated amino acid; (.) indicates that the amino acid is wild type. c Chi hotspot activity in lambda vegetative crosses was determined as described in Materials and Methods 31 . Chi hotspot activity = √(t/c) 1 /(t/c) 2 where t/c is the ratio of turbid (cI + ) to clear (cI857) recombinant plaques among J + R + recombinants in cross 1 (with χ + D123) and cross 2 (with χ + 76). d Frequency of His + [Str R ] recombinants per viable Hfr parent relative to that in the concurrent recBCD + cross. Wild-type frequency was 6.99% ± 0.46% (n = 25). e NA, not applicable.
Scientific Reports | (2020) 10:19415 | https://doi.org/10.1038/s41598-020-73078-0 www.nature.com/scientificreports/ or no Chi hotspot activity but retention of recombination-proficiency and "general" (Chi-independent) nuclease activity [16][17][18]30 . More surprising, however, was recB344: this mutant has significantly reduced Chi hotspot activity, the ratio of recombinant frequency in a genetic interval with Chi to that in the same interval without Chi 31 . Chi activity was 2.1 ± 0.033 in recB344 but 4.9 ± 0.021 in concurrent wt crosses (1 indicates no Chi activity; data are mean ± SEM with n = 3) (Table 1) (see also 18,28 ). It also had reduced cutting of DNA at Chi (Fig. S1), as expected from the genetic assays. The recB344 mutant retains, however, recombination-proficiency in E. coli Hfr crosses (93% of wt) and general nuclease activity (60% of wt) (Tables 1 and 2) 18,28 . Thus, it has the same overall phenotype as RecC tunnel mutants with reduced Chi hotspot activity. Also surprising was the location of the recB344 mutation. DNA sequencing showed that it changes 5′ GCC 3′ to 5′ GTC 3′, which changes alanine 68 to valine (A68V). A68 is part of an α-helix within the RecB helicase domain 14,29 . In the cryoEM structure in which the Chi octamer is in the RecC tunnel, A68 is 24-42 Ǻ from the Chi nucleotides and ~ 65 Ǻ from the RecB nuclease active site 15 . These results show that positions in RecBCD far from either the site of Chi recognition (in the RecC tunnel) or the nuclease active site (in the RecB nuclease domain) are involved in Chi hotspot activity. They also led us to seek additional RecBCD mutants with little or no Chi hotspot activity but with amino acid alterations far from the RecC tunnel or the RecB nuclease active site.
Search for additional wide-spread mutations with the Chi-recognition-deficient phenotype. The recognition of Chi in the RecC tunnel but its stimulation of the RecB nuclease indicates that some "signal" must be transmitted through the enzyme from the tunnel to the nuclease. A search for a Chi-dependent covalent modification, such as phosphorylation, that could account for the Chi-dependent alteration of RecBCD www.nature.com/scientificreports/ activities was negative (AFT, unpublished data). Instead, we found evidence for a Chi-dependent conformational change: a patch on the RecC surface ( Fig. 1B) is sensitive to four proteases before DNA is bound and becomes markedly more resistant when DNA is bound 19 . During unwinding up to Chi this patch remains resistant but becomes protease-sensitive again after Chi's encounter. The recB344 mutation may affect this Chi-dependent conformational change. Other mutations could have a similar effect, by blocking a conformational change from one subunit to another. If so, we reasoned that points of intimate contact between subunits might well be involved in the response to Chi. To test this hypothesis, we examined crystal and cryoEM structures of RecBCD 14,15,29 for points of contact between RecC and RecD (called "CD"), between RecD and RecB ("DB"), and in or near the RecB nuclease domain close to the exit of the RecC tunnel ("BC") ( Fig. 2). (To aid Discussion, we designate each side of the contact as C2 D2, D3 B3, and B4 C4, respectively; Fig. 2.) Contact points for DB and BC were discussed by Wilkinson et al. 29 (see Discussion). We then mutated, by deletion or amino acid substitutions, these points of contact and assayed the genetic and enzymatic activities as for recB344 and other Chi-recognition mutants. (Note that there is no direct assay for Chi recognition, such as binding of Chi DNA to RecBCD, since Chi is recognized only during active unwinding by RecBCD; consequently, only the effect of Chi can be assayed, such as cutting of DNA at Chi and Chi genetic hotspot activity.) As described below, we isolated multiple mutants altered at each contact point. Many of these mutants have reduced or undetectable Chi hotspot and cutting activities, like previously described Chi recognition mutants 16-18,30 . RecC-RecD interaction. In the published crystal and cryoEM structures ( Fig. 2A,B) 14,15,29 , four contiguous amino acids (here named C2) in RecC make intimate contact with three contiguous amino acids (named D2) in RecD (Figs. 2C and S2). C2 is QGEW at positions 541-544 in RecC's helicase-like domain 2B, and D2 is PTP at positions 97-99 in RecD's domain 1 ( Table 1). Deletion of C2 (C2Δ) strongly reduced Chi hotspot activity to 1.5 (from 5.1 in wild type), and deletion of D2 (D2Δ) abolished Chi hotspot activity (Chi activity = 1.0) ( Table 1). As expected, the double mutant C2Δ D2Δ also had no significant Chi activity. Substitution of alanine for the amino acids of C2 or D2 (C2ala or D2ala) also significantly reduced Chi activity, to 3.3 or 2.7, respectively (Table S1). These mutants had slightly reduced recombination proficiency in Hfr matings (~ 40 to 80% of wt); a recBCD null mutant's proficiency is much less (0.7% of wt; Tables 1 and S1). Thus, Chi hotspot activity requires the point of contact (CD) between RecC and RecD.

RecD-RecB interaction.
In the cryoEM structures of RecBCD bound to DNA 15,29 , four contiguous amino acids (QPSR) in RecD with overall positive charge contact an α-helix of 13 amino acids (named B3) in RecB, six with negative charge (D or E; Figs. 2D and S2). We define D3 as SVQPSRLP at positions 521-528 in RecD's helicase domain 2B and B3 as DEHAWDVVVEEFD at positions 634-646 in RecB's helicase domain 2B ( Table 1). As for the CD interaction, complete deletion of B3 or the central part of D3 (QPSR) eliminated Chi hotspot activity ( Table 1). Substitution of alanine for the central four amino acids in D3 or for the six negative amino acids in B3 or both sets strongly reduced Chi activity (to 1.7, 1.7, and 1.5, respectively; Table 1). A second double mutation (D3Δ B3ala) eliminated Chi activity (1.1). Hfr recombination proficiency was reduced to 20-50% of the wildtype value, except in the B3Δ single mutant, which had recombination proficiency similar to that of a recBCD null mutant (Table 1). These results show that the DB point of contact is essential for Chi activity.
As noted above, deletion of the central four amino acids in D3 (QPSR) eliminated Chi activity. To determine which of these four amino acids is critical, we deleted separately the left two (QP) and the right two (SR) amino acids. Surprisingly, both mutants had nearly full Chi activity (4.4 and 5.0, respectively; Table S2A). We then deleted other combinations of amino acids in D3 (SVQPSRLP). Some of these deletions eliminated Chi activity (e.g., deletion of SV, deletion of QP plus LP, or deletion of SRLP); other deletions strongly reduced Chi activity (e.g., deletion of just V, to 1.5). These results show that D3 and its surround are critical for Chi activity and suggest a more complex effect than simple deletion of a few amino acids as the basis for loss of Chi activity. Indeed, except in one case, substitution of alanine for one to four of the amino acids in SVQPSRLP also significantly reduced Chi activity (to 1.4-3.5).
We similarly explored the requirements for amino acids in B3 (DEHAWDVVVEEFD). Deletion of only 3, 5, 8, or 10 amino acids, collectively spanning all points of the 13 amino acids deleted in ΔB3, left significant but reduced Chi activity (2.9-4.4; Table S2B), suggesting that no individual part of B3 is essential. Four or six alanine substitutions distributed throughout B3 substantially reduced Chi activity (to ~ 2.0). Other substitutions in B3 also significantly reduced Chi activity (e.g., ………E..D to ………Q..S had Chi activity of 3.4, and .R…N…QK.S had Chi activity of 3.0. Substitutions of only two D or E residues with K or R left nearly full Chi activity (4.8 and 4.1), but similar K and R substitutions of four or five residues nearly eliminated Chi activity (1.8, 1.6, and 1.3). Thus, both the presence and the amino-acid composition of B3 are critical for Chi activity.
Double D3 B3 mutants were similar to the stronger single mutant, except in one case. An additive effect of ..E.DE.. in D3 and .R…N…QK.S in B3 was observed in the double mutant: Chi activities were 3.0, 3.2, and 1.8, respectively (Table S2C). Thus, the role of DB is more complex than that of CD, but nevertheless this couple also plays an essential role in Chi hotspot activity.

RecB-RecC interaction.
In the published crystal structures of RecBCD 14,32 , a 10-amino-acid α-helix in RecB (named B4) is close to a 25-amino-acid α-helix in RecC; we define C4 as the seven amino acids at the N-terminus of this RecC α-helix plus the adjacent three amino acids, which form a turn (Figs. 2E and S2). B4 is GHGIAQDLMP at positions 913-922 in RecB's nuclease domain, and C4 is FLPDAETEAA at positions 599-608 in RecC's helicase-like domain 2B (Table 1). The B4 α-helix has been postulated to control RecBCD nuclease activity 14 www.nature.com/scientificreports/ Chi-independent nuclease activity (Tables 1 and 2). Deletion of C4, however, eliminated Chi activity, as did the double deletion (B4Δ C4Δ) ( Table 1). Deletion of seven amino acids in C4 also strongly reduced Chi activity (to 2.3) ( Table 1). Alanine substitutions of the ten amino acids in B4 or three of the ten amino acids in C4 slightly reduced Chi activity (to 3.7 and 4.4, respectively; Table S3). These results show that at least part of C4 is essential for Chi activity but that B4 plays at most a minor role on its own. We noted that the B4 and C4 α-helices ordered in the crystal structure are close to a seven-amino-acid loop of RecD (named D4) in the cryoEM structures (PDB 5LD2, 6SJB, 6SJE, 6SJF, 6SJG, 6T2U, and 6T2V; Figs. 2B and S3) 15,29 . D4 is HRHPHSR at positions 469-475 in RecD's helicase domain 2B (Table S3). This loop in RecD and 50 surrounding amino acids are disordered in both molecules in the asymmetric unit of both crystal structures (PDB 1W36 and 3K70; Fig. 2A) 14,32 , and B4 is disordered in the cryoEM structures. This outcome indicates that B4 and the loop in RecD can adopt alternative structures. Deletion of this loop or changing each amino acid to alanine had little if any effect on Chi activity (3.9 or 4.4) or recombination potential (74 or 83% of wild type) ( Table S3). Control of RecBCD thus appears to be largely independent of this loop in RecD.

Recombination proficiency is positively correlated with Chi hotspot activity. The critical role of
Chi in regulating RecBCD's activities predicts that lowering the Chi hotspot activity would also lower recombination proficiency. We tested this hypothesis by plotting the two values for each of the mutants discussed above [RecC tunnel and contact points CD, DB (with recB344), and BC] (Fig. 3). The correlation was significantly positive (R 2 = 0.60, 0.58, 0.46, and 0.65 for these sets, respectively; p = < 0.0001, 0.047, < 0.0001, and < 0.0002, respectively). We noted that the intercept of Hfr recombination proficiency at no Chi hotspot activity (0.27; mean of the four values in Fig. 3) was comparable to the ratio (0.20) of lambda recombination without and with Chi in the Chi hotspot crosses. In other words, removing a Chi site reduces lambda recombination to about the same extent that inactivating any of the four contact points reduces Hfr recombination. Residual recombination reflects RecBCD's innate ability to promote recombination without Chi or the signal it elicits.
New mutants have DNA unwinding activity, general nuclease activity, and assembled RecBCD heterotrimers but have lost Chi cutting. To test more directly the effect of the mutations on RecBCD and its activities, we assayed DNA unwinding, cutting of DNA at Chi, general nuclease activity, and abundance of RecBCD heterotrimeric proteins. Representative mutants altered at each of the contact points were tested. In some cases, the mutants retained approximately wild-type levels of DNA unwinding activity (Figs. 4 and S1), but in other mutants there was significant reduction. As expected, Chi cutting was observed in mutants with full or slightly reduced Chi hotspot activity (> 4.3) but not in mutants with little or no Chi activity (< 1.6). Chi cutting Table 2. RecBCD contact-mutant enzymatic and genetic activities. a The indicated mutation was on a derivative of plasmid pSA607 (recBC 2773 D) in strain V2831 (ΔrecBCD2731). Alleles are described in Tables 1 and S1-S3. b Specific activity in units of ATP-dependent ds DNA nuclease/mg of protein at 25 µM ATP or 1 mM ATP. ND, not determined. c Efficiency of plating (eop) is the phage titer on the indicated strain divided by the titer on strain V2831 (ΔrecBCD2731). d Activity in extracts (Figs. 4 and S1). +, activity present. −, activity absent. ±, activity reduced. e Data from Tables 1 and S1-S3. f ND, not determined.

Contact point
Allele description a Chi-independent nuclease activity was nearly wild-type in some mutants but was low in others (Table 2). In most of the latter cases, nuclease activity was about twofold higher with 1 mM ATP than with 25 µM ATP; wildtype RecBCD activity, by contrast, was about twofold lower with 1 mM ATP, as reported previously 33 . Possible   Figure 3. E. coli Hfr recombination proficiency is positively correlated with Chi hotspot activity. Red data points are for deletion mutants, blue for substitution mutants, and green for mutants with a substitution and deletion; black point is wild type, and star is recB344. Linear regression lines and the coefficient of determination (R 2 ) are shown. Data are from Tables 1, 2, S1, S2, and S3 and refs [16][17][18] 34 . Extracts of all mutants tested contained wild-type abundance of assembled heterotrimeric RecBCD protein (Fig. S4), showing that their nuclease activity reflects low intrinsic activity and not low protein abundance or stability.

Discussion
The results reported here greatly expand the set of mutants with reduced control of RecBCD enzyme by Chi hotspots of recombination. They reveal that points of contact between each of the enzyme's three subunits are essential for Chi activity (Fig. 2C-E), and they provide important material for more direct biophysical assays of the molecular mechanism of this control. www.nature.com/scientificreports/ The 63 mutants studied here have a wide range of effects on Chi activity, from no significant effect to complete loss of Chi activity. At each of the three tested points of intersubunit contact, we found mutants that lacked Chi activity. For two of these contact points (CD and DB), we found such mutations in each of the two interacting subunits (RecC-RecD and RecD-RecB, respectively). For the other contact point (BC), mutations in RecC abolished Chi activity, but none of the RecB or nearby RecD mutants tested dramatically lowered Chi activity. Because the nuclease active site is in RecB 35 and a part of RecC essential for Chi activity (C4 in Table 1 and Fig. 2E) is close to the RecB nuclease domain, it seems likely that RecC and RecB must contact each other to effect Chi hotspot activity. Thus, our choosing points of contact to mutate, based on the crystal and cryoEM structures, successfully identified areas of RecBCD far from the Chi-recognition site (in RecC) and nuclease site (in RecB) critical for Chi's control of RecBCD.
It is noteworthy that two of the points of contact are positioned differently in the crystal and cryoEM structures ( Fig. 2A,B) 14,15 . The RecD-RecB contact points (D3 B3) intimately interact in the cryoEM structures (Figs. 2B,D, and S2) but are > 15 Ǻ apart in the crystal structures ( Fig. 2A). Conversely, the RecB-RecC contact points (B4 C4) intimately interact in the crystal structures ( Figs. 2A,E, and S2) but are > 20 Ǻ apart in the cryoEM structures (Fig. 2B). This is evidence that these points can move relative to each other, in accord with conformational changes of RecBCD being important for Chi hotspot activity. In addition, one of the proposed contact points (B4) is ordered in the crystal structure but not in the cryoEM structures. This outcome also indicates flexibility around these contact points, as expected for parts of the protein that interact differently during steps of the reaction cycle.
Based on the crystal and cryoEM structures of RecBCD bound to a set of forked DNA substrates with increasingly long 5′ single-stranded DNA extensions, Wigley's group has discussed the roles of contact points DB and BC (Figs. 2D,E, and S2). When the 5′ extension of the DNA is elongated from four to ten nucleotides, parts of RecD become more ordered 14,32 . An additional two nucleotides on the 5′ extension result in larger changes 29 . The SH3-like domain of RecD becomes well-ordered, and contact point D3, in the SH3 domain, touches contact point B3. There is also a major change in the B4 α-helix. The C4 α-helix moves a few Ǻ and apparently allows the B4 α-helix to become disordered and to move away from the RecB nuclease active site. They postulated that this movement of ~ 15 Ǻ of what they termed the RecB "linker," which contains B4, was necessary to convert RecBCD from an inactive nuclease (when bound to a ds DNA end) into an active nuclease (during unwinding). Deletion of B4, however, had little effect on RecBCD nuclease or Chi hotspot activity (Tables 1 and 2; Fig. 2E).
Comparison of cryoEM structures of RecBCD bound to a 3′ extension without Chi or with Chi appropriately placed for its recognition by the RecC tunnel revealed additional small changes in the position and orientation of the RecB nuclease domain 15 . These changes were postulated to inactivate the nuclease after making its final cleavage, on the 3′-ended strand upon encountering Chi, a property of purified RecBCD under reaction conditions with high [Mg 2+ ] but not, the evidence indicates, in living cells 1,36,37 . With high [Mg 2+ ], purified RecBCD begins to cut the 5′-ended strand at Chi 38,39 , but the basis for this switch in strand cutting was not addressed by Cheng et al. 15 . The predictable consequences of these structural changes were not tested, for example by the type of mutational analysis used here. Our data here demonstrate the importance of the points of contact in the structures reported by Wigley's group.
A quarter of the mutations altering the contact points (16 of 63 analyzed here) retained nearly full Chi hotspot activity (4.1-5.0), recombination proficiency, and the enzymatic activities tested (Tables 1, 2, and S1-S3; Figs. 4 and S1). Thus, some alterations as great as deletion of ten amino acids [B3Δ (10) in RecB at the DB contact point (Tables 2 and S2)] had little effect. This outcome shows that not all structural alterations, even large ones, block Chi's control of RecBCD and its other activities. It also supports the view that the contact points in mutants that do have an effect have a direct role in Chi activity as proposed in the models discussed below (Fig. 5). Additional points in RecBCD, such as RecB A68 in the helicase domain and changed to V68 in the recB344 mutant (Table 1 and 2), are also likely critical for Chi activity, but more complex analyses, such as molecular dynamics, may be required to reliably predict additional points to mutate.
Here and elsewhere, we have identified five points in RecBCD enzyme at which mutations reduce or abolish Chi activity. These include mutations in the RecC tunnel (Chi recognition) [16][17][18] and in each of the three contact points studied here (CD, DB, and BC) (Tables 1 and S1-S3; Figs. 1 and 2). Mutations in the RecB tether connecting the RecB helicase and nuclease domains show that the tether has to be long enough and sufficiently flexible to allow the nuclease domain to swing and act at Chi (Figs. 5B and S5) 40 . Each of these pairs of contact points spans a distance as great as 80 Ǻ; the maximal distance between points in RecBCD is ~ 140 Ǻ. The sum of distances from Chi recognition in the RecC tunnel to point CD, then to point DB, then to point BC, and finally to the nuclease active site is ~ 185 Ǻ. The signal from Chi to the nuclease domain thus traverses much of the expanse of the enzyme even though in the reported crystal and cryoEM structures the Chi recognition site is only ~ 25 Ǻ from the nuclease active site. This result indicates that a high degree of coordination among the three subunits is required to control the numerous activities of RecBCD. Additional points in RecBCD are also likely required for Chi's control of RecBCD.
We have previously proposed a signal transduction model for Chi's control of RecBCD's multiple activities (Fig. 5A) 41 and a more detailed model (nuclease-swing) for one step (Fig. 5B) 19 . In these models, Chi is recognized in the RecC tunnel (step 1); RecC then signals RecD helicase to stop (step 2). When RecD is stopped, it signals the RecB nuclease domain to swing from its inactive position on the "left" side of RecC to the RecC tunnel exit (step 3). The nuclease then cuts the DNA a few nucleotides 3′ of the Chi octamer and begins loading RecA strandexchange protein onto the newly generated 3′ end (step 4). The mutants described here are consistent with step 2 (CD mutants), step 3 (DB mutants), and step 4 (BC mutants) being disrupted. Alteration of each contact point reduces or eliminates the end result of this "pathway" (or pathways)-Chi hotspot-stimulated recombination. The properties of dozens of mutants in the RecC tunnel and the RecB tether also support these models. www.nature.com/scientificreports/ The mutants described here provide excellent material for biophysical tests of these models. Possible methods include Förster resonance energy transfer (FRET). RecBCD enzyme with fluorescent moieties at two points, for example on the nuclease domain and at the RecC tunnel exit, could show that the nuclease domain swings as inferred from previous studies (Fig. 5B) 19 . Such structural changes might also be visible by cryoEM of RecBCD stopped during active unwinding before and after Chi. Single-molecule unwinding experiments, such as those using DNA curtains 42 , could also show pausing at Chi or not. These methods require advanced equipment not currently available for our studies.
Some mutants studied here unexpectedly had lower "general" (ATP-dependent but Chi-independent) nuclease activity than the wild type (Table 2), although they contained wild-type levels of assembled RecBCD heterotrimer (Fig. S4). These mutants' nuclease activities were higher with 1 mM ATP than with 0.025 mM ATP, the concentration in the standard nuclease assay 33 , whereas the wild-type nuclease activity is lower with 1 mM ATP, which is more nearly physiological (Bochner and Ames, 1982;Bennet et al., 1998;Smith, 2012; these are 1,43,44 . In the nuclease-swing model (Fig. 5B), the nuclease domain can be positioned at the exit of the RecC tunnel, where it can cut DNA, or on the protease-protected patch of RecC (Figs. 1B and 5B), where it cannot cut DNA 19 . We propose that, during unwinding, the nuclease domain remains in these mutants more often than in wild type at the protease-sensitive patch on RecC and thus far from the RecC tunnel exit and unable to cut DNA frequently (Fig. 5B). In this view, the activity of the nuclease reflects the fraction of time the nuclease domain resides at the tunnel exit. This fraction may be altered by the mutations studied here and by the ATP concentration. Other explanations have not been ruled out.
The approach we have used here may be useful to study other complex proteins that undergo large conformational changes during their reaction cycle. Of special interest are mutants altered far from an enzyme's active site(s) that strongly affect its activity. For example, alteration of two amino acids in Thermus thermophilus RNA polymerase β' subunit located ~ 20 Ǻ from the polymerization site reduces transcription rate > 1000-fold 45 . Conversely, a single amino acid change in Saccharomyces cerevisiae RNA polymerase I subunit Rpa135, located ~ 50 Ǻ from the site of RNA synthesis, enhances transcription rate 2-to 3-fold 46 . ATP hydrolysis by myosin is required