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Mechanisms of protein translocation on DNA are differentially responsive to water activity Brigitte S. Naughton, and Norbert O. Reich Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00872 • Publication Date (Web): 29 Nov 2016 Downloaded from http://pubs.acs.org on November 30, 2016
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Mechanisms of protein translocation on DNA are differentially responsive to water activity Brigitte S. Naughton and Norbert O. Reich* Department of Chemistry and Biochemistry, University of California, Santa Barbara, California, 93106 Supporting Information Placeholder ABSTRACT: Water plays important but poorly understood roles in the functions of most biomolecules. We are interested in understanding how proteins use diverse search mechanisms to locate specific sites on DNA; here we present a study of the role of closely associated waters in diverse translocation mechanisms. The bacterial DNA adenine methyltransferase, Dam, moves across large segments of DNA using an intersegmental hopping mechanism, relying in part on movement through bulk water. In contrast, other proteins, such as the bacterial restriction endonuclease EcoRI, rely on a sliding mechanism, requiring the protein to stay closely associated with DNA. Here we probed how these two mechanistically distinct proteins respond to well-characterized osmolytes, DMSO and glycerol. The ability of Dam to move over large segments of DNA is not impacted by either osmolyte, consistent with its minimal reliance on a sliding mechanism. In contrast, EcoRI endonuclease translocation is significantly enhanced by DMSO and inhibited by glycerol, providing further corroboration that these proteins rely on distinct translocation mechanisms. The well-established similar effects on bulk water by these osmolytes, and their differential effects on macromolecule-associated waters, support our results and provide further evidence of the importance of water in interactions between macromolecules and their ligands.
The importance of bound water―water intimately associated with macromolecules―to the function of DNA binding proteins has been well-established for repressors and 1 DNA restriction endonucleases. These functions include sequence-specific and non-specific DNA binding, catalysis, and a limited number of studies looking at the translocation 2,3,4 of proteins along DNA. The structural and dynamic roles of water in such contexts have been investigated by applications of osmotic stress through the introduction of small, neutral osmolytes to induce re-ordering of water around 5,6 biomolecules. Thus, observations that changes in osmotic stress induce changes in protein function provide evidence that bound water contributes to the underlying processes. DNA-binding proteins are essential for maintaining gene expression and directing cellular pathways. Two distinct classes of DNA-modifying enzymes―endonucleases and methyltransferases―are key participants in genome protection and gene regulation. Here we focus on enzymes that rely
on facilitated diffusion to locate binding sites, during which ATP-independent and non-specific DNA binding is followed by translocation to a specific site. The Escherichia coli Type II restriction endonuclease (ENase) EcoRI is part of a restriction-modification system involved in protecting the host genome. Escherichia coli DNA [N(6)-Adenine] methyltransferase (Dam) is an orphan methyltransferase and a crucial regulator of transcription, DNA replication, and nucleoid 7,8,9 structure determination. Consistent with their diverse cellular roles, these proteins exhibit distinct translocation mechanisms. EcoRI ENase relies extensively on a sliding mechanism, involving close association with the DNA backbone as the protein makes single base-pair movements in 10,11,12,14,15 search of its site (TOC Graphic). In contrast, Dam takes advantage of the tendency of DNA ranging from 0.1513 1.0 kb to loop. This allows the protein to transitorily dissociate from one location and “hop” to a distal site on a proximally-looped strand, via a mechanism referred to as in14 tersegmental hopping. The ability of Dam to traverse across vast non-specific regions on a single molecule highlights a unique processivity trend, in which increasing the distance 14 between sites facilitates site location. This mechanism suggests that Dam interacts with bulk-like water to a greater extent than would a protein employing a sliding mechanism, such as EcoRI ENase. Although the precise engagement of associated and bulk water during translocation by Dam and other proteins remains unknown, we hypothesized it requires movement beyond layers of DNA-associated waters, i.e. waters that bind to freely diffusing bulk water. Here we study how DMSO and glycerol, two well-studied osmolytes, differentially alter the 0 .8
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Figure 2. The EcoRI ENase Fp trend increases in the presence of DMSO and decreases in the presence of glycerol. translocation of EcoRI ENase and Dam on various DNA substrates. Both viscogens have numerous biochemical and pharmaceutical applications, including protein storage and compound solvation, and share many physiochemical properties (Table S1). Their differences, however, distinctly im6,16,17 pact waters associated with macromolecules. Enhancements to EcoRI ENase processivity are in agreement with findings that DMSO, a chaotropic amphiphile, effectively increases the diffusivity of macromolecule-associated waters, while decreases to processivity support that glycerol, a kos6,18,19 motropic polyol, dampens diffusivity. Minimal impacts to Dam processivity by either osmolyte agree with findings that DMSO and glycerol dampen the viscosity of bulk water 20 to a similar extent. To model processivity, we apply a multiple-turnover se15 quential reaction mechanism. During k1, the enzyme binds DNA, searches for its specific sequence, and modifies one of two available sites. Catalysis at the second site involves either one of two processes: translocation from the first to second site in a processive event, k2, or a different (or previously dissociated) enzyme approaches from bulk solution to modify the second site in a non-processive event. Here, we include excess DNA relative to enzyme (57-fold to Dam; 200fold to EcoRI ENase), and collect data within 30% conversion to ensure that the majority of k2 values reflect processive events. The constants are applied to the equation for processivity (Fp), describing most simply the enhancement of the 15 second catalytic event relative to the first: Fp: k2/(k1+k2) We first determined how varying DMSO and glycerol from 0.14-0.56 osmolals affects the processivity of EcoRI ENase on a doubly labeled, two-site substrate separated by 335 bp (569 bp total) (Supplemental Materials and Methods and 14,15 Fig. S2). DMSO exceeding 0.56 osmolals diminished activity beyond reasonable analysis (Fig. S4). Our results demonstrate concentration-dependent modulation by each osmolyte; increasing glycerol increasingly disrupts processivity, while increasing DMSO enhances EcoRI ENase processivity nearly to its maximal value (Fp = 0.74 +/- 0.013) (Fig. 1). The contrasting impacts on EcoRI ENase processivity by DMSO and glycerol led us to investigate how―and to what degree―each osmolyte affects the Fp trend. To do so, we included 0.55-0.56 osmolal concentrations in subsequent assays, on substrates with varying intersite distances. EcoRI 10 ENase is processive out to 605 bp intersite distances, and greater osmotic impacts on Fp are expected when the enzyme travels greater distances between sites. Interestingly,
Figure 3. Contrasting osmolyte effects on EcoRI ENase processivity increase with intersite distance. The y-axis is the Fp (DMSO) (glycerol) difference between DMSO and glycerol (Fp -Fp ). Dam Fp remains largely unaffected by DMSO and glycerol. EcoRI ENase maintains its maximal Fp out to 335 bp in the presence of DMSO, followed by only a slight decrease at a 482 bp intersite distance―well above the Fp predicted by a sliding model in the absence of osmolyte (Fig. 2). As base pairs between sites increase, the enhancement to Fp by DMSO relative to no osmolyte increases. Glycerol, in contrast, decreases Fp relative to no osmolyte to a similar extent across each intersite substrate, while still maintaining a fit to a sliding model (Fig. 2). To verify translocation-specific modulation by the cosolvents, we next probed how the processivity trend of Dam is modulated by osmotic stress. Under conditions similar to ENase assays, involving either 0.56 osmolal DMSO or 0.55 osmolal glycerol, we determined Dam’s processivity on substrates with intersite distances ranging from 36 to 484 bp. Assays were carried out as previously described (Supple15 mental Materials and Methods and Fig. S3). As expected of an enzyme that relies on movement through bulk-like water, with minimal translocation along the DNA backbone, Dam Fp is largely unaffected by either osmolyte. Processivity decreases slightly more so in the presence of glycerol than in the presence of DMSO on the 484 bp intersite substrate, resulting in a slightly greater ΔFp (Fig. 3). Effects on individual rate constants can help elucidate the basis for differential processivity shifts. We compared rate constants for EcoRI ENase on the 335 bp intersite substrate, and for Dam on a 284 bp intersite substrate (518 bp total) (Table 1). As anticipated, the osmolytes slowed each rate constant for each enzyme, though to varying degrees. DMSO concentrations as low as 0.014 osmolals weaken initial pro21 tein-ligand binding, as observed by the decrease to k1 for both EcoRI ENase and Dam. Contrasting effects on EcoRI ENase Fp arise through a greater decrease to k1 relative to k2 by DMSO, and a greater decrease to k2 relative to k1 by glycerol. In the presence of DMSO, the initial EcoRI-DNA com-1
Table 1. Osmolyte effects on rate constants (min )
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plex formation is slowed more so than the subsequent, closely bound traverse to the next site; the inverse is true for glycerol. This results in an increase to Fp by DMSO (0.74 +/0.01) and a decrease by glycerol (0.61 +/- 0.02) relative to no osmolyte (0.66 +/- 0.04). In contrast, DMSO and glycerol similarly impact Dam. Dam is predicted to translocate through water dynamics more closely mirroring k1 conditions, and thus we observe rate decreases which yield no advantage―or significant disadvantage―to translocation, only a marginal decrease in Fp by both DMSO (0.48 +/- 0.04) and glycerol (0.46 +/- 0.03), relative to no osmolyte (0.49 +/0.02). The slightly greater decrease to k2 relative to k1 is likely rooted in the availability of only one site following modification of the first, which in turn requires a longer period of time spent site-searching in the osmolyte-impacted viscous solution. Changes in protein batches resulted in high error associated with averaged rates, yet relative k1 and k2 rate shifts remained constant between batches and experiments, resulting in consistent Fp values with low error. Prior work with similar co-solvents indicates that EcoRI ENase dissociation from non-specific DNA is disfavored in the presence of DMSO, and favored in the presence of pol6 ylols such as sucrose (and presumably, glycerol). These effects were suggested to derive from osmolyte-specific interactions with the protein, DNA or both, rather than osmolyteinduced changes in bulk water. Our results with EcoRI ENase are consistent with this prior work. For a protein largely reliant on sliding, factors which allow the protein to remain on the DNA (such as DMSO), particularly non-specific DNA, will enhance Fp. In contrast, factors which enhance dissociation from non-specific DNA (such as glycerol) will logically reduce processivity. In this context, the relative lack of response of Dam to either DMSO or glycerol (Fig. 3 and Table 1) is distinctive. While it may be that these two proteins are differentially impacted by DMSO and glycerol in their interactions with DNA, we suggest that the highly distinctive translocation mechanisms leading to very different Fp profiles contribute to these effects. The minor reliance of Dam on sliding, in combination with its use of intersegmental hopping (TOC graphic), reduces its responsiveness to solutes that alter its interaction with non-specific DNA, and hence 14,15 its processivity. Increases in the bulk water viscosity in 22,23,24, response to the two viscogens dampen water diffusivity 20 and are expected to lower the overall rates of methylation, which is observed (Table 1), but have little impact on Fp. The importance of water in the various functions of DNAbinding enzymes and regulatory proteins is well estab1,3,4,6,22,25 lished. Interstitial waters―waters closely associated with the participating macromolecules―have been observed 26,27,20 in several structural studies. Nevertheless, obtaining clear evidence for the importance of water in dynamic processes―such as initial protein-DNA encounters, site recognition, and translocation―remains more challenging. The use of osmotic stress induced by neutral, small-molecule solutes implicates the direct involvement of water in these processes; indeed, osmotic stress is perceived as being more sensitive than direct structural studies for detecting functionally 1,17 significant waters. In essence, a correlation between changes in osmotic pressure and a biomolecular process implicates a role for bound water in the process. Based on prior studies of protein-DNA interactions using osmolytes to determine the importance of water, we anticipated that since EcoRI ENase
slides extensively, retaining intimate contact over large segments of DNA, these osmolytes would more profoundly impact its translocation mechanism in comparison to Dam, which is much less reliant on sliding. DMSO and glycerol are known to differentially impact bulk and macromolecule17,28,29,30,31, 20 associated waters. A deeper understanding of water behavior―in particular the dynamics in both bulk water and water associated with macromolecules―is emerging from new investigational 18,32,33 methods. For example, Overhauser effect dynamic nuclear polarization (ODNP) measures the translational mobility of water within the vicinity (0.5-1.5 nanometers) of labeled sites. Bulk water tends to be highly mobile, with high diffu19,34 sivity constants. In contrast, water associated with protein and lipid surfaces is 3.5-5 fold less dynamic, with typical protein hydration layers exhibiting five-fold slower mobili23 ty. These properties are likely to play critical roles in biomolecular interactions. Intriguingly, water associated with DNA appears to be only two-fold less dynamic than bulk 18,22,34,35 water. As suggested, similar motilities between bulk and DNA-associated water may result in a smaller entropic drive to displace waters as proteins move along DNA, than the displacement of more tightly associated waters. Because DMSO decreases the mobility of surface waters to a lesser 16, 20 extent than it does bulk, the increase in Fp observed with EcoRI ENase may result from this effect as the water mobility more closely approaches that of bulk water. The often-pleiotropic nature of co-solvents necessitates addressing whether changes in processivity are the result of changes to protein conformation or catalysis, rather than to translocation. Though effects observed on a single protein and on a single substrate suggest potential catalytic or structural changes, we argue that the differential Fp effects on two proteins―both well studied, site-specific and DNA modifying―with notably distinct diffusion mechanisms supports translocation-dependent modulation. The minimal and similar impacts by DMSO and glycerol on Dam reflect similar impacts to bulk water dynamics encountered by Dam during 15,20 both k1 and k2. In contrast, EcoRI ENase processivity enhancement by DMSO and interference by glycerol supports differential impacts to k2, during which EcoRI ENase remains closely associated with DNA. Furthermore, intersitedependence, in which increasing the distance between sites increases EcoRI ENase Fp enhancement by DMSO relative no osmolyte, points toward modulation of translocation via osmotic water perturbation. Our data is further validated by spectroscopic measurements, which show that at concentrations similar to those used here, DMSO and glycerol are excluded from the protein hydration layer, and exert indirect, minimally stabilizing 28,29,39 effects on protein conformation. Osmotic effects on catalysis would result in similar decreases to k1 and k2; though Dam constants decrease, the increasing osmotic impact on Fp relative to intersite distance for EcoRI ENase indicates translocation dependence (Fig. 2 and 3). Reports of DMSO decreasing enzyme activity are likely attributable to 36 decreasing the Km, rather than increasing the kcat, consistent with our results that osmolyte modulation is affecting association-dissociation kinetics. Many studies employ osmolytes to alter the activity of proteins which bind nucleic37 acids. For example, the rates of T7 RNA polymerase and 38 pancreatic deoxyribonuclease are enhanced by DMSO, 34 whereas other proteins are not. Introducing small solutes
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to induce changes to enzymatic activity―particularly enhancements―often provide intriguing but poorly understood results. Here, we attempt to engage biochemical modulations of enzyme kinetics with biophysical studies of water to rationalize a basis for changes in protein functional dynamics. In summary, recent work on the dynamics of bulk water and water associated at biomolecular surfaces reveals that the diffusivity of water is preferentially increased at surfaces 16 by DMSO. Thus, we propose that the greater reliance on scanning of DNA shown by EcoRI ENase results in DMSO enhancing its Fp by attenuating its release from non-specific DNA. In contrast, the ability of polyols like glycerol to favor dissociation of a protein that largely slides enroute to locating its binding site will decrease target-site location kinetics. Of particular interest is that Dam is largely non-responsive to either osmolyte, which we suggest is consistent with its translocation mechanism. Thus, Dam appears to rely to a very limited extent on sliding; rather, its efficient site location involves large jumps mediated by distal regions of DNA looping into proximity. Importantly, such movements most likely involve exposure to bulk water. We suggest that this combination – minimal sliding and greater exposure to bulk water – explains why neither osmolyte has significant impact on Dam’s ability to move between sites on DNA.
ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:. Materials and Methods with relevant assay conditions and procedures, DNA substrates, product and modeling determinations, DNA, Table S1, and Figures S1− S4 (PDF)
AUTHOR INFORMATION Corresponding Author
[email protected] Funding This work was supported by the National Science Foundation (Grant 1413722) and REU awards 1538990 and 1636474.
Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT We thank Dr. Songi Han for comments on this work.
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