The Role of Water in the EcoRI-DNA Binding

Sidorova, Nina Y.; Rau, Donald C.

doi:10.1007/978-3-642-18851-0_12

Cited by 6 publications

(6 citation statements)

References 49 publications

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“…Importantly, however, the similar and minimal impacts on k 1 for EcoRI and Dam, and distinct impacts on k 2 , support osmotic perturbation of translocation, particularly with consideration for cosolute exclusion at the protein−DNA interface. 36 In agreement with our results, spectroscopic measurements of other proteins show DMSO and glycerol, at concentrations comparable to those used here, preferentially excluded. 17,27,28,37 Minimal impacts on Dam F p reflect the similar water dynamics encountered during k 1 and k 2 .…”

supporting

confidence: 92%

“…Future work employing spectral or nuclear magnetic resonance techniques to determine the extent of binding for these proteins remains to be performed. Importantly, however, the similar and minimal impacts on k 1 for Eco RI and Dam, and distinct impacts on k 2 , support osmotic perturbation of translocation, particularly with consideration for cosolute exclusion at the protein–DNA interface . In agreement with our results, spectroscopic measurements of other proteins show DMSO and glycerol, at concentrations comparable to those used here, preferentially excluded. ,,, Minimal impacts on Dam F p reflect the similar water dynamics encountered during k 1 and k 2 . , In contrast, the enhancement of Eco RI ENase processivity by DMSO and interference by glycerol support differential impacts on k 2 , during which Eco RI ENase remains closely associated with DNA.…”

supporting

confidence: 84%

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Mechanisms of Protein Translocation on DNA Are Differentially Responsive to Water Activity

Naughton

Reich

2016

Biochemistry

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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, dimethyl sulfoxide (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 of these osmolytes on bulk water, 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.

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supporting

confidence: 92%

supporting

confidence: 84%

Mechanisms of Protein Translocation on DNA Are Differentially Responsive to Water Activity

Naughton

Reich

2016

Biochemistry

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“…The recognition process in general consists of conformational adaptations of protein and DNA with water and counter-ion release at the protein-DNA interface [119]. This results in a favourable DH contribution from direct protein-DNA recognition interactions and a favourable DS contribution from water and counter-ion release, likely to compensate the unfavourable DS contribution due to the immobilization of amino acid side chains at the protein-DNA interface [120].…”

Section: Recognitionmentioning

confidence: 99%

Type II restriction endonucleases: structure and mechanism

Pingoud

Fuxreiter

Pingoud

et al. 2005

CMLS, Cell. Mol. Life Sci.

441

494

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Type II restriction endonucleases are components of restriction modification systems that protect bacteria and archaea against invading foreign DNA. Most are homodimeric or tetrameric enzymes that cleave DNA at defined sites of 4-8 bp in length and require Mg2+ ions for catalysis. They differ in the details of the recognition process and the mode of cleavage, indicators that these enzymes are more diverse than originally thought. Still, most of them have a similar structural core and seem to share a common mechanism of DNA cleavage, suggesting that they evolved from a common ancestor. Only a few restriction endonucleases discovered thus far do not belong to the PD...D/ExK family of enzymes, but rather have active sites typical of other endonuclease families. The present review deals with new developments in the field of Type II restriction endonucleases. One of the more interesting aspects is the increasing awareness of the diversity of Type II restriction enzymes. Nevertheless, structural studies summarized herein deal with the more common subtypes. A major emphasis of this review will be on target site location and the mechanism of catalysis, two problems currently being addressed in the literature.

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“…However, the slopes for the glycine betaine, Me 2 SO, and sucrose data are higher, ranging from 40 to 60. Variable slopes are common in osmolality studies, but their origin is not clear (37,38,(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54). This issue will be broached under the "Discussion.…”

Section: Role Of Water In K Cat /K M(dhf)mentioning

confidence: 99%

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

Chopra

Dooling

Horner

et al. 2008

Journal of Biological Chemistry

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R67 dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor. This enzyme is a homotetramer possessing 222 symmetry, and a single active site pore traverses the length of the protein. A promiscuous binding surface can accommodate either DHF or NADPH, thus two nonproductive complexes can form (2NADPH or 2DHF) as well as a productive complex (NADPH⅐DHF). The role of water in binding was monitored using a number of different osmolytes. From isothermal titration calorimetry (ITC) studies, binding of NADPH is accompanied by the net release of 38 water molecules. In contrast, from both steady state kinetics and ITC studies, binding of DHF is accompanied by the net uptake of water. Although different osmolytes have similar effects on NADPH binding, variable results are observed when DHF binding is probed. Sensitivity to water activity can also be probed by an in vivo selection using the antibacterial drug, trimethoprim, where the water content of the media is decreased by increasing concentrations of sorbitol. The ability of wild type and mutant clones of R67 DHFR to allow host Escherichia coli to grow in the presence of trimethoprim plus added sorbitol parallels the catalytic efficiency of the DHFR clones, indicating water content strongly correlates with the in vivo function of R67 DHFR.R67 dihydrofolate reductase, a type II DHFR, 2 catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate. The gene for this enzyme is carried by an R-plasmid, and its presence confers resistance to the antibiotic drug, trimethoprim (TMP). Trimethoprim is a competitive inhibitor of chromosomal DHFR with a picomolar K i (1). Although R67 DHFR is not a good catalyst, it is not inhibited by TMP very well, and thus it allows cell growth in the presence of this antibacterial drug (2, 3). R67 DHFR shares no homology in sequence or structure with chromosomal DHFR and has been proposed to be a good model of a primitive enzyme (4).R67 DHFR is a homotetramer with a single active site pore; the overall structure possesses 222 symmetry as seen in Fig. 1 (5). The symmetry of the active site results in overlapping binding sites for substrate, DHF, and cofactor, NADPH. This can be observed as R67 DHFR binds a total of two ligands as follows: either two NADPH molecules or two folate/DHF molecules or one NADPH plus one folate/DHF molecule (6). The first two complexes are dead-end (binary) complexes, whereas the third is the productive ternary complex. Because of the 222 symmetry, binding to either ligand is unlikely to be optimal.The active site pore of R67 DHFR is unusual in its hourglass shape as well as its large size (2938 Å 3 for apoR67 DHFR with hydrogens added, calculated by CASTp (4, 7)). Because of this large volume, DHF and NADPH cannot occupy all the space in the pore and must use water to mediate some contacts with the protein.How do substrate and cofactor bind to R67 DHFR? From NMR, crystallography, and docking studies, the pteridine ring of DHF/fo...

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The Role of Water in the EcoRI-DNA Binding

Cited by 6 publications

References 49 publications

Mechanisms of Protein Translocation on DNA Are Differentially Responsive to Water Activity

Mechanisms of Protein Translocation on DNA Are Differentially Responsive to Water Activity

Type II restriction endonucleases: structure and mechanism

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

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