Sequence-specific DNA Recognition by the SmaI Endonuclease

Withers, Barbara E.; Dunbar, Joan C.

doi:10.1074/jbc.270.12.6496

Cited by 16 publications

(19 citation statements)

References 43 publications

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“…DNA binding assays were carried out by using duplex, synthetic oligonucleotide substrates. The complementary oligonucleotides were annealed, 5Ј-end-labeled with T4 polynucleotide kinase (New England Biolabs), and purified as previously described (68). Experiments used crude extracts containing equivalent concentrations of C ⅐ PvuII FLAG fusion proteins, as determined by Western blot analyses.…”

Section: Methodsmentioning

confidence: 99%

Role and Mechanism of Action of C · Pvu II, a Regulatory Protein Conserved among Restriction-Modification Systems

et al. 2000

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The PvuII restriction-modification system is a type II system, which means that its restriction endonuclease and modification methyltransferase are independently active proteins. The PvuII system is carried on a plasmid, and its movement into a new host cell is expected to be followed initially by expression of the methyltransferase gene alone so that the new host's DNA is protected before endonuclease activity appears. Previous studies have identified a regulatory gene (pvuIIC) between the divergently oriented genes for the restriction endonuclease (pvuIIR) and modification methyltransferase (pvuIIM), with pvuIIC in the same orientation as and partially overlapping pvuIIR. The product of pvuIIC, C ⅐ PvuII, was found to act in trans and to be required for expression of pvuIIR. In this study we demonstrate that premature expression of pvuIIC prevents establishment of the PvuII genes, consistent with the model that requiring C ⅐ PvuII for pvuIIR expression provides a timing delay essential for protection of the new host's DNA. We find that the opposing pvuIIC and pvuIIM transcripts overlap by over 60 nucleotides at their 5 ends, raising the possibility that their hybridization might play a regulatory role. We furthermore characterize the action of C ⅐ PvuII, demonstrating that it is a sequence-specific DNA-binding protein that binds to the pvuIIC promoter and stimulates transcription of both pvuIIC and pvuIIR into a polycistronic mRNA. The apparent location of C ⅐ PvuII binding, overlapping the ؊10 promoter hexamer and the pvuIICR transcriptional starting points, is highly unusual for transcriptional activators.The bacterial type II restriction-modification systems include a DNA modification methyltransferase (MTase) and a restriction endonuclease (REase), both of which act independently on the same DNA sequence (65). The REase cleaves duplex DNA sequences in the absence of sequence-specific DNA modification by the MTase. These systems can defend bacterial cells against viral infection, although other functional roles have also been proposed (45). Restriction-modification systems have provided an important focus for studies of molecular recognition. Biochemical and crystallographic analyses are yielding significant insights into the mechanisms of sequence recognition and catalytic activity of these proteins (3,18,50,66).

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Section: Methodsmentioning

confidence: 99%

Role and Mechanism of Action of C · Pvu II, a Regulatory Protein Conserved among Restriction-Modification Systems

et al. 2000

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“…Note that only a subset of restriction enzymes is able to bind to the correct sequence in the absence of divalent metal ions. Some of these are SmaI and EcoRI, for which the specific and nonspecific binding constants have been determined (12,15). Kinetic tests of -DNA restriction by SmaI using a moving Mg 2ϩ front inside a microchannel has confirmed that the cutting occurs on a time scale shorter than 4 s with our combination of buffer and enzyme concentrations.…”

Section: Resultsmentioning

confidence: 99%

“…The restriction enzymes (New England Biolabs) were used with concentrations of Ϸ3 nM for SmaI, Ϸ3 nM for SacI, and Ϸ13 nM for PacI. We chose these concentrations because of the specific binding constant for SmaI of Ϸ10 9 M Ϫ1 (12). The solution that was filled into the ''entry''-Freely available online through the PNAS open access option.…”

Section: Methodsmentioning

confidence: 99%

Restriction mapping in nanofluidic devices

Riehn

Wang

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

191

183

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We have performed restriction mapping of DNA molecules using restriction endonucleases in nanochannels with diameters of 100 -200 nm. The location of the restriction reaction within the device is controlled by electrophoresis and diffusion of Mg 2؉ and EDTA. We have successfully used the restriction enzymes SmaI, SacI, and PacI, and have been able to measure the positions of restriction sites with a precision of Ϸ1.5 kbp in 1 min using single DNA molecules.R estriction mapping with endonucleases is a central method in molecular biology (1, 2). It is based on the measurement of fragment lengths after digestion, while possibly maintaining the respective order. We present here an approach to restriction mapping that is based on stretching DNA in nanofluidic channels, in which DNA is linearized to a length-independent fraction of its contour length (3, 4).To date, the most powerful method for constructing restriction maps of long DNA molecules (100 kbp and above) is the one developed by Schwartz and coworkers (5, 6). Their technique consists of stretching the DNA on a surface to establish a one-to-one mapping between spatial and genomic position, initiating the restriction by exposing the DNA to restriction enzymes and Mg 2ϩ , and optically observing the lengths of the resulting fragments. An elegant study of the same basic approach showing separation of specific binding and induced cutting was published by Taylor et al. (7).A fundamental principle in determining the error of a measurement, and a common strategy to reduce the experimental error, is to take multiple, statistically independent measurements of the same quantity. Note that the fixing of the molecule to the surface in Schwartz's approach prevents any fluctuations, and hence multiple molecules have to be observed to obtain statistically independent measurements of the same cut position. Moreover, genomic-length DNA molecules stretched on surfaces often exhibit inhomogeneous stretching, including breaks, and thus averaging over multiple molecules becomes imperative (6,8). In contrast, a molecule stretched inside a nanochannel is not subject to any forces other than those causing lateral confinement and thus is able to thermally relax and fluctuate around an equilibrium conformation. The evaluation of a single molecule is thus sufficient if complete digestion is achieved. A detailed description of the statistics and dynamics of DNA molecules in nanochannels is published in ref. 9.The main challenge in employing the concept of stretching and mapping DNA inside a closed fluidic system is to separate the steps of stretching and cutting. We have solved this problem by introducing the DNA electrophoretically into nanochannels and controlling the concentration of the enzyme cofactor Mg 2ϩ in the device shown in Fig. 1. It consists of a microfluidic ''loading'' channel containing the DNA to be analyzed, the restriction enzyme, and EDTA, and a microfluidic ''exit'' channel containing Mg 2ϩ and the restriction enzyme. The two microfluidic channels are linked by 10 n...

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“…What are the features of SmaI and PvuII interaction with DNA that make them respond differently than other restriction endonucleases tested for inhibition by THPs? Withers and Dunbar (50) noticed that PvuII displays certain similarities with SmaI, such as (i) interaction with each of the base pairs within the recognition site, (ii) a potential protein contact to the phosphate 3Ј to the scissile bond, and (iii) no significant bending of the DNA. In addition, SmaI interactions with DNA phosphates are all adjacent and clustered within the recognition site (50), whereas EcoRV endonuclease exhibit phosphate interactions both within and flanking the recognition sequence (51).…”

Section: Discussionmentioning

confidence: 99%

“…Withers and Dunbar (50) noticed that PvuII displays certain similarities with SmaI, such as (i) interaction with each of the base pairs within the recognition site, (ii) a potential protein contact to the phosphate 3Ј to the scissile bond, and (iii) no significant bending of the DNA. In addition, SmaI interactions with DNA phosphates are all adjacent and clustered within the recognition site (50), whereas EcoRV endonuclease exhibit phosphate interactions both within and flanking the recognition sequence (51). In EcoRI, DNA phosphate interactions are delocalized, so the primary clamp occurs at the immediate 5Ј-end of the recognition sequence, and supplementary clamps occur adjacent to the primary and at the center of the recognition sequence (52).…”

Section: Discussionmentioning

confidence: 99%

Effect of Tetrahydropyrimidine Derivatives on Protein-Nucleic Acids Interaction

Malin¹,

Iakobashvili

Lapidot

1999

Journal of Biological Chemistry

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2-Methyl-4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyrimidine (THP(A) or hydroxyectoine) and 2-methyl,4-carboxy-3,4,5,6-tetrahydropyrimidine (THP(B) or ectoine) are now recognized as ubiquitous bacterial osmoprotectants. To evaluate the impact of tetrahydropyrimidine derivatives (THPs) on protein-DNA interaction and on restriction-modification systems, we have examined their effect on the cleavage of plasmid DNA by 10 type II restriction endonucleases. THP(A) completely arrested the cleavage of plasmid and bacteriophage DNA by EcoRI endonuclease at 0.4 mM and the oligonucleotide (d(CGCGAATTCGCG)) 2 at about 4.0 mM. THP(B) was 10-fold less effective than THP(A), whereas for betaine and proline, a notable inhibition was observed only at 100 mM. Similar effects of THP(A) were observed for all tested restriction endonucleases, except for SmaI and PvuII, which were inhibited only partially at 50 mM THP(A). No effect of THP(A) on the activity of DNase I, RNase A, and Taq DNA polymerase was noticed. Gelshift assays showed that THP(A) inhibited the EcoRI-(d-(CGCGAATTCGCG)) 2 complex formation, whereas facilitated diffusion of EcoRI along the DNA was not affected. Methylation of the carboxy group significantly decreased the activity of THPs, suggesting that their zwitterionic character is essential for the inhibition effect. Possible mechanisms of inhibition, the role of THPs in the modulation of the protein-DNA interaction, and the in vivo relevance of the observed phenomena are discussed.Two tetrahydropyrimidine derivatives identified in Streptomyces bacteria (1-3), one a previously unknown metabolite, THP(A), 1 and the other previously identified (as ectoine) in halophilic bacteria (4), THP(B), are now recognized as widely spread osmoprotectants within the bacterial world (5). The role and activities of THPs are of special interest as they represent a limited group of osmoprotectants that are synthesized de novo, in the bacterial cell, in contrast to those transported from the medium (6). Their synthesis in a number of Streptomyces strains as a response to increased salinity and elevated temperature was recently described (7). THPs are small molecules, highly soluble in water and neutral at physiological pH. NMR and x-ray crystallography data show that THPs are zwitterionic molecules with a delocalized charge in the NCN group (Fig. 1) and form the half-chair conformation (8).More information has been accumulated lately on THPs activity in living cells. It was found that exogenously provided ectoine (THP(B)) could reverse growth inhibition, caused by osmotic stress, in Escherichia coli (9), Corynebacterium glutamicum (10), and the soil bacterium Rhizobium meliloti (11). We demonstrated that exogenously provided THP(A), like THP(B), reversed inhibition of E. coli growth by osmotic stress, and moreover, both THP(A) and THP(B) could stimulate growth of E. coli at an elevated temperature (43°C) (7). Recently cloned genes for ectoine synthesis from Halomonas elongata (12) and from Marinococcus halophilus (13) were demonstrated ...

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Sequence-specific DNA Recognition by the SmaI Endonuclease

Cited by 16 publications

References 43 publications

Role and Mechanism of Action of C · Pvu II, a Regulatory Protein Conserved among Restriction-Modification Systems

Role and Mechanism of Action of C · Pvu II, a Regulatory Protein Conserved among Restriction-Modification Systems

Restriction mapping in nanofluidic devices

Effect of Tetrahydropyrimidine Derivatives on Protein-Nucleic Acids Interaction

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