A Model of EcoRII Restriction Endonuclease Action: The Active Complex is Most Likely Formed by One Protein Subunit and One DNA Recognition Site

Karpova, Elizaveta A.; Кубарева, Е. А.; Shabarova, Zoe A.

doi:10.1080/713803460

Cited by 6 publications

(4 citation statements)

References 8 publications

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“…These observations support our previous assumption based on imaging of a bi-lobed shape of the protein in some looped complexes (8) and are in line with the results of gel shift experiments (40, 41). These observations prompted us to propose the following model of the EcoRII-DNA complex formation and dynamics (Fig.…”

Section: Discussionsupporting

confidence: 92%

Single-Molecule Dynamics of the DNA−EcoRII Protein Complexes Revealed with High-Speed Atomic Force Microscopy

et al. 2009

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The study of protein interactions with DNA is important to gain a fundamental understanding of how numerous biological processes occur, including recombination, transcription, repair, etc. In this study, we use the EcoRII restriction enzyme, which employs a three-site binding mechanism in order to catalyze cleavage of a single recognition site. Using high-speed atomic force microscopy (HS-AFM) to image single-molecule interactions in real time, we were able to observe binding, translocation, and dissociation mechanisms of the EcoRII protein. The results show that the protein can translocate along DNA to search for the specific binding site. Also, once specifically bound at a single site, the protein is capable of translocating along the DNA to locate the second specific binding site. Furthermore, two alternative modes of dissociation of the EcoRII protein from the loop structure were observed, which result in the protein stably bound as monomers to two sites or bound to a single site as a dimer. From these observations, we propose a model in which this pathway is involved in the formation and dynamics of a catalytically active three-site complex.The formation of synaptic protein-DNA complexes is central to many biological processes which require communication between two or more DNA regions, including recombination (1,2), replication (3), transcriptional regulation (4), repair (5), transposition (6), and restriction (7,8). Restriction enzymes serve as useful models to study mechanisms by which the intracellular protein machinery functions on DNA, including synapsis. Restriction enzymes (REases), which require binding to two or more cognate recognition sites in order to be catalytically active are widely spread (9). A multi-site mechanism suggests that restriction enzymes serve as evolutionary precursors to many DNA regulatory factors in the cell (10,11). Such a mechanism could also serve an inhibitory function to prevent rare unmethylated recognition sites in the host genome from undergoing restriction (12). In addition to systems involving interactions of two DNA helices, interactions of three or more DNA molecules may occur (13-17).EcoRII is a dimer which recognizes the sequence 5′-CCWGG-3′. It is generally known as a type IIE restriction enzyme. In general, the definition of typeIIE REases is that they bind two DNA recognition sites in order to cleave one of the sites (18). However, recent evidence suggests that the EcoRII protein actually requires three sites to concertedly cleave both strands For this study, we used high speed atomic force microscopy (HS-AFM) to directly image single molecule dynamics of the protein-DNA complexes formed by EcoRII restriction enzyme. It has been used previously to visualize looping and translocation mechanisms of the type III restriction enzyme EcoP15I (31). This HS-AFM relies on a small cantilever design based on the original design by T. Ando (32). This technique has the ability to observe molecular dynamics on a timescale that is 100 times faster than conventional...

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Section: Discussionsupporting

confidence: 92%

Single-Molecule Dynamics of the DNA−EcoRII Protein Complexes Revealed with High-Speed Atomic Force Microscopy

et al. 2009

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“…In agreement with results that were found for other nucleic acid base substitutions (33,34), EcoRII recognized in particular those substrates that were substituted at the 5Ј-C pair and at the central A/T of both strands. Some of the X and Y substitutions that we introduced in the recognition sequence for photocross-linking inhibited or even prevented DNA binding by EcoRII.…”

Section: Discussionsupporting

confidence: 78%

Asymmetric Photocross-linking Pattern of Restriction EndonucleaseEcoRII to the DNA Recognition Sequence

Mücke¹,

Pingoud²,

Grelle³

et al. 2002

Journal of Biological Chemistry

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The EcoRII homodimer engages two of its recognition sequences (5-CCWGG) simultaneously and is therefore a type IIE restriction endonuclease. To identify the amino acids of EcoRII that interact specifically with the recognition sequence, we photocross-linked EcoRII with oligonucleotide substrates that contained only one recognition sequence for EcoRII. In this recognition sequence, we substituted either 5-iododeoxycytidine for each C or 5-iododeoxyuridine for A, G, or T. These iodopyrimidine bases were excited using a UV laser to result in covalent cross-linking products. The yield of EcoRII photocross-linked to the 5-C of the 5-CCAGG strand of the recognition sequence was 45%. However, we could not photocross-link EcoRII to the 5-C of the 5-CCTGG strand. Thus, the contact of EcoRII to the bases of the recognition sequence appears to be asymmetric, unlike that expected for most type II restriction endonucleases. Restriction endonucleases are excellent model systems for the study of specific protein-DNA interactions because of their extraordinary specificity in DNA recognition. Type II restriction endonucleases form a homodimer to recognize their palindromic, double-stranded DNA recognition sequence. Both subunits display a characteristic 2-fold rotational symmetry when they contact their recognition sequence. This has been shown by crystal structure analyzes of several type II restriction endonucleases in complex with their DNA substrates (1, 2). The homodimeric EcoRII is a type IIE restriction endonuclease that differs from type II restriction endonucleases in that it depends on cooperative binding of two of its recognition sequences (5Ј-CCWGG) for DNA cleavage activity (3-7). Type IIE restriction endonucleases, like recombinases, DNA repair enzymes, and transcriptional regulators, interact simultaneously with two recognition sequences (8). Although EcoRII shows sequence homology to the Int family of recombinases (9, 10), it is not exactly clear if EcoRII and integrases also recognize DNA similarly. Another type IIE restriction endonuclease, NaeI, has been shown to have topoisomerase activity when Leu 43 is replaced by Lys (11). The crystal structures of NaeI and of the NaeI⅐DNA complex revealed a NaeI dimer that had two domains per monomer, the endo and the topo domain (12, 13). The endo domain forms a DNA binding motif that is structurally related to other type II restriction endonucleases and harbors the catalytic center. The topo domain contains a catabolite activator protein (CAP) motif for DNA binding (12). Both topo and both endo domains bind one double-stranded DNA recognition sequence (12, 13). Thus, two different structural motifs of NaeI recognize the same DNA recognition sequence and are assigned to an activator and a substrate binding site. Because an activator and a substrate binding site have also been discussed for EcoRII, it is possible that EcoRII possesses a structure similar to that of NaeI (3, 8, 14 -17).It has been shown by membrane-bound peptide libraries that one EcoRII monomer contains a...

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“…These data strengthen our hypothesis of a catalytic inactive enzyme monomer-DNA complex and demonstrate that amino acids essential for dimerization and catalysis are tightly interwoven. However, it contradicts a recently published model that proposed that the EcoRII monomer is catalytically active when complexed with one target DNA sequence (33).…”

Section: Discussioncontrasting

confidence: 47%

Imaging DNA Loops Induced by Restriction Endonuclease EcoRII

Mücke¹,

Mackeldanz²,

Behlke³

et al. 2000

Journal of Biological Chemistry

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EcoRII is a type IIE restriction endonuclease characterized by a highly cooperative reaction mechanism that depends on simultaneous binding of the dimeric enzyme molecule to two copies of its DNA recognition site. Transmission electron microscopy provided direct evidence that EcoRII mediates loop formation of linear DNA containing two EcoRII recognition sites. Specific DNA binding of EcoRII revealed a symmetrical DNase I footprint occupying 16 -18 bases. Single amino acid replacement of Val 258 by Asn yielded a mutant enzyme that was unaffected in substrate affinity and DNase I footprinting properties, but exhibited a profound decrease in cooperative DNA binding and cleavage activity. Because the electrophoretic mobility of the mutant enzyme-DNA complexes was significantly higher than that of the wild-type, we investigated if mutant V258N binds as a monomer to the substrate DNA. Analysis of the molecular mass of mutant V258N showed a high percentage of protein monomers in solution. The dissociation constant of mutant V258N confirmed a 350-fold decrease of the enzyme dimerization capability. We conclude that Val 258 is located in a region of EcoRII involved in homodimerization. This is the first report of a specific amino acid replacement in a restriction endonuclease leading to the loss of dimerization and DNA cleavage while retaining specific DNA binding.Studies on the molecular reaction mechanism of restriction endonucleases (ENases) 1 in the last decade revealed the existence of a subset of unconventional type II ENases. The enzymatic activity of these ENases depends on simultaneous binding of two copies of the recognition site (reviewed in Ref.

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A Model of EcoRII Restriction Endonuclease Action: The Active Complex is Most Likely Formed by One Protein Subunit and One DNA Recognition Site

Cited by 6 publications

References 8 publications

Single-Molecule Dynamics of the DNA−EcoRII Protein Complexes Revealed with High-Speed Atomic Force Microscopy

Single-Molecule Dynamics of the DNA−EcoRII Protein Complexes Revealed with High-Speed Atomic Force Microscopy

Asymmetric Photocross-linking Pattern of Restriction EndonucleaseEcoRII to the DNA Recognition Sequence

Imaging DNA Loops Induced by Restriction Endonuclease EcoRII

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