Asn<sup>141</sup> is essential for DNA recognition by <i>Eco</i>RI restriction endonuclease

Fritz, Andreas; Küster, Wolfgang; Alves, Jürgen

doi:10.1016/s0014-5793(98)01274-5

Cited by 11 publications

(5 citation statements)

References 30 publications

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“…In R.EcoRI, Arg 145 and Arg 200 are engaged in direct sequencespecific interactions with EcoRI recognition sequence (23). Conservative substitutions of these arginines with lysine resulted in decreased affinity for DNA binding, whereas specificity of DNA recognition was saved (19). Similar observations were made for Asn 141 , which forms three hydrogen bonds with two adenines of R.EcoRI sequence (51).…”

Section: Discussionmentioning

confidence: 53%

Functional Analysis of MmeI from Methanol UtilizerMethylophilus methylotrophus, a Subtype IIC Restriction-Modification Enzyme Related to Type I Enzymes

Nakonieczna

Kaczorowski

Obarska-Kosińska

et al. 2009

Appl Environ Microbiol

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MmeI from6 -adenine ␥-class DNA methyltransferases; (iii) the C-terminal portion (aa 610 to 919) containing a putative target recognition domain. Interestingly, all three domains showed highest similarity to the corresponding elements of type I enzymes rather than to classical type II enzymes. We have found that MmeI variants deficient in restriction activity (D70A, E80A, and K82A) can bind and methylate specific nucleotide sequence. This suggests that domains of MmeI responsible for DNA restriction and modification can act independently. Moreover, we have shown that a single amino acid residue substitution within the putative target recognition domain (S807A) resulted in a MmeI variant with a higher endonucleolytic activity than the wild-type enzyme.Restriction-modification (RM) systems are composed of two enzymatic entities: a restriction endodeoxyribonuclease (REase) that cleaves DNA usually within short, specific sequences and a methyltransferase (MTase) that modifies the same sequence in order to protect the host genomic DNA against the action of the cognate restriction enzyme. Based on their molecular structure, functional features, and cofactors requirements, RM systems can be divided into four distinct types (49). The most complex are enzymes of types I and III (and some of type IV), which function as multisubunit molecular machines that exhibit very complex mechanism of action involving NTP hydrolysis and DNA translocation (reviewed in reference 4). On the other hand, type II RM systems comprise relatively simple independent REase and MTase enzymes whose properties, in particular recognition of short specific nucleotide sequences (4 to 8 bp), have made them indispensable tools of modern molecular biology (45). To date, almost 4,000 type II REases have been identified by screening various Bacteria, Archaea, and viruses and characterizing them biochemically (50).DNA MTases of RM systems belong to several different families within the Rossmann fold methyltransferase superfamily (6). Structural conservation is strong across catalytic domains of all DNA MTases, despite sequence divergence between families and frequent fusions with additional, unrelated domains that are involved in, for example, specific recognition of the target DNA sequence (29). Two major families are the m 5 C MTases, a group of proteins with high mutual sequence similarity and only remote similarity to other MTases (48), and the N-MTases (m 6 A and m 4 C MTases), a large and heterogeneous group of enzymes that exhibit very complex mutual relationships (9,34). The N-MTase family contains not only type II enzymes, but also MTase subunits

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Section: Discussionmentioning

confidence: 53%

Functional Analysis of MmeI from Methanol UtilizerMethylophilus methylotrophus, a Subtype IIC Restriction-Modification Enzyme Related to Type I Enzymes

Nakonieczna

Kaczorowski

Obarska-Kosińska

et al. 2009

Appl Environ Microbiol

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“…Attempts to change the specificity of any restriction endonuclease have been uniformly unsuccessful. Exhaustive site-directed mutagenesis of the amino acid residues directly involved in EcoRI-DNA recognition did not yield variants with altered specificity, but rather mutants that have lost (or diminished) ability to cleave the cognate site with no concomitant gain in EcoRI* site cleavage activity (Alves et al, 1989;Needels et al, 1989;Heitman and Model, 1990a;Osuna et al, 1991;Fritz et al, 1998;Ivanenko et al, 1998). These results exemplify the difficulty of the problem: Recognition is not simply the sum of one-to-one interactions between protein side chains and DNA bases, but includes redundant interactions with both members of a base-pair, and conformational factors determined by sequence motifs larger than a single base-pair.…”

Section: Introductionmentioning

confidence: 98%

Structural and Thermodynamic Basis for Enhanced DNA Binding by a Promiscuous Mutant EcoRI Endonuclease

Sapienza

Jen‐Jacobson

2007

Structure

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Promiscuous mutant EcoRI endonucleases bind to the canonical site GAATTC more tightly than does the wild-type endonuclease, yet cleave variant (EcoRI(*)) sites more rapidly than does wild-type. The crystal structure of the A138T promiscuous mutant homodimer in complex with a GAATTC site is nearly identical to that of the wild-type complex, except that the Thr138 side chains make packing interactions with bases in the 5'-flanking regions outside the recognition hexanucleotide while excluding two bound water molecules seen in the wild-type complex. Molecular dynamics simulations confirm exclusion of these waters. The structure and simulations suggest possible reasons why binding of the A138T protein to the GAATTC site has DeltaS degrees more favorable and DeltaH degrees less favorable than for wild-type endonuclease binding. The interactions of Thr138 with flanking bases may permit A138T, unlike wild-type enzyme, to form complexes with EcoRI(*) sites that structurally resemble the specific wild-type complex with GAATTC.

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“…Exhaustive mutation of the amino acid residues directly involved in DNA recognition has not yielded variants with altered specificity, but rather reduction of function mutants [15][16][17][18][19][20][21][22] with reduced ability to cleave the cognate site and no concomitant gain in cleavage activity at other sites. As an alternative approach, Heitman & Model 23 screened for relaxed-specificity point mutations of EcoRI endonuclease, through the ability to induce the SOS DNA-damage response in E. coli despite co-expression of the EcoRI methylase.…”

Section: Introductionmentioning

confidence: 99%

Thermodynamic and Kinetic Basis for the Relaxed DNA Sequence Specificity of “Promiscuous” Mutant EcoRI Endonucleases

Sapienza

Torre

McCoy

et al. 2005

Journal of Molecular Biology

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Asn¹⁴¹ is essential for DNA recognition by EcoRI restriction endonuclease

Cited by 11 publications

References 30 publications

Functional Analysis of MmeI from Methanol UtilizerMethylophilus methylotrophus, a Subtype IIC Restriction-Modification Enzyme Related to Type I Enzymes

Functional Analysis of MmeI from Methanol UtilizerMethylophilus methylotrophus, a Subtype IIC Restriction-Modification Enzyme Related to Type I Enzymes

Structural and Thermodynamic Basis for Enhanced DNA Binding by a Promiscuous Mutant EcoRI Endonuclease

Thermodynamic and Kinetic Basis for the Relaxed DNA Sequence Specificity of “Promiscuous” Mutant EcoRI Endonucleases

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Asn141 is essential for DNA recognition by EcoRI restriction endonuclease

Cited by 11 publications

References 30 publications

Functional Analysis of MmeI from Methanol UtilizerMethylophilus methylotrophus, a Subtype IIC Restriction-Modification Enzyme Related to Type I Enzymes

Functional Analysis of MmeI from Methanol UtilizerMethylophilus methylotrophus, a Subtype IIC Restriction-Modification Enzyme Related to Type I Enzymes

Structural and Thermodynamic Basis for Enhanced DNA Binding by a Promiscuous Mutant EcoRI Endonuclease

Thermodynamic and Kinetic Basis for the Relaxed DNA Sequence Specificity of “Promiscuous” Mutant EcoRI Endonucleases

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Asn¹⁴¹ is essential for DNA recognition by EcoRI restriction endonuclease