Crystallization and preliminary crystallographic analysis of the type IIL restriction enzyme<i>Mme</i>I in complex with DNA

The creation of restriction enzymes with programmable DNA-binding and -cleavage specificities has long been a goal of modern biology. The recently discovered Type IIL MmeI family of restriction-and-modification (RM) enzymes that possess a shared target recognition domain provides a framework for engineering such new specificities. However, a lack of structural information on Type IIL enzymes has limited the repertoire that can be rationally engineered. We report here a crystal structure of MmeI in complex with its DNA substrate and an S-adenosylmethionine analog (Sinefungin). The structure uncovers for the first time the interactions that underlie MmeI-DNA recognition and methylation (5’-TCCRAC-3’; R = purine) and provides a molecular basis for changing specificity at four of the six base pairs of the recognition sequence (5’-TCCRAC-3’). Surprisingly, the enzyme is resilient to specificity changes at the first position of the recognition sequence (5’-TCCRAC-3’). Collectively, the structure provides a basis for engineering further derivatives of MmeI and delineates which base pairs of the recognition sequence are more amenable to alterations than others.

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“…Both the native and selenium-methionine (Se-met) MmeI proteins were expressed and purified as described previously [ 37 ].…”

Section: Methodsmentioning

confidence: 99%

Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities

et al. 2016

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“…The structures of MmeI and NmeAIII have also been solved recently (345), the former with DNA, and are awaiting publication. Type IIC RM proteins possess only one endonuclease catalytic site, yet cleave both DNA strands.…”

Section: Introductionmentioning

confidence: 99%

“…Among Type IIC enzymes, the crystal structure of BpuSI (GGGAC 10/14) has been solved, but without DNA ( 344 ). The structures of MmeI and NmeAIII have also been solved recently ( 345 ), the former with DNA, and are awaiting publication. Type IIC RM proteins possess only one endonuclease catalytic site, yet cleave both DNA strands.…”

Section: Introductionmentioning

confidence: 99%

Type II restriction endonucleases—a historical perspective and more

2014

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This article continues the series of Surveys and Summaries on restriction endonucleases (REases) begun this year in Nucleic Acids Research. Here we discuss ‘Type II’ REases, the kind used for DNA analysis and cloning. We focus on their biochemistry: what they are, what they do, and how they do it. Type II REases are produced by prokaryotes to combat bacteriophages. With extreme accuracy, each recognizes a particular sequence in double-stranded DNA and cleaves at a fixed position within or nearby. The discoveries of these enzymes in the 1970s, and of the uses to which they could be put, have since impacted every corner of the life sciences. They became the enabling tools of molecular biology, genetics and biotechnology, and made analysis at the most fundamental levels routine. Hundreds of different REases have been discovered and are available commercially. Their genes have been cloned, sequenced and overexpressed. Most have been characterized to some extent, but few have been studied in depth. Here, we describe the original discoveries in this field, and the properties of the first Type II REases investigated. We discuss the mechanisms of sequence recognition and catalysis, and the varied oligomeric modes in which Type II REases act. We describe the surprising heterogeneity revealed by comparisons of their sequences and structures.

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“…Although classified as different RM Type, the domain structure of these enzymes may represent an example of highly adaptable evolution in which new RM systems can be produced by gene shuffling, facilitated by horizontal gene transfer, which is a common feature of bacterial host defence mechanisms. Further interpretation of this and of the enzymatic mechanisms will be greatly assisted by structural information on both Type ISP and Type IIL families of RM enzyme (23). …”

Section: Discussionmentioning

confidence: 99%

The Type ISP Restriction-Modification enzymes LlaBIII and LlaGI use a translocation-collision mechanism to cleave non-specific DNA distant from their recognition sites

Šišáková¹,

Aelst²,

Diffin³

et al. 2012

Nucleic Acids Research

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The Type ISP Restriction–Modification (RM) enzyme LlaBIII is encoded on plasmid pJW566 and can protect Lactococcus lactis strains against bacteriophage infections in milk fermentations. It is a single polypeptide RM enzyme comprising Mrr endonuclease, DNA helicase, adenine methyltransferase and target-recognition domains. LlaBIII shares >95% amino acid sequence homology across its first three protein domains with the Type ISP enzyme LlaGI. Here, we determine the recognition sequence of LlaBIII (5′-TnAGCC-3′, where the adenine complementary to the underlined base is methylated), and characterize its enzyme activities. LlaBIII shares key enzymatic features with LlaGI; namely, adenosine triphosphate-dependent DNA translocation (∼309 bp/s at 25°C) and a requirement for DNA cleavage of two recognition sites in an inverted head-to-head repeat. However, LlaBIII requires K+ ions to prevent non-specific DNA cleavage, conditions which affect the translocation and cleavage properties of LlaGI. By identifying the locations of the non-specific dsDNA breaks introduced by LlaGI or LlaBIII under different buffer conditions, we validate that the Type ISP RM enzymes use a common translocation–collision mechanism to trigger endonuclease activity. In their favoured in vitro buffer, both LlaGI and LlaBIII produce a normal distribution of random cleavage loci centred midway between the sites. In contrast, LlaGI in K+ ions produces a far more distributive cleavage profile.

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Crystallization and preliminary crystallographic analysis of the type IIL restriction enzymeMmeI in complex with DNA

Cited by 7 publications

References 30 publications

Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities

Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities

Type II restriction endonucleases—a historical perspective and more

The Type ISP Restriction-Modification enzymes LlaBIII and LlaGI use a translocation-collision mechanism to cleave non-specific DNA distant from their recognition sites

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