Complementation by detached parts of GGCC-specific DNA methyltransferases

Pósfai, György; Kim, Sun C.; Szilák, László; Kovács, Attila; Venetianer, Pál

doi:10.1093/nar/19.18.4843

Cited by 16 publications

(12 citation statements)

References 32 publications

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“…This view is consistent with reports showing that individually inactive segments of MTases, comprising their N-terminal and C-terminal CEs, respectively, can complement each other to form active enzymes. This situation exists naturally in the case of M.AquI (Karreman and de Waard, 1990) and has been artificially generated in the case of M.BspRI (Posfai et al, 1991).…”

Section: Discussionmentioning

confidence: 99%

High plasticity of multispecific DNA methyltransferases in the region carrying DNA target recognizing enzyme modules.

Walter

Trautner²,

Noyer-Weidner³

1992

The EMBO Journal

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Multispecific cytosine C5 DNA methyltransferases (MTases) methylate more than one specific DNA target. This is due to the presence of several target recognizing domains (TRDs) in these enzymes. Such TRDs form part of a variable centre in the MTase primary sequence, which separates conserved enzyme core sequences responsible for general steps in the methylation reaction. By deleting, rearranging and exchanging several TRDs of multispecific MTases, we demonstrate their modular character; they mediate target recognition independent of a particular TRD or core sequence context. We show also that multispecific MTases can accommodate inert material of non‐MTase origin within their variable region without losing their activity. The remarkable plasticity with respect to the material that can be integrated into this region suggests that the enzyme core sequences preceding or following it form separable functional domains. In spite of the documented flexibility multispecific MTases could not be endowed with novel specificities by integration of putative TRDs of monospecific MTases, pointing to differences between multi‐ and monospecific MTases in the way their core and TRD sequences interact.

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Section: Discussionmentioning

confidence: 99%

High plasticity of multispecific DNA methyltransferases in the region carrying DNA target recognizing enzyme modules.

Walter

Trautner²,

Noyer-Weidner³

1992

The EMBO Journal

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“…Finally, each possible cut point is represented by only very few mutants in RGD, while the use of incrementally truncated proteins may result in multiple complementing pairs that are in fact variants of the same cut point. On the other hand, it appears that sometimes protein overlaps might be required for efficient complementation (52). If so, RGD will not identify those overlaps.…”

Section: Discussionmentioning

confidence: 99%

Random Gene Dissection: A Tool for the Investigation of Protein Structural Organization

Sapranauskas¹,

Lubys²

2005

BioTechniques

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To investigate the domain structure of proteins and the function of individual domains, proteins are usually subjected to limited proteolysis, followed by isolation of protein fragments and determination of their functions. We have developed an approach we call random gene dissection (RGD) for the identification of functional protein domains and their interdomain regions as well as their in vivo complementing fragments. The approach was tested on a two-domain protein, the type IIS restriction endonuclease BfiI. The collection of BfiI insertional mutants was screened for those that are endonucleolytically active and thus induce the SOS DNA repair response. Sixteen isolated mutants of the wild-type specificity contained insertions that were dispersed in a relatively large region of the target recognition domain. They split the gene into two complementing parts that separately were unable to induce the SOS DNA repair response. In contrast, all 19 mutants of relaxed specificity contained the cassette inserted into a very narrow interdomain region that connects BfiI domains responsible for DNA recognition and for cleavage. As expected, only the N-terminal fragment of BfiI was required to induce SOS response. Our results demonstrate that RGD can be used as a general method to identify complementing fragments and functional domains in enzymes.

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“…The authors capitalised on previous observations of protein fragment complementation described for some DNA (cytosine‐C5) MTases. These enzymes generally act as monomers, but at least some of them can be split into two (preferably partially overlapping) fragments, which, while inactive by themselves, can assemble to form active enzyme when expressed in the same cell 11. 12 Nomura and Barbas fused an N‐terminal segment of the DNA MTase M.HhaI, encompassing residues 2–240, to a ZFP engineered to recognise a 9 bp sequence.…”

Section: Methodsmentioning

confidence: 99%

Functional Reassembly of Split Enzymes On‐Site: A Novel Approach for Highly Sequence‐Specific Targeted DNA Methylation

Kiss

Weinhold

2008

ChemBioChem

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Assembling the tool on the spot. Split enzymes need to come together to gain activity. Assembly of an artificially split DNA methyltransferase can be directed to a single 5′‐CG‐3′ DNA sequence in vivo by fusing both enzyme fragments to zinc finger proteins; this leads to programmable, highly sequence‐specific DNA methylation. Such tools hold great promise for selective silencing of genes by targeted methylation of their promoters.

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Complementation by detached parts of GGCC-specific DNA methyltransferases

Cited by 16 publications

References 32 publications

High plasticity of multispecific DNA methyltransferases in the region carrying DNA target recognizing enzyme modules.

High plasticity of multispecific DNA methyltransferases in the region carrying DNA target recognizing enzyme modules.

Random Gene Dissection: A Tool for the Investigation of Protein Structural Organization

Functional Reassembly of Split Enzymes On‐Site: A Novel Approach for Highly Sequence‐Specific Targeted DNA Methylation

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