Construction and use of chimeric SPR/phi 3T DNA methyltransferases in the definition of sequence recognizing enzyme regions.

Balganesh, Tanjore S.; Reiners, Luzia; Lauster, Roland; Noyer-Weidner, Mario; Wilke, Kathleen; Trautner, Thomas A.

doi:10.1002/j.1460-2075.1987.tb02681.x

Cited by 93 publications

(42 citation statements)

References 23 publications

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“…Unfortunately, there is only a limited number of direct experimental approaches backing this hypothesis, mostly dealing with the construction of new chimeric proteins by fusing cloned genes (3)(4)(5). These genetic manipulations have opened new insights into studies of the relationships between molecular structure and the biological function of proteins-e.g., it has been demonstrated that reconstruction of an enzyme by domain substitution between two proteins that share extensive amino acid sequence similarity resulted in the switch of substrate or catalytic specificity (6)(7)(8).…”

mentioning

confidence: 99%

Chimeric phage-bacterial enzymes: a clue to the modular evolution of genes.

Dı́az

López

Garcı́a

1990

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

123

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Pneumococcal peptidoglycan amidase (Nacetylmuramoyl-L-alanine amidase, EC 3.5.1.28) and phage CPL1 lysozyme degrade a common substrate (cholinecontaining pneumococcal cell walls); the former hydrolyzes the bond between muramic acid and alanine, whereas the latter breaks down the linkage between muramic acid and glucosamine. The amino acid sequences of their C-terminal domains are homologous. Chimeric genes were constructed by site-

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mentioning

confidence: 99%

Chimeric phage-bacterial enzymes: a clue to the modular evolution of genes.

Dı́az

López

Garcı́a

1990

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

123

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“…The a and ,B ORFs overlap in the middle of the normally poorly conserved region in which, in other modification methylases, the specific recognition site of the protein is thought to be encoded (1). This could mean either that the whole function occurs on just one of the proteins or that the a-and 3-encoded proteins perfectly fit together when active.…”

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confidence: 99%

Agmenellum quadruplicatum M.AquI, a novel modification methylase

Karreman¹,

Waard²

1990

J Bacteriol

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The complete type II modification methylase of Agmenellum quadruplicatum was cloned in Escherichia coli as an R Sau3A fragment of approximately 4.5 kilobases. The coding sequence was contained in a stretch of 1,156 base pairs which was organized into two parallel, partly overlapping open reading frames of 248 and 139 codons. In vivo complementation experiments showed that the synthesis of both predicted peptides was required for full methylase activity. The amino acid sequences were considerably similar to regions of other deoxycytidylate methylases.In procaryotic cells, two fundamentally different restriction-modification (R-M) systems have been found. They can be distinguished by their need for cofactors, as well as by their genetic organization. The R-M complex of the so-called type I systems requires S-adenosylmethionine, ATP, and Mg2+ for its functioning and contains the products of three genes: the first codes for the S unit, which recognizes the specific site, the second directs the synthesis of the R unit, which is responsible for cutting unmethylated DNA, and the last is translated into the M unit, which methylates non-or hemimethylated DNA. The absence of both of the protecting methyl groups at the recognition sites primes the R-M complex for attacking the DNA; the actual cleavage, however, does not occur at the recognition site itself, but somewhere else in the DNA molecule. The type II systems, on the other hand, code for two proteins: an endonuclease needing only Mg2+ for cleavage (which takes place at the recognition site itself) and a modification methylase requiring only S-adenosylmethionine for the protection of DNA. All type II systems characterized at the genetic level so far have been found to be encoded by just two genes: one for the endonuclease and another for the methylase. Only in the case of DpnII have two different methylase genes been discovered (3); both code for a protein that methylates independently of the other.In the cyanobacterium Agmenellum quadruplicatum, an R-M system that consists of isoschizomers of the AvaI (10,13)

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“…A wealth of sequence information (21,27), mutational analysis (41), domain swap experiments (1,20), biochemical studies (4,42), and crystal structures for two enzymes (M.HhaI and M.HaeIII) (19,29) support the view that prokaryotic DNA (cytosine-5) MTases (C5-MTases) share a common architecture and catalytic mechanism. C5-MTases contain 10 conserved amino acid sequence motifs and a variable region between conserved motifs VIII and IX.…”

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confidence: 94%

In Vivo DNA Protection by Relaxed-Specificity SinI DNA Methyltransferase Variants

2008

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The SinI DNA methyltransferase, a component of the SinI restriction-modification system, recognizes the sequence GG(A/T)CC and methylates the inner cytosine to produce 5-methylcytosine. Previously isolated relaxed-specificity mutants of the enzyme also methylate, at a lower rate, GG(G/C)CC sites. In this work we tested the capacity of the mutant enzymes to function in vivo as the counterpart of a restriction endonuclease, which can cleave either site. The viability of Escherichia coli cells carrying recombinant plasmids with the mutant methyltransferase genes and expressing the GGNCC-specific Sau96I restriction endonuclease from a compatible plasmid was investigated. The sau96IR gene on the latter plasmid was transcribed from the araBAD promoter, allowing tightly controlled expression of the endonuclease. In the presence of low concentrations of the inducer arabinose, cells synthesizing the N172S or the V173L mutant enzyme displayed increased plating efficiency relative to cells producing the wild-type methyltransferase, indicating enhanced protection of the cell DNA against the Sau96I endonuclease. Nevertheless, this protection was not sufficient to support long-term survival in the presence of the inducer, which is consistent with incomplete methylation of GG(G/C)CC sites in plasmid DNA purified from the N172S and V173L mutants. Elevated DNA ligase activity was shown to further increase viability of cells producing the V173L variant and Sau96I endonuclease.Type IIP restriction-modification (R-M) systems consist of a sequence-specific endonuclease and a sequence-specific DNA methyltransferase (MTase), which recognize the same DNA sequence (31). MTases transfer a methyl group from the methyl donor S-adenosylmethionine to a cytosine or adenine of the recognition sequence to produce 5-methylcytosine, N4-methylcytosine, or N6-methyladenine (14). The role of the modification MTase is to protect the host DNA from the cognate restriction endonuclease. The exquisite specificity and the availability of two enzymes recognizing the same DNA sequence but carrying out different chemical reactions make R-M systems uniquely interesting for studying sequence-specific DNA-protein interactions.A wealth of sequence information (21, 27), mutational analysis (41), domain swap experiments (1, 20), biochemical studies (4, 42), and crystal structures for two enzymes (M.HhaI and M.HaeIII) (19, 29) support the view that prokaryotic DNA (cytosine-5) MTases (C5-MTases) share a common architecture and catalytic mechanism. C5-MTases contain 10 conserved amino acid sequence motifs and a variable region between conserved motifs VIII and IX. Prokaryotic C5-MTases consist of two domains; the large domain encompasses most of the conserved motifs, whereas the small domain contains the variable region and conserved motif IX. The large domain contains the catalytic site and the cofactor binding site, and the variable region is predominantly responsible for sequence-specific DNA recognition (see also below). The two domains form a cleft, which holds...

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Construction and use of chimeric SPR/phi 3T DNA methyltransferases in the definition of sequence recognizing enzyme regions.

Cited by 93 publications

References 23 publications

Chimeric phage-bacterial enzymes: a clue to the modular evolution of genes.

Chimeric phage-bacterial enzymes: a clue to the modular evolution of genes.

Agmenellum quadruplicatum M.AquI, a novel modification methylase

In Vivo DNA Protection by Relaxed-Specificity SinI DNA Methyltransferase Variants

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