How are Restriction Systems Controlled, and How Do They Recognize DNA Sequences?

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9631137 Blumenthal Restriction endonucleases (REases) are enzymes that cleave both strands of double-stranded DNA at specific nucleotide sequences. Bacteria that produce REases must protect their own DNA, and this is generally done by producing a methyltransferase (MTase) that transfers a methyl group from S-adenosyl-L-methionine to the DNA: the REases do not cleave DNA sequences that have been properly methylated. Two questions stand out. First, the genes for REases and their cognate MTases are often located on mobile genetic elements such as plasmids. When such a plasmid is transferred into a new host cell, how are the genes and enzymes controlled to ensure that the new host's DNA is methylated before REase activity appears? This question has not been fully answered for any REase / MTase pair. One major goal of this project is to answer this question for the PvuII REase / MTase pair. The proposing laboratory has discovered three features affecting PvuII a predicted hairpin in the mRNA for the PvuII REase which may modulate initiation of translation; an apparent transcriptional activator that is required for expression of the PvuII REase gene and a small protein that appears to prevent the two identical subunits of PvuII REase from associating with one another. The second major question regarding REases and MTases is how they recognize their substrate nucleotide sequence amongst all of the other DNA that is present. REases and MTases must not only bind to a specific DNA sequence, but must then bring the relevant portions of the DNA molecule into contact with an active site or sites. Does this impose particular constraints on how the DNA sequence is recognized? One way to approach this is to compare several proteins that recognize the same sequence (or closely related sequences). This project seeks to characterize the DNA sequence recognition by the following proteins "^" indicates cleavage, 5mC and N4mC indicate methylation of the cytosine at the indicated position: PvuII REase CAG^CTG PvuII MTase CAGN4mCTG AluI Ease AG^CT AluI MTase AG5mCT These proteins will be compared to those basic region helix-loop-helix transcription factors, studied by others, which recognize the sequence CAGCTG but do not catalyze any reaction. %%% Certain bacteria produce specialized DNA modifying enzymes, restriction endonucleases (REase), that destroy DNA from other bacteria or viruses. Protection of the organism's DNA is done by a different type of DNA modifying enzymes, methytransferases, that change the chemical nature of the DNA so that the REase does not destroy the DNA of the bacteria that produces the REase. REases are utilized in biological research to cut DNA at precise locations like microscopic lasers to dissect genes. This project will provide insight into the regulation, structure, and function of these modifying enzymes providing information for the eventual design of new molecular biological tools, and the ability to devise mechanisms to manipulate genetic transfer in bacteria. ***