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|Title:||Studies of sequence recognition by the EcoRI restriction/modification system of Escherichia coli|
|Author(s):||Van Cleve, Mark David|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Sequence recognition by the EcoRI restriction/modification system of Escherichia coli is currently the subject of intense study as a model of the general phenomenon of target location by nucleotide sequence-specific enzymes and binding proteins. Models have proposed a variety of means for site location, including formation of hydrogen bond matrices between protein and DNA and ionic contacts between the ribose-phosphate backbone and basic amino acids residues on the protein. We have examined DNA-protein interactions involved in the EcoRI recognition process by using defined oligonucleotide duplex substrates with various alterations inside and outside the recognition site. This model system consists of an endonuclease that cleaves the DNA sequence, GAATTC, between the guanine and adenine residues and a methyltransferase that methylates the 6-amino group of the central adenine residue. The advantages of this model system include the ability to study recognition of the same substrates by two different enzymes and the existence of a large context of information to direct and supplement the investigation.
By determining the steady-state kinetic parameters of the reactions of the endonuclease and methyltransferase with these substrates, we have identified contacts that are important in site recognition by these enzymes. As detailed in Chapters II and III, substituting analogues of the naturally-occurring bases into the recognition site affected the enzymes' kinetic parameters, indicating close interdigitation between enzyme and substrate. In addition, the various analogue substitutions affect recognition by the two enzymes in different ways. This result is interpreted to mean that the enzymes accomplish recognition of GAATTC in different ways.
The importance of contacts outside the recognition sequence was investigated by varying the number of base pairs flanking GAATTC in a series of substrates described in Chapter IV. The results of this study indicate that the endonuclease uses contacts with the two phosphates immediately 5$\sp\prime$ to GAATTC for transition-state binding, and that one or more contacts with the second base pair 5$\sp\prime$ to the site may be used in substrate binding.
|Rights Information:||Copyright 1990 Van Cleve, Mark David|
|Date Available in IDEALS:||2011-05-07|
|Identifier in Online Catalog:||AAI9026341|