Studies of sequence recognition by the EcoRI restriction/modification system of Escherichia coli
Van Cleve, Mark David
This item is only available for download by members of the University of Illinois community. Students, faculty, and staff at the U of I may log in with your NetID and password to view the item. If you are trying to access an Illinois-restricted dissertation or thesis, you can request a copy through your library's Inter-Library Loan office or purchase a copy directly from ProQuest.
Permalink
https://hdl.handle.net/2142/22122
Description
Title
Studies of sequence recognition by the EcoRI restriction/modification system of Escherichia coli
Author(s)
Van Cleve, Mark David
Issue Date
1990
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
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.
Use this login method if you
don't
have an
@illinois.edu
email address.
(Oops, I do have one)
IDEALS migrated to a new platform on June 23, 2022. If you created
your account prior to this date, you will have to reset your password
using the forgot-password link below.
