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 Title: In Vitro and in Vivo Studies of the Recognition of Double-Strand DNA by EcoRI and RsrI Endonucleases Author(s): Fisher, Eric William Doctoral Committee Chair(s): Gumport, Richard I. Department / Program: Biochemistry Discipline: Biochemistry Degree Granting Institution: University of Illinois at Urbana-Champaign Degree: Ph.D. Genre: Dissertation Subject(s): Biology, Genetics Biology, Microbiology Chemistry, Biochemistry Abstract: EcoRI and RsrI endonucleases present rare opportunities to study the mechanisms by which site-specific DNA-binding proteins identify their substrate sequences among an overwhelming number of other sequences to which they also show significant affinity. These proteins have advantages as subjects for the study of protein/DNA recognition in that they are enzymes, permitting the examination of both binding and catalysis. The fact that both enzymes recognize the same sequence and catalyze the same reaction, although they share only 50% amino acid identity and differ physically and chemically, suggests that comparing their recognition properties will be informative with respect to structure/function relationships and to protein evolution. Finally, the effort already expended in characterizing the enzymes leaves them among the best understood of enzymes recognizing specific DNA sequences.Cleavage by EcoRI endonuclease under conditions reducing specificity was examined using steady-state kinetics to determine the hierarchy of preference among sequences other than the canonical, GAATTC. The enzyme suffered a loss of catalytic activity too rapid to permit accurate measurement of kinetic constants (the Michaelis constant, K$\sb{\rm M}$, and the turnover number, k$\sb{\rm cat}$) governing the reaction under the buffer conditions used, and this problem persisted in a variety of different buffers.Because the association constants governing specific and nonspecific binding to large DNA molecules by EcoRI endonuclease in the absence of magnesium ion had been determined, these same values were measured for RsrI. In spite of differing buffer requirements for the two enzymes, RsrI requiring a lower incubation temperature and lower ionic strength, both enzymes show an affinity of approximately 10$\sp$ M$\sp{-1}$ for the GAATTC site in plasmid pBR322, and an average affinity of less than 10$\sp6$ M$\sp{-1}$ for other sequences in the plasmid. When the temperature of RsrI incubation is reduced from 20$\sp\circ$C to 0$\sp\circ$C the specific affinity of RsrI for pBR322 falls and the nonspecific affinity rises slightly. These changes result in a net three-fold decrease in binding selectivity of the enzyme for the specific site over the nonspecific through this temperature range.To begin the task of altering the specificity of EcoRI endonuclease to recognize sequences other than GAATTC, the bacteriophage P22 challenge-phage assay was adapted for use with both EcoRI and RsrI endonucleases. The gene encoding a mutant of EcoRI (E111Q) lacking catalytic activity but retaining binding specificity was mutagenized randomly by several methods, and a mutant was selected with a binding preference for the EcoRI-methylated GAATTC (GA$\sp{\rm m}$ATTC) sequence over the unmethylated sequence. This mutant, isolated after one round of hydroxylamine treatment of cells carrying the ecoRIR(E111Q) gene, possesses binding specificity opposite to wild-type EcoRI endonuclease for methylated and unmethylated GAATTC: wild-type binds tightly to the unmethylated site and several orders of magnitude less to the methylated site, but this mutant appears to bind tightly to the methylated site and almost undetectably to the unmethylated. This mutant will now be examined in vitro with respect to its binding properties, and then sequenced to determine the amino acid change, to rationalize the role of the mutation in conferring a reversal of specificity within the context of the crystallographic model now available for the specific EcoRI endonuclease/DNA complex. (Abstract shortened by UMI.) Issue Date: 1994 Type: Text Description: 164 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1994. URI: http://hdl.handle.net/2142/72365 Other Identifier(s): (UMI)AAI9503183 Date Available in IDEALS: 2014-12-17 Date Deposited: 1994
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