Sequence-specific recognition of DNA by RSRI endonuclease of Rhodobacter sphaeroides, an isoschizomer of EcoRI
Aiken, Christopher Robin
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https://hdl.handle.net/2142/23553
Description
Title
Sequence-specific recognition of DNA by RSRI endonuclease of Rhodobacter sphaeroides, an isoschizomer of EcoRI
Author(s)
Aiken, Christopher Robin
Issue Date
1991
Doctoral Committee Chair(s)
Gumport, Richard I.
Department of Study
Chemistry, Biochemistry
Discipline
Chemistry, Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
In order to study the interaction of RsrI endonuclease with its DNA recognition sequence and to determine whether RsrI recognizes DNA using a mechanism similar to that of EcoRI, I have purified milligram amounts of the enzyme to near homogeneity from its natural source, Rhodobacter sphaeroides strain 630. The enzyme comigrates with EcoRI during SDS-PAGE, with a reduced and denatured molecular weight of 30,000 $\pm$ 2000 Da. RsrI endonuclease is a dimer at concentrations of 0.05 to 1.4 mg/ml. In contrast to EcoRI, no tetrameric form was observed. The isoelectric point of the endonuclease was determined to be 7.0-different than the value of 6.3 reported for EcoRI.
The temperature and salt-dependence of the catalytic activity of RsrI also differ from those of EcoRI. The reaction exhibits Michaelis-Menten kinetics, and values of 14 nM and 6.5 min$\sp{-1}$ were determined for K$\sb{\rm M}$ and k$\sb{\rm cat}$, respectively using plasmid pBR322 as a substrate. RsrI endonuclease, unlike EcoRI, is inactivated by preincubating the enzyme with 10 mM N-ethylmaleimide. This suggests that RsrI but not EcoRI has an essential sulfhydryl. RsrI endonuclease cleaves noncanonical sequences in reactions containing 12.5 mM tris-HCl, 2.5 mM MgCl$\sb2$, and 26% ethylene glycol.
RsrI binding bends DNA by approximately 50$\sp\circ$ and unwinds the DNA helix by 25 $\pm$ 5$\sp\circ$--values similar to those reported for EcoRI endonuclease. RsrI binding protects 12 nucleotides from cleavage by hydroxyl radical and MPE$\cdot$Fe(II) footprinting reagents.
I have tested the RsrI endonuclease with a series of decadeoxyribonucleotides containing base analogues as substrates. The kinetics of RsrI cleavage are affected by each substitution, and the effects are generally more pronounced than with EcoRI, as shown by the greater reduction in the specificity constant k$\sb{\rm cat}$/K$\sb{\rm m}$ for a given substitution. For several substrates, cleavage by RsrI was very slow--less than one-tenth the rate of the corresponding EcoRI reaction. This lower tolerance of the RsrI endonuclease for functional group changes in its recognition site may reflect differences in the mechanisms by which RsrI and EcoRI recognize and cleave DNA.
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