Modulation of the DNA scanning activity of the Micrococcus luteus UV endonuclease
- Vanderbilt Univ. School of Medicine, Nashville, TN (USA)
Micrococcus luteus UV endonuclease incises DNA at the sites of ultraviolet (UV) light-induced pyrimidine dimers. The mechanism of incision has been previously shown to be a glycosylic bond cleavage at the 5'-pyrimidine of the dimer followed by an apyrimidine endonuclease activity which cleaves the phosphodiester backbone between the pyrimidines. The process by which M. luteus UV endonuclease locates pyrimidine dimers within a population of UV-irradiated plasmids was shown to occur, in vitro, by a processive or sliding mechanism on non-target DNA as opposed to a distributive or random hit mechanism. Form I plasmid DNA containing 25 dimers per molecule was incubated with M. luteus UV endonuclease in time course reactions. The three topological forms of plasmid DNA generated were analyzed by agarose gel electrophoresis. When the enzyme encounters a pyrimidine dimer, it is significantly more likely to make only the glycosylase cleavage as opposed to making both the glycosylic and phosphodiester bond cleavages. Thus, plasmids are accumulated with many alkaline-labile sites relative to single-stranded breaks. In addition, reactions were performed at both pH 8.0 and pH 6.0, in the absence of NaCl, as well as 25,100, and 250 mM NaCl. The efficiency of the DNA scanning reaction was shown to be dependent on both the ionic strength and pH of the reaction. At low ionic strengths, the reaction was shown to proceed by a processive mechanism and shifted to a distributive mechanism as the ionic strength of the reaction increased. Processivity at pH 8.0 is shown to be more sensitive to increases in ionic strength than reactions performed at pH 6.0.
- OSTI ID:
- 5224807
- Journal Information:
- Journal of Biological Chemistry; (USA), Vol. 264:29; ISSN 0021-9258
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ENDONUCLEASES
ENZYME ACTIVITY
MICROCOCCUS LUTEUS
GENETIC RADIATION EFFECTS
DNA
ELECTROPHORESIS
PLASMIDS
PYRIMIDINE DIMERS
ULTRAVIOLET RADIATION
BACTERIA
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
CELL CONSTITUENTS
DNA-ASE
ELECTROMAGNETIC RADIATION
ENZYMES
ESTERASES
GENETIC EFFECTS
HYDROLASES
MICROCOCCUS
MICROORGANISMS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PHOSPHODIESTERASES
RADIATION EFFECTS
RADIATIONS
560130* - Radiation Effects on Microorganisms