Modulation of the DNA scanning activity of the Micrococcus luteus UV endonuclease.
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.
Hamilton
R W
RW
Department of Molecular Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Lloyd
R S
RS
D004247:DNA / Q000378:metabolism* / Q000528:radiation effects; D045647:DNA Glycosylases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004706:Endodeoxyribonucleases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D008837:Micrococcus / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000378:metabolism*; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009994:Osmolar Concentration; D010957:Plasmids; D011740:Pyrimidine Dimers / Q000378:metabolism / Q000528:radiation effects; D012965:Sodium Chloride / Q000494:pharmacology; D014466:Ultraviolet Rays
D006898:Hydroxylamines; D009097:Multienzyme Complexes; D011740:Pyrimidine Dimers; D012965:Sodium Chloride; C032219:UV endonuclease; D004247:DNA; C005214:methoxyamine; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase