Excision repair of pyrimidine dimers from simian virus 40 minichromosomes in vitro.
The ability of DNA repair enzymes to carry out excision repair of pyrimidine dimers in SV40 minichromosomes irradiated with 16 to 64 J/m2 of UV light was examined. Half of the dimers were substrate for the DNA glycosylase activity of phage T4 UV endonuclease immediately after irradiation, but this limit decreased to 27% after 2 h at 0 degrees C. Moreover, the apyrimidinic (AP) endonuclease activity of the enzyme did not incise all of the AP sites created by glycosylase activity, although all AP sites were substrate for HeLa AP endonuclease II. The initial rate of the glycosylase was 40% that upon DNA. After incision by the T4 enzyme, excision was mediated by HeLa DNase V (acting with an exonuclease present in the chromatin preparation). Under physiological salt conditions, excision did not proceed appreciably beyond the damaged nucleotides in DNA or chromatin. With chromatin, about 70% of the accessible dimers were removed, but at a rate slower than for DNA. Finally, HeLa DNA polymerase beta was able to fill the short gaps created after dimer excision, and these patches were sealed by T4 DNA ligase. Overall, roughly 30% of the sites incised by the endonuclease were ultimately sealed by the ligase. The resistance of some sites was due to interference with the ligase by the chromatin structure, as only 30-40% of the nicks created in chromatin by pancreatic DNase could be sealed by T4 or HeLa DNA ligases. The overall excision repair process did not detectably disrupt the chromatin structure, since the repair label was recovered in Form I DNA present in 75 S condensed minichromosomes. Although other factors might stimulate the rate of this repair process, it appears that the enzymes utilized could carry out excision repair of chromatin to a limit near that observed at the initial rate in mammalian cells in vivo.
D002875:Chromosomes / Q000378:metabolism* / Q000528:radiation effects; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair; D004279:DNA, Viral / Q000235:genetics*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004706:Endodeoxyribonucleases / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism; D009699:N-Glycosyl Hydrolases; D011740:Pyrimidine Dimers / Q000378:metabolism*; D013539:Simian virus 40 / Q000235:genetics*; D013604:T-Phages / Q000201:enzymology; D014466:Ultraviolet Rays
D004279:DNA, Viral; D009097:Multienzyme Complexes; D011740:Pyrimidine Dimers; C032219:UV endonuclease; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); D009699:N-Glycosyl Hydrolases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D011088:DNA Ligases