Nucleosome rearrangement in vitro. 2. Formation of nucleosomes in newly repaired regions of DNA.
We have reported previously that immediately following nucleotide excision repair in human cells the newly repaired DNA lacks a nucleosome conformation [Smerdon, M. J., & Lieberman, M. W. (1980) Biochemistry 19, 2992-3000]. In this study, we have examined the ability of these nascent DNA regions to acquire a nucleosome structure in vitro by incubating intact or H1-depleted nuclei in buffers containing different salt concentrations (0.025-0.625 M KCl) at 0 or 37 degrees C. Nucleosomes were detected in these regions by an increase in the level of repair-incorporated nucleotides associated with isolated nucleosome core particle DNA. Our results indicate that the nascent DNA is resistant to nucleosome formation during the low-salt transition where the limiting repeat length decreases from approximately 190 to 168 base pairs (bp) [Watkins, J. F., & Smerdon, M. J. (1985) Biochemistry (preceding paper in this issue)]. This result provides further evidence that the nascent DNA is indeed in a nonnucleosomal state. At higher salt concentrations (greater than 0.4 M), where the nucleosome repeat length decreases to a limiting value of approximately 146 bp, there was an increase in nucleosome formation in nascent DNA that correlated with the decrease in limiting repeat length. However, we did not observe a complete randomization of the repair-incorporated nucleotides. Indeed, even at the highest salt concentration used (0.625 M), we never observed more than 50% of the nascent DNA associated with the isolated core particles. This was the case even though a major portion of the nucleosomes had a limiting value repeat length following the high-salt incubation.(ABSTRACT TRUNCATED AT 250 WORDS)
D002250:Carbon Radioisotopes; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000302:isolation & purification / Q000648:ultrastructure; D004260:DNA Repair; D004261:DNA Replication; D005347:Fibroblasts / Q000378:metabolism; D006657:Histones / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D009707:Nucleosomes / Q000378:metabolism / Q000648:ultrastructure*; D014316:Tritium
D002250:Carbon Radioisotopes; D002843:Chromatin; D006657:Histones; D009707:Nucleosomes; D014316:Tritium