We have investigated the distribution in nucleosome core DNA of nucleotides incorporated by excision repair synthesis occurring immediately after UV irradiation in human cells. We show that the differences previously observed for whole nuclei between the DNase I digestion profiles of repaired DNA (following its refolding into a nucleosome structure) and bulk DNA are obtained for isolated nucleosome core particles. Analysis of the differences obtained indicates that they could reflect a significant difference in the level of repair-incorporated nucleotides at different sites within the core DNA region. To test this possibility directly, we have used exonuclease III digestion of very homogeneous sized core particle DNA to "map" the distribution of repair synthesis in these regions. Our results indicate that in a significant fraction of the nucleosomes the 5' and 3' ends of the core DNA are markedly enhanced in repair-incorporated nucleotides relative to the central region of the core particle. A best fit analysis indicates that a good approximation of the data is obtained for a distribution where the core DNA is uniformly labeled from the 5' end to position 62 and from position 114 to the 3' end, with the 52-base central region being devoid of repair-incorporated nucleotides. This distribution accounts for all of the quantitative differences observed previously between repaired DNA and bulk DNA following the rapid phase of nucleosome rearrangement when it is assumed that linker DNA and the core DNA ends are repaired with equal efficiency and the nucleosome structure of newly repaired DNA is identical with that of bulk chromatin. Furthermore, the 52-base central region that is devoid of repair synthesis contains the lowest frequency cutting sites for DNase I in vitro, as well as the only "internal" locations where two (rather than one) histones interact with a 10-base segment of each DNA strand.

Chiral (2S)-[5-13C]leucine was obtained from Escherichia coli deficient in the synthesis of acetolactate when cultures were supplemented with (RS)-[2-13CH3]acetolactate. The carbon-13 nuclear magnetic resonance spectrum showed one strong peak with a chemical shift of 21.4 ppm relative to tetramethylsilane [Sylvester, S. R., & Stevens, C. M. (1979) Biochemistry 18, 4529-4531]. Silver picolinate oxidation of the labeled leucine gave isovaleric acid which was then brominated at the alpha position to give (2RS)-2-bromo[3-13CH3]-isovaleric acid (2-bromo-3-[13C]methylbutanoic acid). Aminolysis afforded (2RS)-[4-13C]valine which was treated with D-amino acid oxidase in the presence of catalase. The final product was identified as (2S,3S)-[4-13C]valine by the specificity of D-amino acid oxidase, by amino acid analysis, and by the persistence of a strong signal at gamma 17.8 in the carbon-13 magnetic resonance spectrum. These results establish the absolute configuration of the biosynthetic leucine to be (2S,4S)-[5-13C]leucine.