Structure of chromatin at deoxyribonucleic acid replication forks: location of the first nucleosomes on newly synthesized Simian virus 40 deoxyribonucleic acid

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Abstract
Exonucleases specific for either 3'' ends (Escherichia coli exonuclease III) or 5'' ends (bacteriophage T7 gene 6 exonuclease) of nascent DNA chains were used to determine the number of nucleotides from the actual sites of DNA synthesis, to the 1st nucleosome on each arm of replication forks in SV40 chromosomes labeled with [3H]thymidine in whole cells. Whereas each enzyme excised all of the nascent [3H]DNA from purified replicating SV40 DNA, only a fraction of the [3H]DNA was excised from purified replicating SV40 chromosomes. The latter result was attributable to the inability of either exonuclease to digest nucleosomal DNA in native replicating SV40 chromosomes. Digestion with either exonuclease did not reduce the amount of newly synthesized nucleosomal DNA released by micrococcal nuclease during a subsequent digestion period. In briefly labeled molecules, as much as 40% of the [3H]DNA was excised from long nascent DNA chains. The fraction of [3H]DNA excised by exonuclease III was reduced in proportion to the actual length of the radiolabeled DNA. The effects of the 2 exonucleases were additive, consistent with each enzyme trimming only the 3'' or 5'' ends of nascent DNA chains without continued excision through to the opposite end. When the fraction of nascent [3H]DNA excised from replicating SV40 DNA by exonuclease III was compared with the fraction of [32P]DNA simultaneously excised from an SV40 DNA restriction fragment, the actual length of nascent [3H]DNA was calculated. From this number, the fraction of [3H]DNA excised from replicating SV40 chromosomes was converted into the number of nucleotides. The average distance from either 3'' or 5'' ends of long nascent DNA chains to the 1st nucleosome on either arm of replication forks was 125 nucleotides. Each exonuclease excised about 80% of the radiolabel in Okazaki fragments, suggesting that less than 1/5 of the Okazaki fragments were contained in nucleosomes. A model for eukaryotic replication forks is presented in which nucleosomes appear rapidly on both the forward and retrograde arms, about 125 and 300 nucleotides, respectively, from the actual site of DNA synthesis. Okazaki fragments may be initiated on nonnucleosomal DNA and then assembled into nucleosomes, generally after ligation to the 5'' ends of long nascent DNA chains is completed.