STRUCTURE OF CHROMATIN AT DEOXYRIBONUCLEIC-ACID REPLICATION FORKS - OKAZAKI FRAGMENTS RELEASED FROM REPLICATING SV40 CHROMOSOMES BY SINGLE-STRAND SPECIFIC ENDONUCLEASES ARE NOT IN NUCLEOSOMES

The structure of replicating chromosomes at the sites of deoxyribonucleic acid (DNA) synthesis was characterized by digestion of replicating simian virus 40 (SV40) chromosomes with either S1 or Neurospora crassa single-strand specific endonucleases. The products of digestion were compared with those from the digestion of nonreplicating SV40 chromosomes and purified replicating and nonreplicating viral DNA. While purified, covalently closed, superhelical DNA [SV40(I) DNA] was cleaved by these nucleases, SV40(I) DNA contained in mature SV40 chromosomes was completely resistant. On the other hand, all replicating SV40 DNA [SV40(RI) DNA], both in the form of purified DNA and replicating chromosomes, was cleaved by these single-strand specific nucleases. Although the DNA contained in replicating chromosomes was cleaved at a rate ~200 times slower than purified SV40(RI) DNA, the initial cleavage of either form of SV40(RI) DNA (25 S) generated a comparatively homogeneous population of 18S DNA. Continued digestion released a maximum of 50-60% of the Okazaki fragments as 4-6S duplex DNA. The 40-50% of the Okazaki fragments which remained with 18S DNA could result from the presence of single-stranded regions of DNA too small to be recognized by S1 nuclease under the conditions used. At least 90% of the Okazaki fragments released by S1 nuclease from formaldehyde-fixed chromosomes were not contained in nucleosomes, but were found as bare DNA. They were digested by micrococcal nuclease at the same rate and to the same extent as purified DNA. They sedimented at 4-6 S before and after treatment with Sarkosyl, proteinase K, and 1 M NaCl, and they had the same buoyant density in CsCl as purified DNA. Therefore, the synthesis of Okazaki fragments and their assembly into nucleosomes need not occur concomitantly. Rather, Okazaki fragments can be synthesized and ligated to longer nascent DNA chains prior to assembly into nucleosomes. © 1979, American Chemical Society. All rights reserved.