Studies on the mode of segregation of histone nu bodies during replication in HeLa cells

Abstract

Two models were tested for the mode of distribution of histone nu bodies at the replication fork. The replication fork was labeled by brief incubation of cells with 3H-thymidine. Nuclei were isolated and digested with low levels of micrococcal nuclease, and the kinetics of cleavage of the pulse-labeled chromatin DNA were compared to the kinetics of clevage of perental chromatin DNA. In chromatin labeled for 30 sec to 10 min, the rate of cleavage of the pulse-labeled region into monomeric nu body-sized units exceeded the rate of cleavage of parental chromatin by a factor of 2, but did not approach the predicted value of 5-6 for random segregation. This value dropped to 1.6 in 15 min and was equivalent to perental chromatin in 20 min labeling experiments. DNA synthesized in the presence of cycloheximide was also digested at twice the rate of parental chromatin DNA. A Poisson analysis of the kinetics of cleavage by micrococcal nuclease further confirmed these observations. The predicted difference in the rate of production of monomeric, dimeric, and trimeric deoxyribonucleoprotein units was very similar to the experimental values of both total chromatin and nascent chromatin. Thus the nu body spacings in newly replicated chromatin closely approximate those in parental chromatin. These results agree well with a conservative or nondispersive model of nucleosome distribution in which the proteins are associated with one of the two daughter chromosomes during replication.