[Identification of the stable DNA-matrix complex (type II) as a site of replication fork]

The two types of DNA-matrix complexes (the weak and tight ones, or type I and type II, respectively) identified in our previous work were studied with respect to their involvement in DNA replication. Nuclei isolated from human fibrosarcoma HT1080 cell line were treated with either restriction endonucleases or ultrasonic desintegrator and afterwards subjected to the triple-gradient Nucleoprotein--Celite chromatography. This permitted fractionation of nuclear DNA into fragments not attached, weakly attached, and tightly attached to the nuclear matrix (DNA 0, DNA I, and DNA II, respectively). It was shown that pulse labelled RNA migrates from DNA II fraction where it resides initially to DNA 0 and further to DNA I during the 2 h chase period. This finding allowed us to consider the tight DNA-matrix complex as the replicative one. The experiments aiming to follow the movements of specific DNA sequences (histone genes) in relation to the DNA-matrix attachment sites were conducted on synchronous HT1080 cells progressing through S phase. The histone sequences appeared to undergo similar movements during the first 30 min of S phase. They reside initially in DNA 0 and DNA I fractions, but as soon as DNA synthesis was restored they migrate consequently to DNA II and DNA 0 fractions. This approach can appear to be a useful tool for studying the schedule of replication of specific genes during S phase.