FIG. 6.

Human Y RNAs are required for semiconservative DNA replication. Late-G₁-phase nuclei were incubated with S100 cytosolic extract in the presence of BrdU triphosphate and [α-³²P]dCTP for 3 h, and purified DNA reaction products were analyzed by density gradient centrifugation and scintillation counting. (A) Specific degradation of hY RNA inhibits semiconservative DNA replication. Cytosolic extract from proliferating HeLa cells was left untreated, treated with RNase A, or treated with anti-hY1 oligonucleotides. Positions of unsubstituted (light-light [LL]), hemisubstituted (heavy-light [HL]), and fully substituted DNA (heavy-heavy [HH]) are indicated as determined by refractive indices. (B) Reconstitution of semiconservative DNA replication by hY RNA. After depletion of hY1 RNA by anti-hY1 oligonucleotides, the treated extract was supplemented with 100 ng of hY3 RNA synthesized in vitro. Representative results of one out of two independent experiments are shown (note that quantitative comparisons of the raw cpm values should not be drawn between the independent experiments of panels A and B).