FIG. 5.

Degradation of hY RNAs in vitro inhibits chromosomal DNA replication in late-G₁-phase template nuclei. n, number of experiments. (A) Specific degradation of hY RNA in vitro. S100 cytosolic extract from proliferating HeLa cells was treated with RNase A or with the DNA antisense oligonucleotides complementary to single-stranded domains of the hY RNAs as shown in Fig. 3. As a negative control, the standard bacteriophage T3 DNA sequencing primer was used. The proportions of the indicated relative RNA amounts remaining in the extract after the treatment were determined by quantitative RT-PCR, using 5S rRNA as the reference. (B) Y RNA degradation reduces the proportion of nuclei replicating in vitro. Late-G₁-phase template nuclei were incubated in untreated and treated S100 cytosolic extract as indicated. Proportions of replicating nuclei were determined as described in the legend to Fig. 1. (C) Y RNA degradation reduces the amount of extract-dependent DNA synthesis in late-G₁-phase nuclei in vitro. Nuclei were incubated in untreated and treated S100 cytosolic extract in the presence of [α-³²P]dCTP as indicated. The incorporation of dNMPs into nascent chromosomal DNA under these conditions was quantitated by precipitation with trichloroacetic acid and scintillation counting. (D) Functional substitution of one depleted hY RNA in the extract by a different hY RNA. After depletion of either hY1 or hY3 RNA by antisense DNA oligonucleotides, the treated extract was supplemented with 100 ng of the indicated hY RNAs synthesized in vitro. Proportions of replicating nuclei were determined as described in the legend to Fig. 1.