We describe a cell-free system from HeLa cells that initiates DNA replication under cell cycle control. G1 but not G2 phase nuclei initiate replication when coincubated with S phase nuclei in cytosolic extracts from S phase but not from G1 or G2 phase HeLa cells. S phase nuclei or an S phase nuclear extract are required for the initiation of semiconservative DNA replication in G1 nuclei but not for elongation in S phase nuclei. S phase nuclear extract could be replaced by recombinant human cyclins A and E complexed to Cdk2 but not by Cdk2 alone or by human cyclin B1 complexed to Cdc2. In S phase cytosol, cyclins A/Cdk2 and E/Cdk2 triggered initiation synergistically.

Noncoding RNAs are recognized increasingly as important regulators of fundamental biological processes, such as gene expression and development, in eukaryotes. We report here the identification and functional characterization of the small noncoding human Y RNAs (hY RNAs) as novel factors for chromosomal DNA replication in a human cell-free system. In addition to protein fractions, hY RNAs are essential for the establishment of active chromosomal DNA replication forks in template nuclei isolated from late-G 1 -phase human cells. Specific degradation of hY RNAs leads to the inhibition of semiconservative DNA replication in late-G 1 -phase template nuclei. This inhibition is negated by resupplementation of hY RNAs. All four hY RNAs (hY1, hY3, hY4, and hY5) can functionally substitute for each other in this system. Mutagenesis of hY1 RNA showed that the binding site for Ro60 protein, which is required for Ro RNP assembly, is not essential for DNA replication. Degradation of hY1 RNA in asynchronously proliferating HeLa cells by RNA interference reduced the percentages of cells incorporating bromodeoxyuridine in vivo. These experiments implicate a functional role for hY RNAs in human chromosomal DNA replication.In recent years, it has become apparent that noncoding RNAs are regulating many biological processes, from gene expression and chromatin dynamics to complex developmental programs (reviewed in references 2, 26, and 35). A fundamental process for which an involvement of noncoding RNAs has not been reported to date is the replication of chromosomal DNA in eukaryotes.Chromosomal DNA replication is initiated at the G 1 -to-S phase transition of the cell division cycle. Regulators for this transition have been identified genetically and biochemically as proteins that interact with chromosomal DNA replication origins during G 1 phase, directing the stepwise formation of preinitiation complexes (reviewed in references 1, 13, 25, 33, and 39). These protein factors are functionally conserved through evolution. The six-protein subunit origin recognition complex is assembled on origin DNA, from which Cdc6 and Cdt1 proteins recruit six minichromosome maintenance proteins (MCM2 to MCM7) to form a prereplicative complex, or replication license, in G 1 phase. Conversion of this complex into active replication forks marks the entry into S phase, which is under the temporal and spatial control of S-phase cyclin-dependent kinase Cdk2 and Dbf4-dependent kinase Cdc7. Additional initiation proteins, including MCM10, Cdc45, GINS complex, Mus101 (Dbp11 and Cut5 in yeasts), and replication protein A (RPA) are recruited in this process to unwind origin DNA (1, 25, 39). Active DNA replication forks are established from there by the stepwise recruitment of DNA polymerase ␣/primase and the replicative DNA polymerases ␦ and ε, together with replication factor C and proliferating nuclear antigen (PCNA). This elaborate pathway has been worked out predominantly in the model systems of amphibian egg extracts and unicellular yeasts; later stage...

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Graphical abstract

Primases synthesise the RNA primers that are necessary for replication of the parental DNA strands. Here we report that the heterodimeric archaeal/eukaryotic primase is an iron-sulfur (Fe-S) protein. Binding of the Fe-S cluster is mediated by an evolutionarily conserved domain at the C terminus of the large subunit. We further show that the Fe-S domain is essential to the unique ability of the eukaryotic primase to start DNA replication.De novo synthesis of RNA primers by primases is essential for cellular and viral DNA replication1,2. Archaeal and eukaryotic primases are heterodimeric enzymes with a small (PriS) and a large (PriL) subunit2. Although the catalytic activity resides within PriS, the PriL subunit is necessary to primase function as disruption of the PriL gene in yeast is lethal3. Reported roles for PriL include stabilisation of PriS, participation in initiation of RNA primer synthesis, determination of product size and transfer of the primer to DNA polymerase α4-11. A recent crystallographic model of the heterodimeric primase from the archaeon Sulfolobus solfataricus provided the first description of the large subunit but did not include its C-terminal domain (PriL-CTD)12. The presence of four conserved cysteines in archaeal and eukaryotic PriL-CTD sequences suggests that the PriL-CTD might be a metal-binding domain (Supplementary Figure 1).We set out to characterise the biochemical and biophysical properties of the PriL-CTD. Freshly purified samples of S. solfataricus PriL-CTD expressed in bacteria as glutathione Stransferase (GST) fusion protein consistently displayed a yellow-brown colour, which turned darker upon concentration of the sample (Supplementary Figure 2). The absorption spectrum of the S. solfataricus GST-PriLCTD showed a broad shoulder around 400 nanometers (nm), next to the expected protein peak at 280 nm (Figure 1a). Fading of the colour under aerobic conditions and a decrease in absorption at 400 nm over time indicated the presence of a chromophore in the PriL-CTD, which is sensitive to air oxidation. Purified Saccharomyces cerevisiae PriL-CTD fused to a maltose-binding protein (MBP) displayed a similar colour and increased absorption at 400 nm as the S. solfataricus GST-PriLCTD (Supplementary * Correspondence (luca@cryst.bioc.cam.ac.uk). Author Contributions SK and LP conceived the project and designed the experiments; SK prepared the recombinant proteins and performed the biochemical experiments; JH performed the EPR analysis; JDM first suggested that the chromophore in the PriL-CTD might be a Fe-S cluster and performed the CD analysis; TK assisted with the primase assays; SK and LP interpreted the data and wrote the paper.

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Noncoding Y RNAs have recently been identified as essential factors for chromosomal DNA replication in human cell nuclei. Here, we investigate the expression of human Y RNAs in tumours and test their requirement for cell proliferation. Relative expression levels of all four human Y RNAs (hY1, hY3, hY4 and hY5 RNA) were determined by quantitative RT -PCR in extracts from human solid tumours, corresponding nonmalignant normal tissues and derived cultured cells. On average, all four hY RNAs are significantly overexpressed in solid tumours between 4-and 13-fold, compared to the corresponding normal tissues. In particular, hY1 and hY3 RNAs are overexpressed in carcinomas (and adenocarcinomas) of the bladder, cervix, colon, kidney, lung and prostate with extremely high statistical significance (ANOVA, between groups, Po10e-22). A functional requirement of all four hY RNAs for cell proliferation was investigated in a systematic survey for loss-of-function by RNA interference (RNAi). Degradation of hY1 and hY3 RNAs in human cell lines resulted in a significant cytostatic inhibition of cell proliferation. We conclude that noncoding hY RNAs have potential both as new cancer biomarkers and as molecular targets for anti-proliferative intervention.

Noncoding Y RNAs are required for the reconstitution of chromosomal DNA replication in late G1 phase template nuclei in a human cell-free system. Y RNA genes are present in all vertebrates and in some isolated nonvertebrates, but the conservation of Y RNA function and key determinants for its function are unknown. Here, we identify a determinant of Y RNA function in DNA replication, which is conserved throughout vertebrate evolution. Vertebrate Y RNAs are able to reconstitute chromosomal DNA replication in the human cell-free DNA replication system, but nonvertebrate Y RNAs are not. A conserved nucleotide sequence motif in the double-stranded stem of vertebrate Y RNAs correlates with Y RNA function. A functional screen of human Y1 RNA mutants identified this conserved motif as an essential determinant for reconstituting DNA replication in vitro. Double-stranded RNA oligonucleotides comprising this RNA motif are sufficient to reconstitute DNA replication, but corresponding DNA or random sequence RNA oligonucleotides are not. In intact cells, wild-type hY1 or the conserved RNA duplex can rescue an inhibition of DNA replication after RNA interference against hY3 RNA. Therefore, we have identified a new RNA motif that is conserved in vertebrate Y RNA evolution, and essential and sufficient for Y RNA function in human chromosomal DNA replication.