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...

BackgroundGenomic mutations caused by cytotoxic agents used in cancer chemotherapy may cause secondary malignancies as well as contribute to the evolution of treatment-resistant tumour cells. The stable diploid genome of the chicken DT40 lymphoblast cell line, an established DNA repair model system, is well suited to accurately assay genomic mutations.ResultsWe use whole genome sequencing of multiple DT40 clones to determine the mutagenic effect of eight common cytotoxics used for the treatment of millions of patients worldwide. We determine the spontaneous mutagenesis rate at 2.3 × 10–10 per base per cell division and find that cisplatin, cyclophosphamide and etoposide induce extra base substitutions with distinct spectra. After four cycles of exposure, cisplatin induces 0.8 mutations per Mb, equivalent to the median mutational burden in common leukaemias. Cisplatin-induced mutations, including short insertions and deletions, are mainly located at sites of putative intrastrand crosslinks. We find two of the newly defined cisplatin-specific mutation types as causes of the reversion of BRCA2 mutations in emerging cisplatin-resistant tumours or cell clones. Gemcitabine, 5-fluorouracil, hydroxyurea, doxorubicin and paclitaxel have no measurable mutagenic effect. The cisplatin-induced mutation spectrum shows good correlation with cancer mutation signatures attributed to smoking and other sources of guanine-directed base damage.ConclusionThis study provides support for the use of cell line mutagenesis assays to validate or predict the mutagenic effect of environmental and iatrogenic exposures. Our results suggest genetic reversion due to cisplatin-induced mutations as a distinct mechanism for developing resistance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0963-7) contains supplementary material, which is available to authorized users.

Loss-of-function mutations in the BRCA1 and BRCA2 genes increase the risk of cancer. Due to their function in homologous recombination repair, much research has focused on the unstable genomic phenotype of BRCA1/2 mutant cells manifest mainly as large scale rearrangements. We used whole genome sequencing of multiple isogenic chicken DT40 cell clones to precisely determine the consequences of BRCA1/2 loss on all types of genomic mutagenesis. Spontaneous base substitution mutation rates increased seven-fold upon the disruption of either BRCA1 or BRCA2, and the arising mutation spectra showed strong and specific correlation with a mutation signature associated with BRCA1/2 mutant tumours. To model endogenous alkylating damage, we determined the mutation spectrum caused by methyl methanesulfonate (MMS), and showed that MMS also induces more base substitution mutations in BRCA1/2 deficient cells. Spontaneously arising and MMS-induced insertion/deletion mutations and large rearrangements were also more common in BRCA1/2 mutant cells compared to the wild type control. A difference in the short deletion phenotypes of BRCA1 and BRCA2 suggested distinct roles for the two proteins in the processing of DNA lesions, as BRCA2 mutants contained more short deletions, with a wider size distribution, which frequently showed microhomology near the breakpoints resembling repair by non-homologous end joining. An increased and prolonged gamma-H2AX signal in MMS-treated BRCA1/2 cells suggested an aberrant processing of stalled replication forks as the cause of increased mutagenesis. The high rate of base substitution mutagenesis demonstrated by our experiments is likely to significantly contribute to the oncogenic effect of the inactivation of BRCA1 or BRCA2.