We are investigating the replication of chromosomal DNA in proliferating human somatic cells. Our research focuses on the regulation of the initiation step of this process.
Initiation of chromosomal DNA replication in vitro
Before a cell can divide, it needs to replicate its chromosomal DNA. A key regulation step for the cell division cycle, therefore, is the initiation of chromosomal DNA replication at the G1-to-S phase transition. To investigate this at molecular level, we have established a cell-free system from human somatic cells that reconstitutes the initiation of chromosomal DNA replication in the test tube. Template nuclei for DNA replication in vitro are isolated from human cells synchronised in the late G1 phase of the cell division cycle. We routinely use the iron-chelating compound mimosine to reversibly arrest proliferating human cells in late G1 phase. Nuclei from these cells have not initiated DNA replication yet, but their incubation in a cytosolic extract from proliferating cells leads to the establishment of replication forks in these nuclei in vitro. These forks then progress predominantly bi-directionally at rates of about 300 nucleotides per minute in vitro, synthesising chromosomal DNA in a semi-conservative manner.
Identification of essential DNA replication initiation factors
The initiation of chromosomal DNA replication in vitro depends on soluble factors present in the extract from human cells. We have been purifying these soluble factors by biochemical fractionation and reconstitution experiments. In addition to cyclin-dependent protein kinases, we have identified replication protein A (RPA), proliferating cell nuclear antigen (PCNA) and the small non-coding human Y RNAs (hY1, hY3, hY4 and hY5) as essential factors required to reconstitute the initiation of DNA replication in this experimental system. Many more essential factors are still present in the extract, or are associated already with the template nuclei. Therefore, this purification project is ongoing.
To see an overview of the current fractionation scheme, please click here.
Non-coding human Y RNAs as DNA replication initiation factors
Human Y RNAs were originally found as the RNA component of soluble ribonucleoproteins (RNPs), termed Ro RNPs. We have since shown that these small non-coding RNAs are essential factors for reconstituting chromosomal DNA replication in the human cell-free system*. Additionally, their degradation in proliferating human cells by RNA interference results in an inhibition of DNA replication and cell proliferation. Furthermore, Y RNAs are significantly over-expressed in solid human tumours.
The molecular mechanisms by which Y RNAs regulate chromosomal DNA replication and cell proliferation are not yet defined. Our current work aims to elucidate these mechanisms. Using the single-molecule technique of molecular combing, backed-up by bulk nascent strand analysis, we have recently shown that Y RNAs are required for the initiation step of chromosomal DNA replication, but not for DNA chain elongation synthesis. Therefore, Y RNAs are required for the activation of chromosomal DNA replication origins and the establishment of DNA replication forks, but not for their progression.
Y RNAs are conserved in all vertebrates. Using a functional survey of known vertebrate Y RNAs and a systematic mutagenesis of human Y1 RNA, we have identified an evolutionarily conserved short double-stranded functional domain preset in all vertebrate Y RNAs. This domain is essential and sufficient for the initiation-promoting function of Y RNAs in the human cell-free system.
Ongoing work now focuses on the identification of proteins which interact with Y RNAs under the experimental conditions that support DNA replication. This is achieved by a combination of Y RNA affinity chromatography, Western blotting and mass spectrometry. Particular focus is given to the analysis of DNA replication initiation proteins in this context, such as the origin recognition complex ORC and associated components of the pre-replication and initiation complexes. Furthermore, we are interested in learning more about the roles of Y RNAs in vertebrate development; and about the regulation of their expression, intracellular localisation and function.
To see an overview of the nucleotide sequences and predicted structures of the four human Y RNAs (hY1, hY3, hY4 and hY5), please click here.
*This research has been highlighted in a press release issued by Cancer Research UK in September 2006. For access, please click here.
Our current research is funded by the Association for International Cancer Research (AICR). In the past, we have also received support from Cancer Research UK, the Human Frontier Science Program (HFSP) and Amersham Pharmacia Biotech. PhD students in the lab receive, or have received scholarships from the Biotechnology and Biological Sciences Research Council (BBSRC), the Medical Research Council (MRC), the Gates Cambridge Trust and the Boehringer Ingelheim Fonds. Summer students are supported, or have been supported, by Vacation Scholarships from the Wellcome Trust, Cancer Research UK and Undergraduate Research Bursaries from the Nuffield Foundation. More details can be found here. We gratefully acknowledge this support.