The location and development of Replicon Cluster Domains in early replicating DNA

Background: It has been known for many years that in metazoan cells, replication origins are organised into clusters where origins within each cluster fire near-synchronously. Despite clusters being a fundamental organising principle of metazoan DNA replication, the genomic location of origin clusters has not been documented. Methods: We synchronised human U2OS by thymidine block and release followed by L-mimosine block and release to create a population of cells progressing into S phase with a high degree of synchrony. At different times after release into S phase, cells were pulsed with EdU; the EdU-labelled DNA was then pulled down, sequenced and mapped onto the human genome. Results: The early replicating DNA showed features at a range of scales. Wavelet analysis showed that the major feature of the early replicating DNA was at a size of 500 kb, consistent with clusters of replication origins. Over the first two hours of S phase, these Replicon Cluster Domains broadened in width, consistent with their being enlarged by the progression of replication forks at their outer boundaries. The total replication signal associated with each Replicon Cluster Domain varied considerably, and this variation was reproducible and conserved over time. We provide evidence that this variability in replication signal was at least in part caused by Replicon Cluster Domains being activated at different times in different cells in the population. We also provide evidence that adjacent clusters had a statistical preference for being activated in sequence across a group, consistent with the ‘domino’ model of replication focus activation order observed by microscopy. Conclusions: We show that early replicating DNA is organised into Replicon Cluster Domains that behave as expected of replicon clusters observed by DNA fibre analysis. The coordinated activation of different Replicon Cluster Domains can generate the replication timing programme by which the genome is duplicated.</p

Biologic markers of risk in nipple aspirate fluid are associated with residual cancer and tumour size

We previously demonstrated that nipple aspirate fluid (NAF) can be obtained from virtually all non-Asian women between the ages of 30 and 72. The focus of this report is to (1) determine the association of candidate markers of breast cancer risk in NAF obtained from fresh mastectomy specimens with residual breast carcinoma, and (2) evaluate the association of the markers with breast tumour progression. Nipple aspiration was performed on 97 specimens. Cytology, DNA index (including % hypertetraploid cells), cell cycle parameters (S phase fraction, % cells in G2/M), prostate-specific antigen (PSA), epidermal growth factor (EGF), testosterone, carcinoembryonic antigen (CEA) and prostaglandin D synthase (PGDS) were evaluated in NAF for their association with (1) residual ductal carcinoma in situ (DCIS) or invasive cancer, and (2) pathologic tumour size. NAF was obtained from 99% (96/97) of specimens. Atypical and malignant NAF cytology were significantly associated with residual DCIS or invasive cancer (P = 0.001) and with larger tumours (P = 0.004). One hundred per cent and 88% of subjects with malignant and atypical NAF cytology, respectively, had residual carcinoma. The percentage of cells in G2/M and DNA index were associated both with risk of residual carcinoma (P = 0.01 for each) and larger tumour size (DNA index, P = 0.03; G2/M, P = 0.05), although neither biomarker improved the ability of NAF cytology, to predict residual breast cancer. Higher DNA index was associated with atypical cytology (P = 0.0001). In summary, atypical and malignant NAF cytology are associated with larger tumour size, and are highly predictive of residual carcinoma after needle or excisional biopsy of the breast. © 1999 Cancer Research Campaig

The location and development of Replicon Cluster Domains in early replicating DNA

Background: It has been known for many years that in metazoan cells, replication origins are organised into clusters where origins within each cluster fire near-synchronously. Despite clusters being a fundamental organising principle of metazoan DNA replication, the genomic location of origin clusters has not been documented.Methods: We synchronised human U2OS by thymidine block and release followed by L-mimosine block and release to create a population of cells progressing into S phase with a high degree of synchrony. At different times after release into S phase, cells were pulsed with EdU; the EdU-labelled DNA was then pulled down, sequenced and mapped onto the human genome.Results: The early replicating DNA showed features at a range of scales. Wavelet analysis showed that the major feature of the early replicating DNA was at a size of 500 kb, consistent with clusters of replication origins. Over the first two hours of S phase, these Replicon Cluster Domains broadened in width, consistent with their being enlarged by the progression of replication forks at their outer boundaries. The total replication signal associated with each Replicon Cluster Domain varied considerably, and this variation was reproducible and conserved over time. We provide evidence that this variability in replication signal was at least in part caused by Replicon Cluster Domains being activated at different times in different cells in the population. We also provide evidence that adjacent clusters had a statistical preference for being activated in sequence across a group, consistent with the ‘domino’ model of replication focus activation order observed by microscopy.Conclusions: We show that early replicating DNA is organised into Replicon Cluster Domains that behave as expected of replicon clusters observed by DNA fibre analysis. The coordinated activation of different Replicon Cluster Domains can generate the replication timing programme by which the genome is duplicated