Abstract
DNA combing allows the investigation of DNA replication on genomic single DNA molecules, but the lengths that can be analysed have been restricted to molecules of 200–500 kb. We have improved the DNA combing procedure so that DNA molecules can be analysed up to the length of entire chromosomes in fission yeast and up to 12 Mb fragments in human cells. Combing multi-Mb-scale DNA molecules revealed previously undetected origin clusters in fission yeast and shows that in human cells replication origins fire stochastically forming clusters of fired origins with an average size of 370 kb. We estimate that a single human cell forms around 3200 clusters at mid S-phase and fires approximately 100,000 origins to complete genome duplication. The procedure presented here will be adaptable to other organisms and experimental conditions.
Highlights
DNA replication initiates at origins of replication located at specific sites along chromosomes and cells utilise a subset of available origins to complete genome duplication[13]
We used DNA prepared from fission yeast to develop improvements in the DNA combing procedure that can extend the length of molecules that can be analysed
To improve the effectiveness of DNA combing method we considered the factors that could influence the mechanical stability of double-stranded DNA molecules such as the ionic strength and pH of the combing buffer, the temperature, the base composition and the pulling velocities exerted on the DNA molecule[25,26,27,28]
Summary
DNA replication initiates at origins of replication located at specific sites along chromosomes and cells utilise a subset of available origins to complete genome duplication[13]. DNA combing has been used to investigate the pattern of replication origin firing and replication fork velocities on genomic single DNA molecules[4,10,16,17,18] This reveals cell-to-cell differences in origin usage important for understanding how genomes are replicated during S-phase. We report a modified DNA combing protocol that allows the analysis of DNA replication on single DNA molecules up to 12 Mb in length We have developed this methodology in the fission yeast Schizosaccharomyces pombe where we can analyse molecules whose length average is 2 Mb with some molecules occasionally reaching 5.6 Mb, allowing the entire length of all three fission yeast chromosomes to be investigated. Our findings suggest that the spatial organisation of fired origins is similar in different eukaryotes
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