Combing DNA from in vitro Replication Experiments

As discussed earlier, CDK deregulation induces DNA replication stress (Section 1.7). The results described in Chapter 3 demonstrated that in in vitro replication models Ciz1-N471 can both broaden and amplify the DNA replication initiation activity of recombinant cyclin A/CDK2 (Figure 3.7; 3.9). This activity perturbation could constitute CDK activity deregulation if it acts the same in vivo allowing cells to enter S phase at canonically non-permissive kinase levels it could induce DNA replicative stress. Combining cell-free DNA replication assays and DNA combing by repeating cyclin A/CDK2 titration experiments and investigating the effects of replication at non-permissive kinase levels could help identify whether Ciz1 can induce replication stress, which could help to explain its apparent oncogenic function.

Combining cell-free experiments using replication competent nuclei from quiescent release synchronised cells could be a powerful tool for investigating replication stress in mammalian replication. Replication fork deceleration and replication fork stalling are the definition of replication stress (Zeman & Cimprich., 2014). To be able to measure this directly is key in identifying factors that induce stress. To test if DNA from nuclei harvested post quiescence release cell-free replication assays were prepared as in chapter 3. DNA was purified from 20µl of isolated nuclei, DNA was combed onto silanized coverslips and stained with YOYO1 (Figure 4.12).

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DNA combed from in vitro replication assays and stained with YOYO1 consistently resulted in short fragmented stretches of DNA, typically shorter than 20 µm, this is displayed in figure 4.8. DNA of this quality could not be used for analysis of DNA replication. To successfully use in

vitro replication experiments in DNA combing the technique will need to be modified to

ensure that DNA purified is of a high enough quality to get enough data to measure replication dynamics. It is not clear why DNA from post quiescent nuclei would produce fragmented DNA, this could be due to DNA damage introduced during the nuclei isolation, or storage.

4.5 Chapter Discussion

DNA combing is a powerful tool for the study of DNA replication origin usage, fork rates and for investigating replicative stress. Here, the basic steps in establishing the technique were completed and a rudimental analysis of DNA fork progression rate determined. In addition, measurements of replication fork stalling, and initial attempts to combine post-quiescence in

vitro replication method and DNA combing.

We have established DNA combing as a tool for the investigation of DNA replication dynamics we can begin to obtain quantitative data to study DNA replication stress in the future. Here the replication fork speed of NIH3T3 cells was estimated using DNA combing to be 1.31 kbp/min. This seems a plausible replication fork speed with other studies providing a fork rate between 1-2.1 kbp/min, other replication fork rates seem to vary around this amount.

Figure 4.12 –Combing DNA from in vitro Replication Experiments: Combed genomic DNA purified from nuclei isolated from NIH3T3 cells and stained with YOYO1 (green).

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Hamster V79 fibroblast cells display a fork speed between around 1.6 kbp/min and 2.1 kbp/min (Wilhelm et al., 2016) and HeLa cell replication fork speed varies from less than 1 kbp/min through to 2 kbp/min (Técher et al., 2013). This highlights that this replication fork speed is plausible, but also highlights the high variability in replication fork speed even within the same cell line, meaning comparing specific fork speed results should only be done within experiments, to ensure meaningful results.

The analysis here has demonstrated that determination of fork progression fits with observed rates of other cell lines and species (Table 4.1). The results generated to date suggest that the IOD in an unperturbed system has guided few measurable inter-origin distances. DNA fibres are typically too short, and only a few IOD are detected. The technique should be modified to yield longer DNA fibres.

The DNA is likely to be sheared after it has been released into buffered solution due to low forces applied during movement of the solution. Modification of the procedure can be performed in several ways to reduce denaturation of DNA and DNA shearing. These included reducing movement between release of DNA from agarose plugs by melting plugs directly into the Teflon blocks, altering NaCl concentrations of DNA buffer solution and using a combing apparatus with lower friction. Each method saw a large increase in the length of DNA

visualised by combing (Kaykov et al., 2016). Modifying the technique could allow for combing of longer DNA strands to determine more parameters of replication dynamics. Similar

approaches can be trialled in the system used in this study to increase fibre length in the future. These modifications should also be used when attempting to increase DNA fibre length from in vitro DNA replication experiments.

Once the technique for combing of DNA from post quiescence in vitro replication experiments has been developed, it could be used to investigate replication stress in this system. An advantage of cell-free replication experiments is that it allows for incorporation of modified

Species Mean Replication Fork Speed

(kb/min)

Reference Human Primary Normal

Keratinocytes

1.46 (Conti et al., 2007)

Hamster V-79 Cells 2.09 (Willhelm et al., 2016)

Saccharomyces cerevisiae 2.9 (median 2.3 (Raghuraman et al., 2001)

Schizosaccharomyces pombe 2.8 (Heichinger et al., 2006)

HeLa cells 1-2 ( Técher et al., 2013)

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nucleotides that have large tags such as biotin-16-dUTP and digoxigenin-11-dUTP. This allows for detection without denaturation of DNA, by counterstaining dsDNA with Yoyo1, reducing the number of antibody incubations, saving time. It would also be of interest to investigate replication dynamics in isolated nuclei from mitotically cycling cell compared to post- quiescent release nuclei.

There are a number of way nuclei could be duel labelled, Marheineke et al. (2005) duel labelled DNA from late G1 nuclei arrested by mimosine using biotin-16-dUTP and digoxygenin- dUTP and detected using fluorescently tagged avidins for biotin-16-dUTP and using

fluorescent antibodies to detect digoxygenin dUTP. Another modified nucleotide often used to measure DNA replication is EdU; EdU was used in chapter 3 to monitor number of cells entering S phase, EdU can be fluorescently labelled using click it chemistry. EdU has been used in DNA combing experiments as a substitute for BrdU to avoid the DNA denaturation stage (Bianco et al., 2012). Due to no DNA denaturation being required for any of these detection methods DNA can be counterstained with YOYO1 or another fluorescent dsDNA binding fluorophore which would save significant amounts of time.

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