Techniques used in the Nouspikel lab

We use a whole range of classical laboratory techniques:

Cell biology: cell culture, transfection
Molecular biology: DNA/RNA purification, cloning, PCR, siRNA.
Proteomics: Western blots, 2D gels, protein purification.

But we also use a few specialized techniques to study DNA repair, which are briefly described below.

Global genome repair (GGR) of UV-induced lesions
GGR of benzopyrene chemical adducts
Gene specific repair of UV-induced lesions (TCR and DAR)
In-vitro assay for cisplatin crosslinks

Click on any picture to enlarge it.

Global repair of UV-induced lesions

UV light causes two types of DNA lesions: cyclobutane-pyrimidine dimers (a.k.a. CPDs) and (6-4)pyrimidine-pyrimidone photoproducts (a.k.a (6-4)PPs). Both are characterized by the formation of illegitimate bonds between two pyrimidines adjacent on the same DNA strand.

We can generate these lesions very easily by briefly irradiating cells with a germicidal lamp. The big advantage of this technique over chemical agents is that we can control very precisely the amount of damage and the moment when it is introduced.

We can quickly and easily measure the amount of each lesion in bulk genomic DNA thanks to antibodies produced by Dr Toshio Mori, which specifically recognize CPDs or (6-4)PPs. DNA is simply blotted on a membrane, and the amount of lesions is measured in a "western blot" type of experiment.

Alternatively, we can use an assay relying on the property of the phage enzyme T4 endonuclease V (TEV) to nick the damaged strand in proximity of a CPD. We simply digest DNA with TEV, denature it to separate the strands, and measure the size of the fragments (for instance with sucrose gradients). The more lesions, the smaller the fragments.

Example

Removal of (6-4)PPs from hNT neurons (in red), compared with their precursor cells (NT2, in blue), measured with the antibody assay. Click to enlarge.

For more details:
Terminally differentiated human neurons repair transcribed genes but display attenuated global DNA repair Nouspikel,T. & Hanawalt,P.C. Mol. Cel. Biol. 20:1562-1570 (2000) (download pdf file 138 K)

Global repair of BPDE adducts

Benzopyrenes are chemicals present in cigarette smoke, which forms benzo[a]pyrene diol-epoxide (BPDE) adducts on purines. To reveal the lesions, we use an assay developed by Dr Dave Philips, in which DNA is digested to the nucleotide level with nucleases. The presence of a BPDE adduct inhibits the nuclease and results in the formation of di-nucleotides, which can be post-labeled with 32P and revealed by 2-dimensional thin layer chromatography.

Example

Repair of BPDE adducts in NT2 precursors (blue) and hNT neurons (red). The initial dose of BPDE is comparable to what is found in the blood of a heavy smoker...

For more details:
p53-dependent global genomic repair of benzo[a]pyrene-7,8-diol-9,10-epoxide adducts in human cells. Lloyd,D.R. & Hanawalt, P.C. Cancer Res. 60:517-521 (2000)

TCR and DAR assay

Transcription-coupled repair (TCR) and differentiation-associated repair (DAR) are much more difficult to measure, because we need to work at the level of a single gene. These experiments are time-consuming (about 2 weeks) and require a lot of DNA (50 ug per condition). The assay, developed in the 80s in the Hanawalt lab, is basically a Southern blot with a couple of twists.

First of all, in addition to the required restriction enzyme, DNA is also treated with TEV, which will chop any restriction fragment containing a CPD. Thus, on the final autoradiogram, only those fragments that did not contain any CPD will appear as a nice band. By quantifying this band, and by knowing the initial amount of fragments (which we get from a mock-digested control), we can deduce the amount of CPDs by using the Poisson expression:

Amount of lesions = -ln(intact fragments/total fragments)

The other trick is to use RNA probes so as to reveal one strand at a time. This allows us to visualize differences in repair between the transcribed strand and the non-transcribed strand. Typically, TCR will cause a strand bias in an active gene, with the TS being repaired faster than the NTS. DAR is defined as the proficient repair of the NTS in cells that have little or no global repair of CPDs.

Example

TCR and DAR in the cMyc gene in HL60 cells.

For more details
Determination of damage and repair in specific DNA sequences. Spivak,G. & Hanawalt,P.C. Methods 7:147-161 (1995)

In-vitro assay for cisplatin crosslinks

This assay was developed by the laboratory of Rick Wood. In its most basic version, a plasmid is build, which contains an unique cisplatin crosslink in the vicinity of a 32P label. The plasmid is then incubated with whole cell extract, prepared according to Manley et al. If NER occurs, it will excise a short (around 29 nt) oligonucleotide containing both the lesion and the label. These excision fragments are very specific and can be revealed on a sequencing gel.

Repair-deficient extracts can then be complemented with various protein fractions, to try and identify the cause of the deficiency.

For more details
Mammalian DNA nucleotide excision repair reconstituted with purified protein components. Aboussekhra,A. et al. Cell 80:859-868 (1995)

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Research interests
Thierry Nouspikel's CV