Modelo taxonómico de Anderson y Krathwohl

Interphase egg extract from X. laevis can recapitulate a number of cellular events, including replication, DNA repair, checkpoint control and chromatin remodelling and has been therefore widely used to study molecular principles of these processes. In particular, DNA-induced ATM signalling can be reconstituted in interphase extracts depleted of the endogenous xlMRN complex and supplemented with the wild-type recombinant human complex (Costanzo et al. 2001). This experimental set-up could be employed to monitor ATM signalling in the presence of the mutant hMRN complex and determine the link between structural features of the complex and its function as the signalling mediator.

4.5.1. Preparation of the recombinant hMRN

Expression and purification of the full-length recombinant human MRN complex was first reported in 1999 (Paull and Gellert 1999). This protocol, with minor modifications, was used to prepare the hMRN complex in this work. In contrast to the original protocol, the complex carried only one polyhistidine tag at the C-terminus of Rad50. It was shown though, that this modification did not affect complex formation and purification. Also, hMRN was expressed in HighFive insect cells for 72 hours, rather than in Sf9 insect cells for 48 hours and the cells were infected with a single baculovirus carrying all three genes of the MRN complex. In the original work cells were co-infected with three separate baculoviruses. Again, it was shown that this modification did not alter the expression level or the behaviour of the complex during purification.

The complex was first purified on NiNTA resin. High DNA contamination could be easily removed during the ion exchange step and finally the complex was subjected to gel filtration on Superose 6 column. As shown in the gel filtration elution profile (Figure 30), the complex formed a number of species. This result was contradictory to the previously published study where the authors claimed to obtain a complex of approximately 1.2 MDa (Paull and Gellert 1999). Furthermore, the stoichiometry of the complex was clearly disturbed, with Nbs1 being underexpressed relative to Mre11 and Rad50 (Figure 30).

Results

71

Figure 30.Elution profile of the recombinant human full-length MRN complex in gel filtration. The complex forms several oligomeric species. Nbs1 is at substoichiometric level.

Attempts to obtain a more homogenous sample with less impurities and an elevated Nbs1 level failed. These included expression of the complex in HighFive cells co-infected with separate Mre11/Rad50- and Nbs1-baculoviruses at a high excess of the latter one. Also, placing the polyhistidine tag at the C-terminus of Nbs1 instead of Rad50 or Mre11 did not improve the purity and stoichiometry of the complex.

4.5.2. Interphase X. laevis egg extracts

X. laevis egg extract suitable for studies of mitotic DSBs should be blocked at the early stage of interphase, preferably in phase G1. Such extracts are still able to proceed to S- and G2-phases of the cell cycle. In order to test whether extracts were indeed halted in G1 phase, demembrated sperm nuclei were introduced to the extract. Responsive extracts (i. e. G1-phase extracts of good quality) should in this case recapitulate replication. As shown in Figure 31, extract prepared in this work were fully capable of restoring replication on sperm DNA, as monitored by incorporation of the Cy3-labelled dCTP in the newly synthesized DNA.

Results

72

Figure 31. Replication of demembrated sperm nuclei in X. laevis egg extract. Chromatin in sperm nuclei changes from compact (A) towards a more loose and relaxed form (B and C). Incorporation of the Cy3-dCTP into the newly synthesized DNA is well visible after 60 min upon the nuclei addition.

4.5.3. DNA-induced ATM activation in interphase X. laevis egg extracts

In order to monitor checkpoint signalling elicited by hMRN mutants, interphase extract from Xenopus laevis unfertilized eggs was used. In the assay, the extract was first depleted of the endogenous cellular MRN complex with the anti-xlMre11 polyclonal serum. The efficiency of the immunodepletion was very high, as monitored by Western blot analysis (Figure 32).

Figure 32. Immunodepletion of the endogenous MRN complex from X. laevis egg extract.

Immunodepletion efficiency with polyclonal anti-xlMre11 serum is very high as monitored by a Western blot analysis performed against xlMre11. ∆: depleted extract, M: mock extract.

DNA damage was mimicked by the introduction of exogenous blunt-ended DNA to the extract and the signalling response was monitored by the appearance of the phopshorylated species of ATM (autophosphorylation site S1981). Optimization of this experimental setup included type of DNA to induce the response (poly(A)/poly(T) duplex, phage λ DNA-BstEII digest, 200 bp blunt- ended PCR product), DNA concentration in the extract, reaction time and reaction temperature. In established conditions, a clear phosphorylation of the ATM was detected in mock extract, whereas no ATM phosphorylation could be visible in the xlMRN-depleted extract (Figure 33). The response was very fast, with a partial ATM-phosphorylation already detectable after 2 minutes upon stimulation with DNA.

Results

73

Figure 33. ATM phosphorylation in X. laevis egg extract. DNA-induced ATM phosphorylation is visible only in mock extract (M), but not in xlMRN-immunodepleted extract (∆). ATM is also not phosphorylated in extract not stimulated with DNA.