Discussion

3.7 Discussion

3.7.1 Cloning of chicken Nse2

The chicken Nse2 cDNA was successfully cloned allowing the generation of anti-chicken Nse2 antibodies, and preliminary biochemical and localisation analysis of the Nse2 protein. In addition, we confirmed that the predicted protein from the G. gallus non-redundant protein sequences database similar to S. cerevisiae MMS21, is indeed a chicken homologue of Nse2. We found that the size and sequence of the chicken Nse2 cDNA are in agreement with information published in the NCBI database. Comparison of Nse2 protein sequences from different species revealed that chicken Nse2 is significantly similar to vertebrate but not to its unicellular homologues. The highest similarity has been observed in the region of Nse2 catalytic domain, which is required for Nse2 SUMO ligase activity. Despite the fact that the overall Nse2 sequence has changed from yeast to human, the sequence required for Nse2 SUMO ligase activity has been conserved throughout evolution. Additionally, the structure of the chicken Nse2 gene is very similar to its human homologue, suggesting that the gene itself is also evolutionarily conserved, at least between chicken and human. This analysis confirmed that we have cloned the chicken homologue of Nse2 protein.

3.7.2 Generation of antisera against chicken Nse2

We have generated an anti-chicken Nse2 (SIE009AP) antibody and used it in immunoblotting experiments. The SIE009AP antibody detects the endogenous Nse2 protein as single band in wild-type DT40 cells, showing that there is only one form of Nse2 protein expressed in these cells. This is consistent

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with the data from human experiments, in which a single form of Nse2 was detected in HeLa cells (Behlke-Steinert et al., 2009; Potts et al., 2006; Taylor et al., 2008). The observed size of chicken Nse2 (27 kDa) is similar to the mass predicted from its amino acid sequence. Additionally, the SIE009AP antibody does not detect any protein in the Nse2-deficient cells, confirming that indeed it is Nse2 that we are detecting in wild-type DT40 cells. Unfortunately, the SIE009AP is not able to detect Nse2 protein in immunofluorescence microscopy experiments; therefore we concluded that SIE009AP can be used only for immunoblotting.

3.7.3 Biochemical properties of myc-tagged Nse2

The cloned Nse2 cDNA was used to generate a 3myc fusion protein to analyse chicken Nse2. The fusion protein has been successfully expressed in Nse2-deficient cells. Human Nse2 co-immunoprecipitates with Smc5, Smc6 and Nse1 (Potts and Yu, 2005; Taylor et al., 2008). We found that 3myc-Nse2 co-immunoprecipitated with Smc5, indicating that as in human cells, chicken Nse2 interacts with Smc5 in vivo. Furthermore, this confirms that the cDNA we have cloned codes for a component of the Smc5-Smc6 complex. The N-terminal 3myc tag on the Nse2 protein does not interfere with its binding to Smc5, suggesting that the fusion is a functional protein in vivo.

In our immunoblot experiments, the anti-myc antibody detects 3myc-Nse2 as a double band. Because it is expressed ectopically from the cDNA sequence it is unlikely that these represent alternative forms of Nse2. Extensive studies in yeast and human models identified post translational modification (PTM) of several components of the Smc5-Smc6 complex; Smc6 phosphorylation and Smc5, Smc6, Nse2, Nse3 and Nse4 SUMOylation (Andrews et al., 2005; Pebernard et al., 2004; Potts and Yu, 2005; Taylor et al., 2008; Taylor et al., 2001; Zhao and Blobel, 2005). The SUMO peptide is highly charged and its addition to a target protein results in an increase of observed molecular mass by 10-20 kDa. We hypothesised that the extra band may be a phospho-form of Nse2. However, phosphatase treatment did not abolish presence of the slower migrating form of 3myc-Nse2. Therefore, we concluded that the extra band is not a phosphorylated form of Nse2. In addition, the SIE009

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antibody detects a double band of 3myc-Nse2 but not of the endogenous protein.

This raises two possibilities; (1) myc tag is part of the protein being modified and (2) we do not detect modification of endogenous Nse2, because of its low abundance compared to over-expressed 3myc-Nse2. We believe that it is rather unlikely that the myc tag is specifically modified, as no such PTM has been reported in the literature. Therefore, we concluded that the extra band that we detected is a yet unidentified PTM of over-expressed chicken Nse2.

3.7.4 Localisation of chicken Nse2

We expressed an Nse2-GFP fusion in Nse2-deficient cells and used it to study the localisation of Nse2. Experiments in human and yeast cells have shown a nuclear localisation of Nse2 (Potts and Yu, 2007; Zhao and Blobel, 2005). We observed both cytoplasmic and nuclear localisation of the Nse2-GFP. In interphase cells, Nse2-GFP co-localised with the DNA signal but not in mitotic cells, suggesting a cell cycle-dependent localisation of Nse2. We also detected a centrosomal localisation of the Nse2 protein, which has not been previously reported. To confirm our observation we performed a set of experiments which suggest that this localisation of Nse2-GFP may be an artefact of paraformaldehyde fixation. We believe that the relatively abundant Nse2-GFP protein can be detained at the centrosome through pareformaldehyde cross-linking activity. In addition, we have not detected 3myc-Nse2 at the centrosome.

However, we cannot rule out the possibility that Nse2 is present at the centrosomes, but that our microscopy experiments performed failed to reveal such localisation. We have observed 9myc-Smc5 and Smc6-GFP localisation at centrosomes, suggesting that Nse2 might be present at this organelle (Stephan, 2007). Recently, the human cohesin complex has been found on the centrosomes (Guan et al., 2008). The Smc5-Smc6 complex co-localises with cohesin at chromosomes and has been reported to be required for cohesin recruitment to double strand breaks (Lindroos et al., 2006; Potts et al., 2006). In addition, both complexes share a similar chromatin loading mechanism which relies on interaction with the Scc2/Scc4 heterodimer (Lindroos et al., 2006; Michaelis et al., 1997). This suggests that, similar to the cohesin complex, Smc5-Smc6 complex may be a component of the centrosome.

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Chapter 4 Generation and characterisation of