Generation of Nse2 -/-/- Smc5 - knockout cells

Our analysis of the Smc5-Smc6 complex by characterisation of Nse2-deficient cells (this study) and Smc5 knockouts (Stephan et al., 2011a) revealed distinct phenotypes (Table 5.1). We observed different responses of Nse2- and Smc5-deficient cells towards IR and MMS. Chromosomal aberrations are increased after IR in Smc5^- cells but not in Nse2^-/-/- cells. Conversely, Nse2^-/-/- but not Smc5-deficient cells show elevated chromosomal aberrations after MMS.

Loss of Smc5 results in depletion of Smc6 and Nse2, whereas disruption of Nse2 leads only to loss of Smc5 but not Smc6.

Fission yeast smc6.T2nse2.SA double mutants show slower growth kinetics and budding yeast smc6-9mms21-sp demonstrate increased sensitivity towards UV-induced DNA damage than either of single mutants (Andrews et al., 2005; Chavez et al., 2010b). Hazbun et al. have identified two different Smc5-Smc6 complexes and recently, two complexes, Nse2-Smc5-Smc5-Smc6 in interphase and Nse2-Smc5 in mitotic cells, were detected by gel filtration in human cells (Behlke-Steinert et al., 2009; Hazbun et al., 2003). In vitro studies with recombinant Smc5 protein showed its preferential binding to single stranded DNA, independently of its Smc partner Smc6 (Roy et al., 2011). These data suggest that components of the Smc5-Smc6 complex may have separable functions. Taking these observations into consideration, we hypothesised that the differences observed between Nse2- and Smc5-deficient cells are due to (1) some non-overlapping functions of Nse2 and Smc5 proteins, (2) defective regulation of Smc5-Smc6 the in absence of Nse2 SUMO ligase, (3) or that different Smc5- and Nse2- containing subcomplexes exist that have distinct functions. As shown in Table 5.1, chromosomal aberrations seem to be specifically Nse2-dependent and IR sensitivity is only observed in the absence of Smc5 supporting the first hypothesis.

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Table 5.1 Comparison of Smc5^- and Nse2^-/-/- phenotypes

Smc5^- cells* Nse2^-/-/- cells**

Viability Viable Viable

Growth rate Wild-type Wild-type

Mitotic index/aberrations Higher than wild-type Higher than wild-type Destabilisation of Smc5-Smc6

Chromosomal aberrations Increased after IR but not MMS

Increased after MMS and 4-NQO

Sister chromatid cohesion

↑

Targeting assay frequencies Wild-type Slightly increased Sister chromatid exchange

Smc5 mutants showed more severe phenotypes than the Nse2^-/-/- cells in most of the experiments. Assuming that an Smc5-Smc6 heterodimer is still formed within the Nse2 mutants (but not in Smc5 background) and if this

Smc5-172

Smc6 complex lacks Nse2-mediated SUMOylation, the more severe phenotypes support the idea that the observed phenotypic differences between Nse2- and Smc5-deficient cells are due to mis-regulation of the Smc5-Smc6 complex. To investigate this notion, we disrupted the Smc5 gene in Nse2 background. We hypothesised that if Smc5 and Nse2 proteins have separate functions, the loss of Smc5 protein in the Nse2-deficient cells would result in a phenotype stronger that that observed in Smc5^- cells.

The targeting strategy and vectors for Smc5 disruption were as described (Stephan et al., 2011a). Nse2^-/-/- cells were transfected with an Smc5 targeting vector to disrupt Smc5 (Stephan et al., 2011a). We successfully targeted Smc5 in the Nse2^-/-/- background and obtained several viable clones (Figure 5.1).

Figure 5.1 Southern blot analysis of the Nse2^-/-/-Smc5^- mutants.

Genomic DNA was isolated from wild-type and Nse2^-/-/-Smc5^- cells and digested with EcoRI (Smc5 probe), XmnI (Nse2 probe) and analysed by Southern blotting with the indicated probes.

As expected, the Smc5 probe detects replacement of the wild-type band (6.9 kb) with a targeted band of 4.9 kb (Figure 5.1). We also looked at the Nse2 locus with the Nse2 probe. The 6.3 kb wild-type band was absent in the double mutant and it had been replaced by two 14.7 kb (double intensity) and 5.8 kb (single intensity) mutant bands, showing that we have targeted Smc5 in the Nse2-deficient cells (Figure 5.1).

Next, we isolated total mRNA from the Nse2^-/-/-Smc5^- cells and tested whether the mRNA of Nse2 and Smc5 genes was still present in these cells (Figure 5.2). Reverse transcriptase PCR revealed that there is no Nse2 and Smc5

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mRNA present in the double knockout cells. The Smc5 and Nse2 deficient cells were used here as negative controls.

Figure 5.2 Analysis of Smc5 and Nse2 mRNA expression in Nse2^-/-/-Smc5^- cells.

Reverse transcriptase-polymerase chain reaction with total mRNA isolated from the wild-type, Smc5^--, Nse2^-/-/--, and two Nse2^-/-/-Smc5^--targeted clones and the indicated gene specific primers.

We then investigated the levels of Smc5 and Nse2 proteins to confirm their absence in these cells (Figure 5.3). Immunoblotting revealed depletion of Smc5 and Nse2 proteins in the double knockout background.

Figure 5.3 Immunoblot analysis of Smc complexes Nse2^-/-/-Smc5^-.

Proteins from wild-type, Nse2-, Smc5-deficient and Nse2^-/-/-Smc5^- cells were extracted, separated by SDS-PAGE and analysed by immunoblotting with the indicated antibodies.

Our previous analysis had shown that loss of the Smc5 results in depletion of Smc6 and Nse2, whereas disruption of Nse2 proteins leads to reduction of only Smc5 protein (Stephan et al., 2011a). We found that the loss of Nse2 and Smc5

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results in depletion of Smc6 to levels similar to those observed in Smc5-deficient cells. The levels of cohesin and condensin complexes did not change upon disruption of Nse2 and Smc5, indicating that only Smc5-Smc6, but not other Smc complexes, is destabilised in the absence of Nse2 and Smc5. The lack of Nse2 and Smc5 mRNAs and respective proteins in the Nse2^-/-/-Smc5^- cells further confirms that the Smc5-Smc6 complex is not essential for chicken cell viability.

5.1.2 Nse2

Smc5

cells show growth retardation and mitotic