Host cell reactivation assessment of XPG mutants

Utilizing the UDS (see chapter 3.5.2) assay, I was able to investigate the immediate DNA synthesis after irradiation in dependency of multiple XPG mutants. However, I further analyzed how these mutants complement the XPG deficiency in XP20BE fibroblast cells via transient overexpression and performance of the host cell reactivation (HCR) assay three days after transfection (see Figure 23) (see chapter 3.5.1).

Figure 23: Host cell reactivation of XP20BE cells by XPG mutants

The black bars indicate the relative NER capacity of an UV irradiated Firefly reportergene plasmid compared to the unirradiated expression level (normalized to the cotransfected Renilla luciferase, see chapter 3.5.1) in dependency of various XPG mutants, three days after transfection. The error bars are calculated using error propagation of the mean values of normalized Firefly, either irradiated or not, due to statistical standard techniques. Significance levels are calculated using the Student’s T-Test (comparison of two groups). The small fat vertical lines, in the left of each significance comparison, mark the reference group. Abbreviations: ***=p<0.001, **=p<0.01, *=p<0.05, n.s. = not significant; e.v. = empty vector; WT = healthy donor; DM = double mutant).

XP20BE cells transfected with the empty vector showed no complementation ability at all, whereas the transfection of wildtype XPG complemented this defect, measured three days after transfection.

Wildtype cells of a healthy donor displayed an even more increased NER capacity, which is due to the fact that recombinant XPG in cured XP20BE cells, in contrast to endogen XPG in wt cells, first has to be expressed and modified etc. to a certain extent before it is able perform NER. Furthermore, the transient transfection approach supplies 50-60% transfection efficacy (data not shown) (see chapter 3.3.4).

All full-length mutants (FF, E791A, D77A and their respective double mutants) increase the repair rate of the XPG deficient fibroblasts excessively in a highly significant manner compared to the empty vector (p<0.001; Student’s T-test). The truncated Isoform VI, preselected for NER activity, confirms that strong enhancement compared to the empty vector control (p=0.002; Student’s T-test). In contrast the artificially truncated XPG version Del is not able to complement.

Both FF-mutation containing clones were able to complement XP20BE cells to the same extent compared to wildtype XPG. Furthermore the D77A endonuclease defective mutant (mutation in N-domain) displayed also no significant difference. In contrast, the E791A mutation in the I-domain of XPG reduces the NER capacity compared to wt XPG, whether alone or in combination with D77A (E791A: p<0.01, DM_E/D: p=0.06; Student’s T-test). However, due to the relatively low sample size in the case of DM_E/D (p-value 0.006), significance was only achieved in the case of E791A alone. The fact that the DM_E791A/FF double mutant behaves like wt XPG indicates the cure of E791A-caused impaired NER by the lack of the C-terminally located PIP-box. This accounts possibly for another factor, which is favorable able to recruit PCNA and to perform endonuclease cleavage.

The endonuclease defective mutant E791A itself in turn revealed an increased NER activity compared to the truncated versions Del and IsoVI (p<0.001; Student’s T-test). IsoVI represents the weakest XPG variant, but is significantly enhanced capable of performing NER compared to Del (p<0.05; Student’s T-test).

Taken together, these results indicate a strong influence on NER capability by the essential E791 mutation to alanine and a detectable but significantly decreased activity of Isoform VI. The next figure displays the HCR analysis of several XPG PIP- and UBM mutants (see Figure 24, see chapter 4.2.1).

Figure 24: HCR of PIP- and UBM mutants as well as the respective double- and triple mutants A) XPG b) FF c) E791A and d) IsoVI were used as template for additional mutagenesis of the PIP-N and UBM domain of XPG (see chapter 4.2.1). The black bars indicate the relative NER capacity of an UV irradiated Firefly Reportergene plasmid compared to the unirradiated expression level (normalized to the cotransfected Renilla luciferase, see chapter 3.5.1) in dependency of various XPG mutants, three days after transfection. The error bars are calculated using error propagation of the mean values of normalized Firefly, either irradiated or not, due to statistical standard techniques. Significance levels are calculated using the Student’s T-Test (comparison of two groups). The small fat vertical lines, in the left of each significance comparison, mark the reference group. Abbreviations: ***=p<0.001,

**=p<0.01, *=p<0.05, n.s. = not significant; e.v. = empty vector; WT = healthy donor).

Both, the PIP- and UBM mutation, decrease the XP20BE complementation ability of wt XPG (see Figure 24a) in a significant manner (XPG-UBM: p<0.001, all other: p<0.05; Student’s T-test). The displayed difference of reduced significance levels of all PIP mutants compared to the UBM mutant, are due to the relative low sample size of HCR measurements of the PIPs compared to all others.

Methodical difficulties during mutagenesis of this specific region lead only to the successful creation at the very end of the thesis. The PIP-, UBM-, and the respective double mutants all behaved in the same fashion like the endonuclease defective E791A mutant (no significant difference, Student’s T-test).

In comparison to the PIP-box mutated FF mutant (see Figure 24b), all further PCNA interaction impairing mutations, PIP- and UBM, particularly decrease the complementation activity of the FF clone to a very significant extent in the double mutants (p<0.001; Student’s T-test). Most strikingly, the triple mutant FF-PIP-UBM is not able to perform NER, whereas the respective single mutants complement on E791A level.

The E791A mutation was combined with PIP- and UBM mutagenesis and also analyzed utilizing HCR (see Figure 24c). The PIP mutation had no influence on the behavior, indicated by the E791A-PIP double mutant compared to E791A alone. Though the E791A-UBM mutant and the triple mutant E791A-UBM-PIP decrease the observed NER capability in a weak and strongly significant manner (E791A-UBM: p<0.05, E791A-UBM-PIP: p<0.001; Student’s T-test), respectively. However, the E791-PIP-UBM triple mutant shows no significant difference to the E791A-UBM or FF-E791-PIP-UBM mutants, and behaved like the empty vector control (Student’s T-test).

The XPG splice variant Isoform VI was also mutated via quick change mutagenesis in order to create single- as well as double mutants and analyzed with the HCR assay (Figure 24d). The performed mutagenesis displayed no significant difference of the respective mutants compared to Isoform VI (Mann-Whitney-U-test). However, the PIP- and UBM single mutants still statistically complement the XP20BE XPG deficiency (IsoVI-PIP: p<0.01, IsoVI-UBM: p<0.001; Student’s T-test) in contrast to the corresponding double mutant IsoVI-PIP-UBM which is not significantly different from the empty vector control (Student’s T-test).

Taken together, these results indicate that FF sites are dispensable for complementation, but not for immediate DNA repair synthesis (see chapter 4.2.4). The mutagenesis of PIP-N and UBM confers complementation deficiency to XPG on the same level like E791A mutation. The simultaneous mutagenesis of three sites (FF-PIP-UBM, E791A-PIP-UBM) leads to the prevention or strong restriction of NER activity. Additionally, the N-terminal mutations of PIP-N and UBM display a synergistic effect, which leads to the suggestion that they act in concert.

4.2.4 Short summary of the quantitative results