Nucleotide excision repair (NER) is a versatile pathway involved in the repair
of various DNA lesions. Patients with defective NER can suffer from disorders
like xeroderma pigmentosum (XP) or Cockayne syndrome (CS). The NER
enzyme XPG accounts in its various mutated forms for XP or a combined
XP/CS phenotype depending on the nature of the mutations in the XPG gene.
Currently still little is known about the molecular origin of CS and XP/CS and
the involvement of XPG. There is growing evidence that XPG exerts functions
in non-NER DNA metabolisms, which may be responsible for prevention of
CS.
Our project aimed at studying the various roles of the XPG homologue Rad 13
in fission yeast as a model system, thereby focussing on identifying NER-independent functions of Rad13. In a qualitative epistasis analysis with the
NER mutants swi10 and rhp14, we found that rad13 is epistatic to these
genes in response to UV and the anticancer drug cisplatin. In contrast, upon
treatment with MMS and mitomycin C, double mutants showed an increased
sensitivity compared with the single mutants. This indicates that Rad1 3 has a
NER-independent function in response to these agents.
To determine the function of defined regions of Rad1 3, point mutations have
been introduced into highly conserved domains. rad13 strains with mutations
in the nuclease domains were sensitive to UV, MMS and cisplatin, but clearly
less than a rad13 deletion mutant. P72H, a point mutation found in XP-G/CS
patients, believed to destabilise the protein, rendered the cells as sensitive as
a deletion mutant.
By gel filtration experiments we showed that Rad 13 is part of a high-molecular
weight complex in NER-proficient and -deficient strain backgrounds. In a pull-down experiment we identified putative protein interaction partners of Rad13.