A C. elegans homolog of the Cockayne syndrome complementation group A gene

Abstract

Cockayne syndrome (CS) is a debilitating and complex disorder that results from inherited mutations in the CS complementation genes A and B, CSA and CSB. The links between the molecular functions of the CS genes and the complex pathophysiology of CS are as of yet poorly understood and are the subject of intense debate. While mouse models reflect the complexity of CS, studies on simpler genetic models might shed new light on the consequences of CS mutations. Here we describe a functional homolog of the human CSA gene in Caenorhabditis elegans. Similar to its human counterpart, mutations in the nematode csa-1 gene lead to developmental growth defects as a consequence of DNA lesions.

Keywords: C. elegans; Cockayne syndrome; Nucleotide excision repair.

Figure 1. Bioinfomatical identification of D2101.3 as a CSA homolog

(A) A dendrogram showing the alignment of C. elegans proteins and known CSA sequences, calculated using the neighbour-joining method. The uncharacterized C. elegans ORF D2013.3 clusters with the CSA sequences. (B) Representation of the genomic architecture of D2013.3. Green boxes represent exons, black lines represent introns, and untranslated regions are in gray. The region deleted in the corresponding alleles is represented in red.

Figure 2. D2013.3 is required for TC-NER but dispensable for GG-NER

(A) The percentage of larval stages 65 hours after UV-treatment administered at the L1 larval stage. The tm4539 and tm5232 alleles display UV-sensitivity similar to the TC-NER-defective csb-1 mutant strain. A minimum of 40 worms were used for each UV-dose, and each treatment was conducted in triplicate. Error bars represent SD. *=p<0.05, **=p<0.01, ***=p<0.001, two-tailed t-test, compared to wildtype. (B) Egg-laying activity of the indicated mutants 16 hours after UV-treatment at young adult stage. Timed egg-laying was conducted with three worms per UV-dose for 3 hours. Each treatment was conducted in triplicate. (C) Percentage of hatched eggs after UV-treatment. Egg-laying and hatching of csa-1 mutant worms are unaffected, unlike GG-NER deficient xpc-1 mutant worms. B,C-Error bars represent SD. Statistical significance was calculated using two-tailed t-test, comparing treatments to 0mJ/cm².

Figure 3. csa-1 mutants are illudin-M sensitive

Percentage of larval stages 65 hours after 4μM or 40μM illudin-M treatment. Illudin-M induces a developmental delay at 4μM and a developmental arrest at 40μM in csa-1 and csb-1 mutants. A minimum of 40 worms was used for each illudin-M dose and each treatment was conducted in triplicate. Error bars represent SD. *=p<0.05, **=p<0.01, ***=p<0.001, two-tailed t-test, compared to wildtype.

Figure 4. csa-1 is epistatic to csb-1 and TC-NER specific

Percentage of larval stages 65 hours after UV-treatment during the L1 larval stage of the indicated genotypes. csa-1;csb-1 double mutants display similar UV-sensitivity as csa-1 and csb-1 single mutants. csa1;xpc-1 double mutants display enhanced UV-sensitivity compared to csa-1 and xpc-1 single mutants. A minimum of 40 worms was used for each UV-dose, and each treatment was conducted in triplicate. Error bars represent SD. ns = not significant, two-tailed t-test, compared to csb-1.