Academic journal article Genetics

Mutator Phenotype of Caenorhabditis Elegans DNA Damage Checkpoint Mutants

Academic journal article Genetics

Mutator Phenotype of Caenorhabditis Elegans DNA Damage Checkpoint Mutants

Article excerpt

ABSTRACT

DNA damage response proteins identify sites of DNA damage and signal to downstream effectors that orchestrate either apoptosis or arrest of the cell cycle and DNA repair. The C. elegans DNA damage response mutants mrt-2, hus-1, and clk-2(mn159) displayed 8- to 15-fold increases in the frequency of spontaneous mutation in their germlines. Many of these mutations were small- to medium-sized deletions, some of which had unusual sequences at their breakpoints such as purine-rich tracts or direct or inverted repeats. Although DNA-damage-induced apoptosis is abrogated in the mrt-2, hus-1, and clk-2 mutant backgrounds, lack of the apoptotic branch of the DNA damage response pathway in cep-1/p53, ced-3, and ced-4 mutants did not result in a Mutator phenotype. Thus, DNA damage checkpoint proteins suppress the frequency of mutation by ensuring that spontaneous DNA damage is accurately repaired in C. elegans germ cells. Although DNA damage response defects that predispose humans to cancer are known to result in large-scale chromosome aberrations, our results suggest that small- to medium-sized deletions may also play roles in the development of cancer.

THE genome is under continuous surveillance for DNA damage by checkpoint sensor proteins, which, if activated, can elicit temporary arrest of the cell cycle and repair of the damaged lesion. In multi-cellular eukaryotes, an alternative fate for cells that reach a critical threshold of DNA damage is apoptosis. Malfunction of DNA damage response proteins can stimulate tumorogenesis in humans (V enkitaraman 2002), probably as a consequence of improper processing of endogenous or exogenous forms of DNA damage that results in alterations to the genome. An understanding of the precise relationship between genome instability and the development of cancer is currently a topic of intense study and includes analysis of gross chromosomal rearrangements (GCRs) for many forms of cancer. While GCRs are commonly observed in cancer cells and may contribute to tumorogenesis, it is unclear if they herald the presence of other forms of DNA damage that are also relevant. We have chosen to address this issue by studying DNA damage response mutants in the nematode Caenorhabditis elegans.

Proteins that respond to DNA damage are thought to function via a tiered network of biochemical interactions. Putative sensors of DNA damage such as the phosphatidyl inositol-3 (PI-3)-like kinases ATR and ATM and their respective partners ATRIP and the MRE11/ RAD50/NBS1 complex help to initiate a DNA damage response (DDR) (SANCAR et al. 2004). A complex of checkpoint proteins that is independently recruited to sites of DNA damage is the RAD9/RAD1/HUS1 (9-1-1) proliferating cell nuclear antigen (PCNA)-like sliding clamp, which is loaded onto single-stranded DNA by the RAD17 clamp loader and its four replication factor C subunits (GRIFFITHS et al. 1995; KOSTRUB et al. 1998; BURTELOW et al. 2000; CASPARI et al. 2000; ZOU et al. 2002; BERMUDEZ et al. 2003). The ATR and ATM checkpoint kinases respond to DNA damage by phosphorylating members of the 9-1-1 damage sensor complex as well as downstream "mediators" such as BRCA1, CLASPIN, 53BP1, the signaling kinases CHK1 and CHK2, and ef-fectors such as p53 (SANCAR et al. 2004).

Genetic studies in mice and yeast suggest that DNA damage response proteins may suppress genome instability, in part, by facilitating the repair of endogenous DNA double-strand breaks (DSBs) (PATEL et al. 1998; MOYNAHAN et al. 1999, 2001; DENG and SCOTT 2000; MYUNG et al. 2001a,b; HOWLETT et al. 2002; KRAAKMAN-VAN DER ZWET et al. 2002; VENKITARAMAN 2002; D'ANDREA and GROMPE 2003; PENNANEACH and KOLODNER 2004). Examination of genome stability in haploid yeast mutants defective for the 9-1-1 checkpoint complex has revealed modestly elevated levels of GCRs (MYUNG et al. 2001b). Mutation of vertebrate 9-1-1 complex subunits results in genome instability and lethality (WEISS et al. …

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