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Abstract:
Neocarzinostatin (NCS) is an acidic, single-chain polypeptide of 109 amino acids that has shown some antitumor activity in clinical trials. NCS is mutagenic in recA+ strains of Escherichia coli, but not in recA strains; on the other hand, a defect in the nucleotide-excision-repair pathway has no effect on the mutagenicity of NCS in E. coli. Similar results are seen in mammalian cells. Excision-repair-deficient xeroderma pigmentosum (XP) cells repair NCS-induced DNA damage at the same rate as repair-proficient XP heterozygotes, and X-ray-sensitive ataxia telangiectasia fibroblasts are also sensitive to NCS. I have investigated the mutagenicity of NCS in the ad-3 forward-mutation test in nucleotide excision-repair-sufficient and -deficient heterokaryons of Neurospora crassa. Resting conidia from a repair-sufficient strain, H-12, and a nucleotide-excision-repair-deficient strain (uvs-2) H-59, were exposed to NCS. These conidia were assayed for survival and ad-3 forward mutation. The results show that H-59 is more sensitive to the killing and mutagenic activities of NCS than is H-12. These data indicate, in contrast to E. coli and mammalian cells, that the nucleotide-excision-repair pathway of N. crassa does repair NCS-induced lesions. In other experiments, ad-3 mutants induced by NCS in H-59 were characterized to determine the spectrum of NCS-induced mutation. The results show that NCS induces both intracistronic mutations and multilocus deletions in H-59.
Insights
Neocarzinostatin (NCS) is mutagenic, but Neurospora crassa
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Neocarzinostatin (NCS) is a polypeptide with antitumor properties.
- NCS exhibits mutagenicity in Escherichia coli and mammalian cells, independent of nucleotide excision repair.
- Previous studies suggest DNA repair mechanisms influence NCS mutagenicity.
Purpose of the Study:
- To investigate the mutagenicity of Neocarzinostatin (NCS) in Neurospora crassa.
- To determine if the nucleotide excision repair pathway in N. crassa repairs NCS-induced DNA damage.
- To characterize the spectrum of mutations induced by NCS in N. crassa.
Main Methods:
- Utilized the ad-3 forward-mutation assay in Neurospora crassa heterokaryons.
- Exposed nucleotide excision-repair-sufficient (H-12) and -deficient (uvs-2, H-59) strains to NCS.
- Assayed conidia for survival and ad-3 forward mutation, and characterized induced mutants.
Main Results:
- The repair-deficient strain (H-59) showed increased sensitivity to NCS's killing and mutagenic effects compared to the repair-sufficient strain (H-12).
- This indicates that the nucleotide excision repair pathway in N. crassa actively repairs NCS-induced DNA damage.
- NCS induced both intracistronic mutations and multilocus deletions in the repair-deficient strain.
Conclusions:
- Contrary to findings in E. coli and mammalian cells, the nucleotide excision repair pathway in Neurospora crassa plays a role in repairing Neocarzinostatin-induced DNA damage.
- Neocarzinostatin is mutagenic in N. crassa and can induce various types of genetic alterations, including deletions.