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Bacteriophage T4 mutants hypersensitive to an antitumor agent that induces topoisomerase-DNA cleavage complexes
1Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Abstract:
Many antitumor agents and antibiotics affect cells by interacting with type II topoisomerases, stabilizing a covalent enzyme-DNA complex. A pathway of recombination can apparently repair this DNA damage. In this study, transposon mutagenesis was used to identify possible components of the repair pathway in bacteriophage T4. Substantial increases in sensitivity to the antitumor agent m-AMSA [4'-(9-acridinylamino)methanesulfon-m-anisidide] were found with transposon insertion mutations that inactivate any of six T4-encoded proteins: UvsY (DNA synaptase accessory protein), UvsW (unknown function), Rnh (RNase H and 5' to 3' DNA exonuclease), alpha-gt (alpha-glucosyl transferase), gp47.1 (uncharacterized), and NrdB (beta subunit of ribonucleotide reductase). The role of the rnh gene in drug sensitivity was further characterized. First, an in-frame rnh deletion mutation was constructed and analyzed, providing evidence that the absence of Rnh protein causes hypersensitivity to m-AMSA. Second, the m-AMSA sensitivity of the rnh-deletion mutant was shown to require a drug-sensitive T4 topoisomerase. Third, analysis of double mutants suggested that uvsW and rnh mutations impair a common step in the recombinational repair pathway for m-AMSA-induced damage. Finally, the rnh-deletion mutant was found to be hypersensitive to UV, implicating Rnh in recombinational repair of UV-induced damage.
Insights
Bacteriophage T4 mutants lacking RNase H (Rnh) show increased sensitivity to antitumor drugs and UV radiation. This suggests Rnh is crucial for repairing DNA damage caused by these agents.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Type II topoisomerases are cellular targets for many antitumor agents and antibiotics.
- These agents stabilize covalent enzyme-DNA complexes, leading to DNA damage.
- Recombinational repair pathways are known to mend this type of DNA damage.
Purpose of the Study:
- To identify components of the DNA repair pathway in bacteriophage T4 involved in repairing topoisomerase-mediated damage.
- To investigate the specific role of the T4 Rnh (RNase H) gene in sensitivity to the antitumor agent m-AMSA and UV radiation.
Main Methods:
- Transposon mutagenesis was employed to screen for bacteriophage T4 mutants with increased sensitivity to m-AMSA.
- Characterization of mutations involved constructing in-frame deletion mutants (rnh deletion) and analyzing double mutants.
- Sensitivity assays were performed using the antitumor agent m-AMSA and UV radiation.
Main Results:
- Mutations inactivating six T4 proteins (UvsY, UvsW, Rnh, alpha-gt, gp47.1, NrdB) led to substantial increases in m-AMSA sensitivity.
- An rnh deletion mutant exhibited hypersensitivity to m-AMSA, dependent on a functional T4 topoisomerase.
- The rnh deletion mutant was also hypersensitive to UV radiation, indicating Rnh's role in repairing UV-induced DNA damage.
Conclusions:
- The T4 Rnh protein plays a significant role in the recombinational repair of DNA damage induced by agents like m-AMSA.
- Rnh is implicated in the repair pathway for UV-induced DNA damage, suggesting a broader role in DNA repair.
- The study identified several T4 proteins involved in this repair pathway, highlighting the complexity of DNA damage response mechanisms.