Mechanism of DNA polymerase II-mediated frameshift mutagenesis

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Escherichia coli utilizes three SOS-inducible DNA polymerases (Pol II, IV, and V) for translesion synthesis and mutagenesis.
• The choice of polymerase for DNA lesion bypass depends on the lesion type and local DNA sequence context.
• Error-free bypass of N-2-acetylaminofluorene adducts at NarI sites requires Pol V (umuDC), whereas mutagenic -2 frameshift bypass depends on Pol II (polB).

Purpose of the Study:

• To investigate the role of purified DNA Pol II in vitro in generating the -2 frameshift bypass product observed in vivo.
• To elucidate the competition dynamics between Pol II and Pol V in translesion synthesis pathways.

Main Methods:

• In vitro biochemical assays using purified DNA Pol II.
• Genetic analysis of Escherichia coli strains with specific gene deletions (Delta polB) and plasmid-based gene complementation.
• Comparative analysis of mutation frequencies under varying polB and umuDC gene copy numbers.

Main Results:

• Purified DNA Pol II demonstrated the ability to generate the -2 frameshift bypass product in vitro.
• A Delta polB strain was completely defective in the -2 frameshift mutation pathway, which was restored by introducing the polB gene.
• Altering the relative copy numbers of polB and umuDC genes modulated the preference for frameshift versus error-free translesion pathways.

Conclusions:

• DNA Pol II directly mediates the -2 frameshift bypass pathway at NarI sites.
• A competitive model between Pol II and Pol V for translesion synthesis at specific DNA lesions is proposed.
• The relative expression levels of competing polymerases can dictate the outcome of DNA damage tolerance pathways.