Multiple solutions to inefficient lesion bypass by T7 DNA polymerase

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA replication can stall at damaged sites.
• Translesion synthesis (TLS) is a mechanism to bypass DNA lesions.
• Understanding polymerase switching during TLS is crucial for DNA repair fidelity.

Purpose of the Study:

• To test the hypothesis that enzymatic switching during TLS balances efficiency and fidelity.
• To quantitatively determine the lesion bypass capabilities of replicative polymerases.
• To model TLS using T7 DNA polymerase to bypass TT dimers and abasic sites.

Main Methods:

• Measured efficiency and fidelity of DNA lesion bypass using T7 DNA polymerase.
• Investigated the role of exonuclease activity in preventing TLS.
• Analyzed TLS fidelity including template-primer strand rearrangements and insertions.

Main Results:

• T7 DNA polymerase showed no bypass of TT dimers or abasic sites in a single round.
• Exonuclease activity was critical in preventing TLS; exonuclease-deficient enzyme showed limited bypass after thousands of cycles.
• TLS fidelity was influenced by sequence context, and lesions affected adjacent base copying fidelity.

Conclusions:

• Lesion bypass efficiency and fidelity are influenced by polymerase choice and sequence context.
• TT dimers and abasic sites pose distinct challenges to DNA polymerases.
• The developed methods can guide the selection of polymerases for specific lesion bypass in biological systems.