Probing the Effect of Bulky Lesion-Induced Replication Fork Conformational Heterogeneity Using...

Area of Science:

• Biochemistry
• Molecular Biology
• Toxicology

Background:

• Bulky organic carcinogens form DNA adducts in vivo.
• These DNA adducts can adopt multiple conformations, influencing mutagenicity and repair.
• Conformation-specific replication inhibition is poorly understood due to lesion dynamics.

Purpose of the Study:

• To correlate in vitro polymerase activity inhibition with specific conformations of a single bulky DNA adduct.
• To investigate the impact of different DNA adduct conformations on polymerase binding affinity and inhibition.
• To provide insights into conformation-specific replication inhibition relevant to cell survival.

Main Methods:

• Utilized surface plasmon resonance (SPR) to assess binding affinity.
• Employed HPLC-based steady-state kinetics to measure polymerase inhibition.
• Studied two distinct conformations of a FABP-modified DNA lesion in different sequence contexts.

Main Results:

• Quantified differences in polymerase binding affinity between the two DNA adduct conformations (67%B:33%S and 100%B).
• Revealed distinct levels of polymerase inhibition associated with each conformation.
• Demonstrated that sequence context influences DNA adduct conformation and polymerase interaction.

Conclusions:

• Conformation-specific differences in polymerase binding and inhibition were observed for the FABP-modified DNA lesion.
• These findings highlight the importance of considering DNA adduct conformation in understanding replication fidelity and cell survival.
• The study provides a foundation for further research into the biological consequences of DNA adduct conformational heterogeneity.