Replication block by an enediyne drug-DNA deoxyribose adduct

Area of Science:

• Molecular Biology
• Medicinal Chemistry
• Biochemistry

Background:

• Neocarzinostatin chromophore (NCS) is an enediyne antibiotic.
• Under anaerobic conditions, NCS forms a covalent drug-DNA adduct at the 5' carbon of deoxyribose within a 2-nucleotide bulge.
• This adduct formation involves general base-catalyzed intramolecular activation to a reactive radical species, distinct from DNA strand cleavage.

Purpose of the Study:

• To investigate the impact of a site-specific NCS-DNA adduct on DNA replicative synthesis.
• To analyze the primer extension properties of various DNA polymerases in the presence of the adduct.
• To understand how different polymerase mechanisms interact with drug-induced DNA lesions.

Main Methods:

• Preparation of a single-stranded oligonucleotide containing a single NCS adduct at a thymine (T) residue.
• 5'-32P-labeled primer extension assays using multiple DNA polymerases, including Klenow fragment (with and without 3'-5' exonuclease activity), T4, herpes simplex virus, cytomegalovirus, Sequenase (exo-minus T7 DNA polymerase), and human DNA polymerase beta.
• Varied reaction conditions, including substitution of Mn2+ for Mg2+ and creation of gaps opposite the lesion.

Main Results:

• Klenow fragment (exo-) DNA polymerase synthesis stops at the base 3' to the adduct, while the exo+ version allows one additional nucleotide incorporation.
• Mn2+ enhances the effect observed with Klenow polymerase.
• T4, herpes simplex virus, and cytomegalovirus DNA polymerases exhibit similar behavior to Klenow polymerase.
• Sequenase (exo-minus T7 DNA polymerase) is more efficient in nucleotide insertion opposite the lesion compared to exo-minus Klenow.
• DNA polymerase beta extends primers to the nucleotide opposite the lesion, and can add up to two nucleotides when a gap is present.
• Base incorporation fidelity opposite the lesion remains unimpaired.

Conclusions:

• The NCS-DNA adduct acts as a specific block or pause site for various DNA polymerases during replication.
• The enzyme's exonuclease activity and cofactor influence its ability to bypass or incorporate nucleotides opposite the adduct.
• DNA polymerase beta demonstrates a unique ability to accommodate the lesion, especially in gapped DNA.
• Unlike DNA base adducts, the NCS-DNA adduct does not impair the fidelity of base incorporation opposite the lesion.