The eukaryotic replisome tolerates leading-strand base damage by replicase switching

Area of Science:

• Molecular Biology
• DNA Replication
• Genetics

Background:

• High-fidelity replicative DNA polymerases (Pol ε and Pol δ) are generally considered ill-equipped for damaged DNA replication.
• Low-fidelity translesion synthesis (TLS) polymerases are typically required for complete replication of damaged DNA templates.

Purpose of the Study:

• To investigate the inherent tolerance of a reconstituted yeast replisome to oxidative DNA lesions.
• To elucidate the mechanism of DNA lesion bypass by replicative polymerases.

Main Methods:

• Reconstitution of a yeast replisome using purified proteins.
• In vitro assays to assess DNA lesion bypass efficiency.
• Analysis of polymerase switching during replication of damaged DNA templates.

Main Results:

• The yeast replisome demonstrated inherent tolerance to thymine glycol (Tg), an oxidative DNA lesion.
• Leading-strand bypass of Tg occurred efficiently, independent of TLS polymerases.
• A switch from Pol ε to Pol δ facilitated rapid, efficient, and error-free bypass of Tg and 8-oxoguanine.

Conclusions:

• Replicative DNA polymerases Pol ε and Pol δ possess inherent capabilities for bypassing DNA damage.
• A replicase switch mechanism, involving a shift from Pol ε to Pol δ, enables efficient leading-strand synthesis past oxidative DNA lesions.
• This mechanism may represent a conserved strategy for maintaining genome integrity during DNA replication stress.