Translesion synthesis DNA polymerases promote error-free replication through the minor-groove DNA adduct...

Area of Science:

• Molecular Biology
• DNA Repair Mechanisms
• Genetics

Background:

• N3-Methyladenine (3-MeA) is a DNA adduct formed by methyl donors and alkylating agents.
• 3-MeA protrudes into the DNA minor groove, significantly hindering replicative DNA polymerases (Pols).
• The precise mechanisms for replicating 3-MeA-damaged DNA in human cells were previously unidentified.

Purpose of the Study:

• To investigate the roles of translesion synthesis (TLS) polymerases in replicating 3-MeA-damaged DNA in human cells.
• To elucidate the distinct pathways and fidelity of TLS polymerases involved in bypassing the 3-MeA lesion.

Main Methods:

• Utilized the stable 3-deaza analog, 3-deaza-3-methyladenine (3-dMeA), to mimic 3-MeA's DNA minor groove blockage.
• Analyzed the involvement of specific TLS polymerases (Polι, Polκ, Polθ, Polζ) in replication through the 3-dMeA adduct.
• Performed steady-state kinetic analyses to assess nucleotide insertion efficiency and fidelity.

Main Results:

• Replication through 3-dMeA occurs via three distinct pathways involving Polι/Polκ, Polθ, or Polζ.
• Polι/Polκ pathway: Polι inserts a nucleotide, and Polκ extends synthesis.
• Polθ pathway: Polθ performs both insertion and extension.
• Polζ pathway: Polζ extends synthesis after an unknown polymerase inserts a nucleotide.
• Polι and Polθ showed reduced efficiency and high propensity for incorrect nucleotide insertion opposite 3-dMeA.
• Despite low polymerase fidelity, overall TLS replication of the 3-dMeA lesion in human cells is highly error-free.

Conclusions:

• Human cells employ multiple TLS polymerase pathways to replicate DNA containing the 3-MeA lesion.
• Distinct TLS polymerases contribute differently to nucleotide insertion and extension, ensuring efficient bypass.
• The cellular context ensures high-fidelity replication of 3-MeA damage, despite the inherent low fidelity of individual TLS polymerases.