5-Formylcytosine mediated DNA-peptide cross-link induces predominantly semi-targeted mutations in both Escherichia...

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Histone proteins can form toxic DNA-protein conjugates (DpCs) with 5-formylcytosine (5fC).
• Repair of these lesions may involve proteolytic digestion, leading to DNA-peptide cross-links (DpCs).
• Understanding DpC replication is crucial for DNA repair and genome stability.

Purpose of the Study:

• To investigate the replication of a model DpC containing a 5fC-conjugated peptide.
• To analyze the sequence context effects (CXG and CXT) on DpC replication fidelity.
• To identify the translesion synthesis (TLS) polymerases involved in DpC mutagenesis.

Main Methods:

• Replication of synthetic DNA constructs containing a model DpC in Escherichia coli and HEK 293T cells.
• Mutation analysis to determine error rates and mutation types.
• Knockdown studies using cells deficient in specific human TLS polymerases (hPol η, hPol ι, hPol ζ).
• Enzyme kinetics studies with purified human DNA polymerase η (hPol η).

Main Results:

• DpC replication resulted in low viability in E. coli (<10%) but high TLS efficiency in HEK 293T cells (72%).
• In E. coli, DpCs were bypassed largely error-free (2-3% mutations), with semi-targeted mutations (G→T, G→C) dominating at the 3' base.
• In HEK 293T cells, 7-9% mutations occurred, predominantly semi-targeted (G→T for CXG, T→G for CXT), reduced by deficiencies in hPol η, hPol ι, or hPol ζ.
• hPol η kinetics confirmed its role in inducing G→T and T→G transversions at the base 3' to the DpC.

Conclusions:

• 5fC-conjugated DpCs exhibit a unique replication pattern, being bypassed with high fidelity in both prokaryotic and eukaryotic systems.
• Replication of DpCs primarily induces semi-targeted mutations at the 3' position to the lesion.
• Human TLS polymerases, particularly hPol η, play a significant role in the mutagenic bypass of these DNA-protein cross-links.