5-Formylcytosine mediated DNA-protein cross-links block DNA replication and induce mutations in human cells

Area of Science:

• Epigenetics and DNA repair
• Molecular biology
• Biochemistry

Background:

• 5-Formylcytosine (5fC) is an epigenetic DNA modification.
• 5fC can form reversible DNA-protein conjugates (DPCs) with histone proteins.
• The impact of 5fC-mediated DPCs on DNA replication is not fully understood.

Purpose of the Study:

• To investigate the effects of 5fC-mediated DPCs on DNA replication.
• To determine how translesion synthesis DNA polymerases interact with 5fC-mediated DPCs.
• To elucidate the mutagenic consequences of 5fC-mediated DPCs during replication.

Main Methods:

• Primer extension assays using synthetic DNA duplexes with site-specific 5fC-protein DPCs.
• Replication experiments with plasmids containing 5fC-peptide cross-links in HEK 293T cells.
• Molecular simulations of polymerase-DNA-peptide complexes.

Main Results:

• Histone H2A or H4 DPCs completely inhibited DNA replication, but were bypassed upon proteolytic digestion.
• Translesion synthesis polymerases η and κ bypassed 5fC-peptide cross-links in an error-prone manner.
• Replication of 5fC-peptide cross-linked plasmids in HEK 293T cells induced targeted C→T transitions and -1 deletions.
• Molecular simulations showed peptides fitting into the DNA major groove and stable mismatches at the polymerase active site.

Conclusions:

• 5fC-mediated DPCs pose a significant barrier to DNA replication.
• Translesion synthesis polymerases can bypass these DPCs, leading to specific mutations.
• These findings provide insights into the mutagenic potential of epigenetic modifications during DNA replication.