Long-Patch Base Excision Repair of 5'-DNA-Peptide Cross-Links Derived from Abasic DNA Lesions

Area of Science:

• Molecular Biology
• DNA Repair Mechanisms
• Biochemistry

Background:

• DNA single-strand breaks (SSBs) can form covalent DNA-protein cross-links (DPCs) at 5'-termini.
• These 5'-DPCs arise from oxidized abasic sites (2-deoxyribonolactone) and impede SSB repair by preventing 5'-phosphate ligation.
• While proteasome-mediated proteolysis of 5'-DPCs is known, the mechanism for removing the remaining DNA-peptide cross-link (DpC) is unclear.

Purpose of the Study:

• To investigate the repair mechanism of 5'-DNA-peptide cross-links (5'-DpCs) in DNA single-strand breaks.
• To identify the enzymes involved in the excision of 5'-DpCs.
• To elucidate the pathway for the complete repair of SSBs containing 5'-DPCs.

Main Methods:

• Chemical synthesis of site-specific 5'-DpC and model 5'-DPC adducts.
• Incubation of synthesized adducts with HeLa cell nuclear extracts.
• Enzymatic assays to assess repair activity and identify key enzymes, including proteolysis and flap endonuclease activity.
• Use of trypsin for controlled proteolysis of cross-linked proteins.

Main Results:

• HeLa cell nuclear extracts efficiently repaired chemically synthesized and site-specific 5'-DpC.
• Human flap-endonuclease 1 (hFEN1) was identified as a crucial enzyme for the excision of the 5'-DpC.
• Proteolysis of the cross-linked protein by trypsin significantly enhanced DPC repair in nuclear extracts.
• A model 5'-DPC repaired via reductive amination also showed enhanced repair after proteolysis.

Conclusions:

• 5'-DPCs within SSBs are repaired through a multi-step process involving proteolysis.
• Human flap-endonuclease 1 (hFEN1) plays a key role in excising the remaining DNA-peptide cross-link.
• The repair pathway likely involves proteolysis followed by the long-patch base excision repair pathway for complete SSB repair.