Novel mechanisms for the removal of strong replication-blocking HMCES- and thiazolidine-DNA adducts in humans.
Nucleic Acids Res
; 51(10): 4959-4981, 2023 06 09.
Article
in En
| MEDLINE
| ID: mdl-37021581
ABSTRACT
Apurinic/apyrimidinic (AP) sites are DNA lesions created under normal growth conditions that result in cytotoxicity, replication-blocks, and mutations. AP sites are susceptible to ß-elimination and are liable to be converted to DNA strand breaks. HMCES (5-hydroxymethylcytosine binding, ES cell specific) protein interacts with AP sites in single stranded (ss) DNA exposed at DNA replication forks to generate a stable thiazolidine protein-DNA crosslink and protect cells against AP site toxicity. The crosslinked HMCES is resolved by proteasome-mediated degradation; however, it is unclear how HMCES-crosslinked ssDNA and the resulting proteasome-degraded HMCES adducts are processed and repaired. Here, we describe methods for the preparation of thiazolidine adduct-containing oligonucleotides and determination of their structure. We demonstrate that the HMCES-crosslink is a strong replication blocking adduct and that protease-digested HMCES adducts block DNA replication to a similar extent as AP sites. Moreover, we show that the human AP endonuclease APE1 incises DNA 5' to the protease-digested HMCES adduct. Interestingly, while HMCES-ssDNA crosslinks are stable, the crosslink is reversed upon the formation of dsDNA, possibly due to a catalytic reverse reaction. Our results shed new light on damage tolerance and repair pathways for HMCES-DNA crosslinks in human cells.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
DNA Adducts
/
DNA Repair
Limits:
Humans
Language:
En
Journal:
Nucleic Acids Res
Year:
2023
Type:
Article
Affiliation country:
Japan