Language：English   Publishing type：Research paper (scientific journal)  

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 β-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.

DOI： 10.1093/nar/gkad246

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DNA damage tolerance pathways are regulated by proliferating cell nuclear antigen (PCNA) modifications at lysine 164. Translesion DNA synthesis by DNA polymerase η (Polη) is well studied, but less is known about Polη-independent mechanisms. Illudin S and its derivatives induce alkyl DNA adducts, which are repaired by transcription-coupled nucleotide excision repair (TC-NER). We demonstrate that in addition to TC-NER, PCNA modification at K164 plays an essential role in cellular resistance to these compounds by overcoming replication blockages, with no requirement for Polη. Polκ and RING finger and WD repeat domain 3 (RFWD3) contribute to tolerance, and are both dependent on PCNA modifications. Although RFWD3 is a FANC protein, we demonstrate that it plays a role in DNA damage tolerance independent of the FANC pathway. Finally, we demonstrate that RFWD3-mediated cellular survival after UV irradiation is dependent on PCNA modifications but is independent of Polη. Thus, RFWD3 contributes to PCNA modification-dependent DNA damage tolerance in addition to translesion DNA polymerases.

DOI： 10.26508/lsa.202201584

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Authorship：Lead author   Language：Japanese  

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DOI： 10.1074/jbc.RA118.005167

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DOI： 10.1093/nar/gky943

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