Determination of DNA lesion bypass using a ChIP-based assay.
DNA Repair (Amst)
; 108: 103230, 2021 12.
Article
em En
| MEDLINE
| ID: mdl-34571449
ABSTRACT
DNA lesion bypass facilitates DNA synthesis across bulky DNA lesions, playing a critical role in DNA damage tolerance and cell survival after DNA damage. Assessing lesion bypass efficiency in the cell is important to better understanding of the mechanism of carcinogenesis and chemoresistance. Here we developed a chromatin immunoprecipitation (ChIP)-based method to measure lesion bypass activity across cisplatin-induced intrastrand crosslinks in cancer cells. DNA lesion bypass enables the replication to continue in the presence of replication blocks. Thus, the successful lesion bypass should result in the coexistence of DNA lesions and the newly synthesized DNA fragment opposite to this lesion. Using ChIP, we precipitated the cisplatin-induced intrastrand crosslinks, and quantitated the precipitated newly synthesized DNA that was labeled with BrdU. We validated this method on ovarian cancer cells with inhibited TLS activity. We then applied this method to show that ovarian cancer stem cells exhibit high lesion bypass activity relative to bulk cancer cells from the same cell line. In conclusion, this novel ChIP-based lesion bypass assay can detect the extent to which cisplatin-induced DNA lesions are bypassed in live cells. Our study may be applied more broadly to the study of other DNA lesions, as specific antibodies to these specific lesions are available.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
DNA
/
DNA Polimerase Dirigida por DNA
Idioma:
En
Revista:
DNA Repair (Amst)
Assunto da revista:
BIOLOGIA MOLECULAR
/
BIOQUIMICA
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Estados Unidos