2 research outputs found
Fluorescently-labelled CPD and 6-4PP photolyases: new tools for live-cell DNA damage quantification and laser-assisted repair
UV light induces cyclobutane pyrimidine dimers
(CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PPs), which can result in carcinogenesis
and aging, if not properly repaired by nucleotide excision repair (NER). Assays to determine DNA damage load and repair rates are invaluable tools for fundamental and clinical NER research. However, most
current assays to quantify DNA damage and repair
cannot be performed in real time. To overcome this
limitation, we made use of the damage recognition
characteristics of CPD and 6-4PP photolyases (PLs).
Fluorescently-tagged PLs efficiently recognize UVinduced DNA damage without blocking NER activity, and therefore can be used as sensitive live-cell
damage sensors. Importantly, FRAP-based assays
showed that PLs bind to damaged DNA in a highly
sensitive and dose-dependent manner, and can be
used to quantify DNA damage load and to determine repair kinetics in real time. Additionally, PLs
can instantly reverse DNA damage by 405 nm laserassisted photo-reactivation during live-cell imaging,
opening new possibilities to study lesion-specific
NER dynamics and cellular responses to damage
removal. Our results show that fluorescently-tagged
PLs can be used as a versatile tool to sense, quantify and repair DNA damage, and to study NER kinetics and UV-induced DNA damage response in living
cells