We observed more increase in the GFP-XPC mobilization upon PR of 6-4PPs compared to CPDs. used to quantify DNA damage load and to DPP4 determine repair kinetics in real time. Additionally, PLs can instantly reverse DNA damage by 405 nm laser-assisted 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. INTRODUCTION Our genome is continuously exposed to various types of DNA damage. If not repaired correctly, DNA lesions may result in mutations, cellular senescence or cell death, which can eventually lead to various pathological conditions including carcinogenesis and aging (1). To counteract these deleterious effects of DNA damage, cells have evolved a variety of mechanisms, including several DNA repair pathways (2). Nucleotide excision repair (NER) is one of the most versatile DNA repair pathways, as it removes a wide variety of DNA helix-destabilizing lesions. Prominent examples of NER substrates are the UV-induced cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PPs). The biological importance of NER is illustrated by the severe clinical symptoms of human disorders caused by inherited NER defects, including the cancer-prone xeroderma pigmentosum (XP) syndrome or the premature aging disorder Cockayne’s syndrome (CS) (3). NER is initiated by two sub-pathways that differ in their mode of damage recognition. Global genome NER (GG-NER) detects lesions in the entire genome, by the main DNA damage binding protein XPC (4). XPC recognizes DNA-helix distortions such as induced by 6-4PP lesions, but needs the activity of the UV-DDB complex, composed of DDB1 and DDB2, to detect mildly helix-destabilizing CPD lesions (5,6). Transcription-coupled NER (TC-NER) is initiated when DNA damage located in the actively transcribed strand blocks elongating RNA polymerase II, which results in the recruitment of the TC-NER factors CSA, CSB and UVSSA (7,8). Once the DNA lesion is recognized, general transcription factor II H (TFIIH) is recruited (9,10) to unwind the DNA surrounding the damage (11) and to verify the lesion together with XPA (12,13). The endonucleases XPG and ERCC1/XPF subsequently remove a GNE-140 racemate 30 nucleotide long fragment of DNA around the lesion (14). Finally, the DNA is restored back to its original state by DNA synthesis and ligation steps (15,16). Recent studies have shown that NER is a tightly regulated, multistep pathway that requires many proteins and post-translational modifications for the efficient and accurate transition between the successive reaction steps (3,17C19). Additionally, as NER takes place in the complex chromatin and nuclear environment, many factors involved in chromatin remodeling (3,20,21), transcription (22), or replication (23) influence NER activity, and most likely many other involved factors are awaiting their discovery. Therefore, assays to quantify DNA damage and repair rates are invaluable tools to investigate the roles of such factors and to obtain new fundamental insights into the molecular mechanism of NER. Moreover, assays to detect impairments or deficiencies in NER activity have been crucial for the diagnosis of NER-deficient patients and can be used as indicators for predispositions to mutations, the onset of cancer, or DNA damage-induced aging (24C27). Over the years, several assays were developed to quantitatively monitor UV-induced DNA damage and NER-mediated repair. Traditionally, NER activity is measured by determining the rate of UV-induced DNA repair synthesis, the last step of the NER reaction (28C30), or by determining the levels of CPDs in the DNA in time using T4 endonuclease V (31). Over the years, several other assays have been developed to monitor upstream NER activity, including UV-damage removal (32), NER-induced incisions (33) or quantification of GNE-140 racemate excision products (34). TC-NER is often determined indirectly by quantifying the recovery of RNA synthesis (RRS) (35,36), GNE-140 racemate or by using host cell reactivation assays (37). Alternatively, TC-NER can be measured in a direct manner by strand-specific repair assays (38), or by more recently developed single-cell assays, such as the modified COMET-FISH.
