Single cell analysis of human RAD18-dependent DNA post-replication repair by alkaline bromodeoxyuridine comet assay.
Damage to DNA can block replication progression resulting in gaps in the newly synthesized DNA. Cells utilize a number of post-replication repair (PRR) mechanisms such as the RAD18 controlled translesion synthesis or template switching to overcome the discontinuities formed opposite the DNA lesions and to complete DNA replication. Gaining more insights into the role of PRR genes promotes better understanding of DNA damage tolerance and of how their malfunction can lead to increased genome instability and cancer. However, a simple and efficient method to characterise gene specific PRR deficiencies at a single cell level has not been developed. Here we describe the so named BrdU comet PRR assay to test the contribution of human RAD18 to PRR at a single cell level, by which we kinetically characterized the consequences of the deletion of human RAD18 on the replication of UV-damaged DNA. Moreover, we demonstrate the capability of our method to evaluate PRR at a single cell level in unsynchronized cell population.
Institute of Genetics, Biological Research Centre of Hungarian Academy of Sciences, Szeged, Hungary.
Mónika
M
Mórocz
Himabindu
H
Gali
István
I
Raskó
C Stephen
CS
Downes
Lajos
L
Haracska
D001973:Bromodeoxyuridine / Q000378:metabolism; D020552:Comet Assay / Q000379:methods*; D004247:DNA / Q000096:biosynthesis* / Q000235:genetics*; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000528:radiation effects; D004261:DNA Replication / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004307:Dose-Response Relationship, Radiation; D055786:Gene Knockout Techniques; D045325:HCT116 Cells; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D016196:S Phase / Q000235:genetics / Q000528:radiation effects; D059010:Single-Cell Analysis / Q000379:methods*; D044767:Ubiquitin-Protein Ligases; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; C485954:RAD18 protein, human; D004247:DNA; D044767:Ubiquitin-Protein Ligases; D001973:Bromodeoxyuridine