Kinetics of endogenous mouse FEN1 in base excision repair.
The structure specific flap endonuclease 1 (FEN1) plays an essential role in long-patch base excision repair (BER) and in DNA replication. We have generated a fluorescently tagged FEN1 expressing mouse which allows monitoring the localization and kinetics of FEN1 in response to DNA damage in living cells and tissues. The expression of FEN1, which is tagged at its C-terminal end with enhanced yellow fluorescent protein (FEN1-YFP), is under control of the endogenous Fen1 transcriptional regulatory elements. In line with its role in processing of Okazaki fragments during DNA replication, we found that FEN1-YFP expression is mainly observed in highly proliferating tissue. Moreover, the FEN1-YFP fusion protein allowed us to investigate repair kinetics in cells challenged with local and global DNA damage. In vivo multi-photon fluorescence microscopy demonstrates rapid localization of FEN1 to local laser-induced DNA damage sites in nuclei, providing evidence of a highly mobile protein that accumulates fast at DNA lesion sites with high turnover rate. Inhibition of poly (ADP-ribose) polymerase 1 (PARP1) disrupts FEN1 accumulation at sites of DNA damage, indicating that PARP1 is required for FEN1 recruitment to DNA repair intermediates in BER.
Centre for Molecular Biology and Neuroscience and Institute of Clinical Medicine, Oslo University Hospital, Rikshospitalet, Norway.
Liv
L
Kleppa
Pierre-Olivier
PO
Mari
Elisabeth
E
Larsen
Guro Flor
GF
Lien
Camille
C
Godon
Arjan F
AF
Theil
Gaute J
GJ
Nesse
Hege
H
Wiksen
Wim
W
Vermeulen
Giuseppina
G
Giglia-Mari
Arne
A
Klungland
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001921:Brain / Q000378:metabolism; D002478:Cells, Cultured; D004249:DNA Damage; D004260:DNA Repair; D045585:Flap Endonucleases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D055879:Gene Knock-In Techniques; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D000071137:Poly (ADP-Ribose) Polymerase-1; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D016196:S Phase
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D018809:Proliferating Cell Nuclear Antigen; C054562:yellow fluorescent protein, Bacteria; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; C572473:Fen1 protein, mouse; D045585:Flap Endonucleases