Human FAN1 promotes strand incision in 5'-flapped DNA complexed with RPA.
Fanconi anaemia (FA) is a human infantile recessive disorder. Seventeen FA causal proteins cooperatively function in the DNA interstrand crosslink (ICL) repair pathway. Dual DNA strand incisions around the crosslink are critical steps in ICL repair. FA-associated nuclease 1 (FAN1) is a DNA structure-specific endonuclease that is considered to be involved in DNA incision at the stalled replication fork. Replication protein A (RPA) rapidly assembles on the single-stranded DNA region of the stalled fork. However, the effect of RPA on the FAN1-mediated DNA incision has not been determined. In this study, we purified human FAN1, as a bacterially expressed recombinant protein. FAN1 exhibited robust endonuclease activity with 5'-flapped DNA, which is formed at the stalled replication fork. We found that FAN1 efficiently promoted DNA incision at the proper site of RPA-coated 5'-flapped DNA. Therefore, FAN1 possesses the ability to promote the ICL repair of 5'-flapped DNA covered by RPA.
Laboratory of Structural Biology, Graduate School of Advanced Science & Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan and.
Daisuke
D
Takahashi
Laboratory of Structural Biology, Graduate School of Advanced Science & Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan and.
Koichi
K
Sato
Laboratory of Structural Biology, Graduate School of Advanced Science & Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan and.
Emiko
E
Hirayama
Laboratory of DNA Damage Signaling, Department of Late Effects Studies, Radiation Biology Center, Kyoto University, Yoshida-konoe, Sakyo-ku, Kyoto 606-8501, Japan.
Minoru
M
Takata
Laboratory of Structural Biology, Graduate School of Advanced Science & Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan and kurumizaka@waseda.jp.
Hitoshi
H
Kurumizaka
D004247:DNA / Q000737:chemistry*; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication / Q000235:genetics*; D004706:Endodeoxyribonucleases; D004720:Endonucleases / Q000737:chemistry; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005199:Fanconi Anemia / Q000235:genetics / Q000473:pathology; D006801:Humans; D064251:Multifunctional Enzymes; D051716:Replication Protein A / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction
D064251:Multifunctional Enzymes; D051716:Replication Protein A; D004247:DNA; D004706:Endodeoxyribonucleases; D004720:Endonucleases; D005090:Exodeoxyribonucleases; C551063:FAN1 protein, human
DNA interstrand crosslinking repair; Fanconi anaemia; Fanconi anaemia-associated nuclease 1 (FAN1); replication protein A (RPA); structure-specific endonuclease