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Human FAN1 promotes strand incision in 5'-flapped DNA complexed with RPA
Daisuke Takahashi1, Koichi Sato1, Emiko Hirayama1
1Laboratory of Structural Biology, Graduate School of Advanced Science & Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan and.
Abstract:
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.
Insights
Fanconi anaemia-associated nuclease 1 (FAN1) effectively incises DNA at stalled replication forks, even when coated by Replication protein A (RPA). This finding is crucial for understanding DNA interstrand crosslink repair in Fanconi anaemia.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Fanconi anaemia (FA) is a recessive disorder impacting DNA repair.
- Efficient DNA interstrand crosslink (ICL) repair requires dual DNA strand incisions.
- FA-associated nuclease 1 (FAN1) is implicated in DNA incision at stalled replication forks.
Purpose of the Study:
- To investigate the effect of Replication protein A (RPA) on FAN1-mediated DNA incision.
- To characterize the endonuclease activity of purified human FAN1.
- To determine FAN1's role in repairing RPA-coated DNA structures.
Main Methods:
- Purification of bacterially expressed recombinant human FAN1.
- Assay of FAN1 endonuclease activity on 5'-flapped DNA substrates.
- Analysis of FAN1 incision efficiency on RPA-coated 5'-flapped DNA.
Main Results:
- Purified human FAN1 demonstrated robust endonuclease activity on 5'-flapped DNA.
- FAN1 efficiently promoted DNA incision at the correct site on RPA-coated 5'-flapped DNA.
- RPA binding did not inhibit, but rather facilitated, FAN1's DNA incision activity.
Conclusions:
- FAN1 can effectively process RPA-coated 5'-flapped DNA structures.
- FAN1 plays a significant role in the DNA interstrand crosslink (ICL) repair pathway.
- These findings elucidate FAN1's function in maintaining genomic stability in the context of FA.
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