Abstract
Fanconi anemia (FA) is a multigenic disease of bone marrow failure and cancer susceptibility stemming from a failure to remove DNA crosslinks and other chromosomal lesions. Within the FA DNA damage response pathway, DNA-dependent monoubiquitinaton of FANCD2 licenses downstream events, while timely FANCD2 deubiquitination serves to extinguish the response. Here, we show with reconstituted biochemical systems, which we developed, that efficient FANCD2 deubiquitination by the USP1-UAF1 complex is dependent on DNA and DNA binding by UAF1. Surprisingly, we find that the DNA binding activity of the UAF1-associated protein RAD51AP1 can substitute for that of UAF1 in FANCD2 deubiquitination in our biochemical system. We also reveal the importance of DNA binding by UAF1 and RAD51AP1 in FANCD2 deubiquitination in the cellular setting. Our results provide insights into a key step in the FA pathway and help define the multifaceted role of the USP1-UAF1-RAD51AP1 complex in DNA damage tolerance and genome repair.
Highlights
Cells from patients with Fanconi anemia (FA) exhibit hypersensitivity to mitomycin C (MMC), reactive aldehydes, and other agents that cause interstrand DNA crosslinks (ICLs) or interfere with DNA replication[1,2]
Given that ID2 mono-ubiquitination requires DNA9,30 and that modified ID2 remains associated with chromatin in cells[10], we hypothesized that ID2 deubiquitination may be dependent on DNA
We have employed reconstitution biochemistry and cell-based tests to define the importance of the DNA binding attribute of UAF1 and RAD51AP1 in FANCD2 deubiquitination (Fig. 4e)
Summary
Cells from patients with Fanconi anemia (FA) exhibit hypersensitivity to mitomycin C (MMC), reactive aldehydes, and other agents that cause interstrand DNA crosslinks (ICLs) or interfere with DNA replication[1,2]. Owing to the broader involvement of UAF1 in DNA damage repair and other biological processes, its loss engenders a more severe phenotype than USP1 ablation, as evidenced by early embryonic lethality in mice and a higher degree of cellular sensitivity to DNA damaging agents[20,21,28,29]. Both the unmodified and mono-ubiquitinated forms of ID2 bind DNA avidly, and there is good evidence that modified ID2 remains associated with DNA lesions until damage repair or translesion DNA synthesis has occurred[10]. It was suggested recently that DNA shields ubiquitinated ID2 from the DUB activity of USP1-UAF1, such that the removal of DNA would lead to a marked enhancement of the efficiency of ID2 deubiquitination[11]
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