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Published on: June 26, 2020
FANCM of the Fanconi anemia core complex is required for both monoubiquitination and DNA repair
Yutong Xue1, Yongjiang Li, Rong Guo
1Laboratory of Genetics, National Institute on Aging, National Institutes of Health, 333 Cassell Drive, TRIAD Center Room 3000, Baltimore, MD 21224, USA.
Abstract:
In response to DNA damage, the Fanconi anemia (FA) core complex functions as a signaling machine for monoubiquitination of FANCD2 and FANCI. It remains unclear whether this complex can also participate in subsequent DNA repair. We have shown previously that the FANCM constituent of the complex contains a highly conserved helicase domain and an associated ATP-dependent DNA translocase activity. Here we show that FANCM also possesses an ATP-independent binding activity and an ATP-dependent bi-directional branch-point translocation activity on a synthetic four-way junction DNA, which mimics intermediates generated during homologous recombination or at stalled replication forks. Using an siRNA-based complementation system, we found that the ATP-dependent activities of FANCM are required for cellular resistance to a DNA-crosslinking drug, mitomycin C, but not for the monoubiquitination of FANCD2 and FANCI. In contrast, monoubiquitination requires the entire helicase domain of FANCM, which has both ATP dependent and independent activities. These data are consistent with participation of FANCM and its associated FA core complex in the FA pathway at both signaling through monoubiquitination and the ensuing DNA repair.
Insights
The Fanconi anemia (FA) core complex, particularly FANCM, participates in DNA repair beyond signaling. Its ATP-dependent activities are crucial for cellular resistance to DNA crosslinking agents.
Area of Science:
- Molecular Biology
- Genetics
- DNA Repair Mechanisms
Background:
- The Fanconi anemia (FA) core complex is essential for DNA damage response, primarily known for FANCD2/FANCI monoubiquitination.
- The FANCM protein within the complex possesses helicase and DNA translocase activities, but its role in downstream DNA repair is unclear.
Purpose of the Study:
- To investigate the DNA binding and translocation activities of FANCM.
- To determine the role of FANCM's ATP-dependent activities in cellular resistance to DNA crosslinking agents and FANCD2/FANCI monoubiquitination.
Main Methods:
- Biochemical assays to characterize FANCM's DNA binding and translocation on synthetic four-way junction DNA.
- siRNA-based complementation system to assess cellular resistance to mitomycin C and FANCD2/FANCI monoubiquitination.
Main Results:
- FANCM exhibits ATP-independent binding and ATP-dependent bi-directional branch-point translocation activity on four-way junction DNA.
- ATP-dependent activities of FANCM are essential for mitomycin C resistance but not for FANCD2/FANCI monoubiquitination.
- Monoubiquitination of FANCD2/FANCI requires the entire helicase domain of FANCM, encompassing both ATP-dependent and independent activities.
Conclusions:
- FANCM and the FA core complex are involved in both the signaling (monoubiquitination) and DNA repair steps of the FA pathway.
- FANCM's distinct enzymatic activities contribute to different aspects of the DNA damage response, highlighting its multifaceted role.
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