Structural mechanism of DNA interstrand cross-link unhooking by the bacterial FAN1 nuclease

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA interstrand cross-links (ICLs) pose significant threats to genomic stability, impeding DNA replication and transcription.
• The FANCD2-FANCI-associated nuclease (FAN1) is a key enzyme involved in resolving ICLs, functioning independently of the Fanconi anemia pathway.
• Previous studies suggested specific structural features, like a basic pocket or dimerization, are crucial for human FAN1's ICL unhooking activity.

Purpose of the Study:

• To investigate the mechanism of ICL resolution by Pseudomonas aeruginosa FAN1 (PaFAN1), particularly its ability to resolve ICLs despite lacking previously identified human FAN1 features.
• To elucidate the role of specific basic regions within PaFAN1 in substrate recognition and DNA cleavage during ICL repair.
• To compare the ICL unhooking mechanisms of bacterial and human FAN1 and identify conserved functional elements.

Main Methods:

• X-ray crystallography was employed to determine the structures of PaFAN1 bound to various DNA substrates, including those mimicking ICLs.
• Site-directed mutagenesis was used to substitute key residues within the identified basic regions of PaFAN1 and human FAN1.
• In vitro nuclease assays were performed to assess the endonuclease and exonuclease activities of wild-type and mutant FAN1 enzymes on ICL-containing DNA substrates.

Main Results:

• PaFAN1 possesses a conserved Arg/Lys patch that recognizes phosphate groups near the 5' terminus of DNA substrates with flaps or nicks, facilitating cleavage at approximately 3-nt intervals.
• Disruption of Lys-260 in PaFAN1 impaired its exonuclease activity and ICL unhooking, but not initial endonuclease activity.
• Simultaneous disruption of the Arg/Lys patch and the basic pocket in human FAN1 significantly reduced its ICL-resolving activity, indicating a complementary function.

Conclusions:

• The Arg/Lys patch in PaFAN1 plays a crucial role in recognizing DNA termini and guiding the enzyme's catalytic activity for ICL resolution.
• Two distinct basic regions in FAN1, the Arg/Lys patch and the basic pocket, work complementarily to efficiently unhook DNA interstrand cross-links.
• These findings highlight a conserved mechanism involving basic regions in FAN1 enzymes across species for maintaining genomic stability.