MCM8/9 and FANCD2 interact within a shared pathway in response to replication stress caused by DNA crosslinks

Area of Science:

• Molecular Biology
• Genetics
• DNA Repair Mechanisms

Background:

• Genomic integrity is maintained by proteins like Fanconi anemia (FA) pathway proteins and MCM8/9 helicase complex, especially under replication stress.
• The precise relationship and functional cooperation between FANCD2 and MCM8/9 in DNA repair remain largely uncharacterized.

Purpose of the Study:

• To elucidate the functional relationship and interaction between the MCM8/9 helicase complex and FANCD2 in the context of DNA interstrand crosslink (ICL) repair.
• To determine the molecular mechanisms underlying their cooperation and their roles in maintaining genomic stability.

Main Methods:

• Immunofluorescence and co-immunoprecipitation assays to investigate protein interactions and localization.
• Functional assays including γH2AX DNA damage assessment and cell survival studies.
• Genetic manipulation using knockout and knockdown approaches for MCM8, MCM9, and HROB.

Main Results:

• MCM8/9 directly interacts with the FANCD2 complex and is recruited to ICL-induced nuclear foci, but independently of FANCD2 monoubiquitination.
• FANCD2 is essential for MCM8/9 recruitment, while MCM8/9 foci formation requires ATPase activity, the BRCv motif, and HROB, but not for FANCD2 binding.
• Loss of MCM8/9 or HROB leads to increased FANCD2 foci, suggesting MCM8/9 mitigates replication stress. Combined loss of MCM9 and FANCD2 shows epistatic effects, indicating they function in the same pathway.

Conclusions:

• MCM8/9 acts as a downstream interactor and effector of FANCD2 in the DNA ICL repair pathway.
• The findings clarify the functional cooperation between MCM8/9 and FANCD2, highlighting their combined importance in resolving DNA damage and maintaining genomic stability.