Abstract
MUS81-EME1 is a DNA endonuclease involved in replication-coupled repair of DNA interstrand cross-links (ICLs). A prevalent hypothetical role of MUS81-EME1 in ICL repair is to unhook the damage by incising the leading strand at the 3′ side of an ICL lesion. In this study, we report that purified MUS81-EME1 incises DNA at the 5′ side of a psoralen ICL residing in fork structures. Intriguingly, ICL repair protein, Fanconi anemia complementation group A protein (FANCA), greatly enhances MUS81-EME1-mediated ICL incision. On the contrary, FANCA exhibits a two-phase incision regulation when DNA is undamaged or the damage affects only one DNA strand. Studies using truncated FANCA proteins indicate that both the N- and C-moieties of the protein are required for the incision regulation. Using laser-induced psoralen ICL formation in cells, we find that FANCA interacts with and recruits MUS81 to ICL lesions. This report clarifies the incision specificity of MUS81-EME1 on ICL damage and establishes that FANCA regulates the incision activity of MUS81-EME1 in a damage-dependent manner.
Highlights
Interstrand cross-links (ICLs) covalently tether both strands of a DNA helix and block essential DNA transactions including replication and transcription
When an ICL blocks the replication machinery, a protein complex termed as the Fanconi anemia core complex is recruited to stalled replication forks and monoubiquitinates another two Fanconi anemia proteins FANCD2 and FANCI
Fanconi anemia cells are hypersensitive to DNA cross-linking compounds including mitomycin C, cisplatin and diepoxybutane, indicating that they are defective in repairing ICLs [5,11,12,13,14,15,16,17,18,19]
Summary
Interstrand cross-links (ICLs) covalently tether both strands of a DNA helix and block essential DNA transactions including replication and transcription. Using nuclear protein extracts and complementation analysis, it was demonstrated that FANCA is required for efficient incisions at the sites of psoralen-mediated ICLs [30] These data imply that FANCA may function outside the Fanconi anemia core complex and directly participate in ICL incision. Kanaar and colleagues found that MUS81 is not involved in the generation of DSBs from DNA damage that affects only one strand of the DNA duplex [48] These results indicate that the structure-specific DNA endonuclease MUS81-EME1 is involved in incision of ICLs, but not non-ICL DNA damage, residing in a replication fork. We found that FANCA regulates the endonuclease activity of MUS81-EME1 in a damagedependent manner
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