Abstract
DNA interstrand cross-links (ICLs) block the progress of the replication and transcription machineries and can weaken chromosomal stability, resulting in various diseases. FANCD2-FANCI-associated nuclease (FAN1) is a conserved structure-specific nuclease that unhooks DNA ICLs independently of the Fanconi anemia pathway. Recent structural studies have proposed two different mechanistic features for ICL unhooking by human FAN1: a specific basic pocket that recognizes the terminal phosphate of a 1-nucleotide (nt) 5' flap or FAN1 dimerization. Herein, we show that despite lacking these features, Pseudomonas aeruginosa FAN1 (PaFAN1) cleaves substrates at ∼3-nt intervals and resolves ICLs. Crystal structures of PaFAN1 bound to various DNA substrates revealed that its conserved basic Arg/Lys patch comprising Arg-228 and Lys-260 recognizes phosphate groups near the 5' terminus of a DNA substrate with a 1-nt flap or a nick. Substitution of Lys-260 did not affect PaFAN1's initial endonuclease activity but significantly decreased its subsequent exonuclease activity and ICL unhooking. The Arg/Lys patch also interacted with phosphates at a 3-nt gap, and this interaction could drive movement of the scissile phosphates into the PaFAN1-active site. In human FAN1, the ICL-resolving activity was not affected by individual disruption of the Arg/Lys patch or basic pocket. However, simultaneous substitution of both FAN1 regions significantly reduced its ICL-resolving activity, suggesting that these two basic regions play a complementary role in ICL repair. On the basis of these findings, we propose a conserved role for two basic regions in FAN1 to guide ICL unhooking and to maintain genomic stability.
Highlights
DNA interstrand cross-links (ICLs) block the progress of the replication and transcription machineries and can weaken chromosomal stability, resulting in various diseases
Because Homo sapiens FAN1 (HsFAN1) most efficiently binds to a DNA with a 1-nt 5Ј flap and a 4 – 8-nt 3Ј flap, we examined the activity of Pseudomonas aeruginosa FAN1 (PaFAN1) on a 1-nt 5Ј flap DNA [33]
Mammalian FAN1 can bind to the proximal site of replisome stalling via interaction with the ID complex, the nuclease directly recognizes the substrate DNA and processes replication-like structures and ICLs [26, 32,33,34,35]
Summary
We have used a DNA substrate with a 4-nucleotide 5Ј flap [23]. because HsFAN1 most efficiently binds to a DNA with a 1-nt 5Ј flap and a 4 – 8-nt 3Ј flap, we examined the activity of PaFAN1 on a 1-nt 5Ј flap DNA [33]. This interaction scrunches the three nucleotides in the gap, facilitating the translocation of the post-nick duplex toward the NTD, positioning the scissile phosphate of the post-nick into the active site Based on this structure, we propose that the binding of the gap to the Arg/Lys patch can be used as a driving force to reposition the post-nick strand for successive exonuclease activities by PaFAN1 following the initial endonuclease incision. The K260A and R228A/K260A proteins showed initial endonuclease activity (product a) similar to that of the WT PaFAN1, the exonuclease activity (product b to d) of these two mutants was slightly but reproducibly decreased This result suggests that Lys-260 is important for exonucleolytic activity, likely through an interaction with terminal phosphate(s) of the initially cleaved DNA substrate, which could facilitate the translocation of the post-nick DNA with respect to the active site. We do not rule out the possibility that processive binding can occur and may be detected under different assay conditions because both NTD and CTD quite interact with the pre-nick and post-nick duplex, respectively
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