Structural Basis Of Substrate Recognition And Methvlation Regulation Of Human Flap Endonuclease 1

Human flap endonuclease 1(FEN1)is a conserved structure-specific nuclease,which has 5' flap endonuclease(FEN),5' exonuclease(EXO)and gap endonuclease(GEN)activities.In human cell,FEN1 is essential for eukaryotic Okazaki fragment maturation in DNA replication process.In addition,FEN1 is also involved in the long-patch base excision repair pathway,which is critical for the maintenance of genome stability.Thus,the nuclease activities and the cellular localization of FEN1 must be properly regulated.Deficiency of FEN 1 impaired the DNA replication and repair,which is the underlying cause of many disease including Huntington's disease and cancer.Current studies suggest that at least two mechanisms contribute to the regulation of FEN1:namely the substrate recognition and protein post-translational modifications(PTMs).Despite the weak exonuclease activity in vitro,FEN1 is mainly an endonuclease in vivo.Biochemical experiments suggest that double flap DNA with a characteristic single nucleotide 3' flap,which could be accurately and efficiently digested,is the preferred substrate for FEN 1.Two different mechanisms of FEN1 cleavage have been proposed,which were both supported by experimental observations.In the "tracking model",FEN1 protein firstly entered the 5' end of DNA and then slided along the flap to the junction for cleavage.The "threading" model suggests that FEN 1 binded to DNA junction first,followed by 5' flap recognition and cleavage.On the other hand,the nuclease activities of FEN 1 can be finely regulated by PTMs including methylation,phosphorylation and ubiquitination.The methylation of FEN 1 at arginine 192 site enhanced its interaction with PCNA and is essential for its recruitment into the nucleus.The phosphorylation at serine 187 site is critical for FEN1 dissociation from replication fork.Interestingly,the phosphorylation of FEN1 is strongly inhibited by its methylation,suggesting the cross-talk between these two types of PTMs.However,the detailed mechanism is still unclear.In this study,FEN1 was co-crystallized with substrate double flap DNA.Combined with biochemical assays and kinetics measurements,we proposed a model for FEN1 regulation.The main conclusions are:1.We were able to express,purify and co-crystallize codon optimized human FEN1 in apo and binary complex.The crystals were diffracted to beyond to 1.9-2.3 A and structures were determined by molecular replacement.2.The FEN1 apo structure could be superimposed well with published FEN1-DNA structure and the DNA binding regions were disordered due to the lack of substrate DNA.Two divalent metal ions were observed in the active site,which suggests that FEN1 used a two-metal-ion mechanism for catalysis.3.In the pre-cleavage FEN1-DNA complex,the 5' flap of the substrate double flap DNA was observed,for the first time,which supports the "threading" model for its substrate binding.Arginine 47 was observed to interact with 5' flap,suggesting its role in both substrate DNA recognition and helical gateway formation.4.Methylation-mimic mutant(R192F)FEN1 protein was used for co-crystallization with double flap DNA.The FEN1(R192F)-DNA binary structure exhibited identical overall structure and the active-site configuration,which is consistent with the unaffected nuclease activity of methylated FEN1.The ?-pin region is a loop region involved in the substrate binding in the wild-type binary complex.However,this region formed an a helix in FEN1(R192F)-DNA binary structure,which inhibits the interaction between FEN1 and cy cl in-dependent kinase.