Mechanistic Insights into the Cleavage Kinetics of the Superfamily of 5’ Nucleases: A Case of GEN1 and XPG

  • Amer Bralic

Student thesis: Doctoral Thesis


Nucleases encompass a group of enzymes that cleave the phosphodiester bonds of nucleic acids and as such are indispensable for life. We investigated the catalytic mechanisms of two members of the structure-specific FEN1-like 5’ exo- and endonuclease superfamily, namely Flap endonuclease GEN homolog 1 (GEN1) and Xeroderma Pigmentosum Group G DNA repair protein (XPG). While cleavage by the nucleases is necessary in many pathways, unwanted cleavage is detrimental and needs to be strongly regulated and suppressed. Human GEN1 is a cytosolic homologous recombination protein that resolves persisting four-way Holliday junctions (HJ). Through single-molecule FRET (smFRET) we show that the GEN1 monomer generally follows the isomer bias of the HJ in its initial binding and subsequently distorts it for catalysis. It remains tightly bound but has no catalytic activity until a GEN1 dimer is formed. Fast on- and slow off-rates of GEN1 dimer and its increased affinity to the singly-cleaved HJ enforce the forward reaction to always ensure complete HJ resolution. GEN1, therefore, catches singly-cleaved HJs and leads to dimerization which seemingly cleaves without fail. Another vital pathway relying on nucleases is the Nucleotide Excision Repair (NER). It is a multi-protein and multi-step mechanism culminating in the excision of a damage-harboring ≈25-27nt DNA fragment. XPG is among the first proteins recruited during NER, yet it is the last to execute. We used FeS cluster Induced Fluorescence Quenching as part of our smFRET assays to show that the TFIIH XPG complex unwinds ≈11 fold better and cleaves ≈100 fold better than TFIIH and XPG on their own. Interestingly, this stimulation requires the positioning of XPD on a stretch of ≈15-20 nts of ssDNA. While translocating, the incision activity of TFIIH-XPG seems to be strongly suppressed. Upon damage encounter and stalling, however, TFIIH-XPG is triggered for incision. Our results, therefore, suggest an intrinsic DNA-mediated protein-protein interaction between TFIIH and XPG which facilitates NER steps up to the second incision. Therefore, both in the case of GEN1 and XPG, the mode of operation is to strongly suppress the nuclease activity until activated by DNA-mediated protein-protein interactions.
Date of AwardDec 2022
Original languageEnglish (US)
Awarding Institution
  • Biological, Environmental Sciences and Engineering
SupervisorSamir Hamdan (Supervisor)


  • Nucleases
  • DNA repair
  • GEN1
  • FEN1
  • XPG
  • Nucleotide Excision Repair

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