Living organisms must keep their DNA content intact to assure the integrity of their genomic information. DNA replication and DNA damage repair are the main efforts to accomplish this task. Hence, the failure of these mechanisms could lead to genomic instability and subsequently development of diseases such as cancers could occur. The DNA2 enzyme was shown to have essential roles in DNA metabolism. In human cells DNA2 was implicated in the completion of DNA replication and the repair of DNA double- strand breaks via the homologous recombination pathway. In addition, DNA2 was shown to be important for telomeric and mitochondrial DNA maintenance as well as Okazaki fragment processing and cell cycle checkpoint act... More
Living organisms must keep their DNA content intact to assure the integrity of their genomic information. DNA replication and DNA damage repair are the main efforts to accomplish this task. Hence, the failure of these mechanisms could lead to genomic instability and subsequently development of diseases such as cancers could occur. The DNA2 enzyme was shown to have essential roles in DNA metabolism. In human cells DNA2 was implicated in the completion of DNA replication and the repair of DNA double- strand breaks via the homologous recombination pathway. In addition, DNA2 was shown to be important for telomeric and mitochondrial DNA maintenance as well as Okazaki fragment processing and cell cycle checkpoint activation in yeast cells. My PhD project started with the preparation of recombinant proteins that allowed biochemical investigations of their functions. I could purify human DNA2 and its variants in an active state and in concentrations sufficient for our assays. Further I produced DNA2 partners WRN and BLM helicases and various single-stranded DNA binding proteins such as the replication protein A (RPA). In my experiments, I could recapitulate DNA2’s nuclease activity and its regulation by RPA. While in the absence of RPA DNA2 degrades 3’ or 5’ terminated single-stranded DNA, the nuclease is restricted to degrade 5’ terminated DNA in the presence of RPA.I was able to describe the helicase activity of human DNA2 nuclease- deficient mutant that had been a matter of contradiction in the field. We observed that the helicase activity of hDNA2 is cryptic and it is masked by its nuclease activity. Studying DNA end resection processes, we found that DNA2 functionally integrates with BLM and WRN helicases. We showed that the helicase activity of DNA2 acts as a motor speeding up the degradation of ssDNA produced by its helicase partners WRN or BLM. We propose that the DNA2 motor acts as a translocase on single-stranded DNA accelerating the degradation by the nuclease domain of hDNA2. I also participated in several collaborative projects showing that DNA2 cooperates with WRN/BLM in DNA end resection and the restart of reversed replication forks with WRN but not BLM in vitro and in vivo. Finally, we showed that RPA possesses double-stranded DNA melting capacity and characterized its dynamics at DNA forks.
