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Dmgen Shows A Flap Endonuclease Activity, Cleaving The Blocked-flap Structure And Model Replication Fork

Tác giả: Yoshihiro Kanai, Gen Ishikawa, Ryo Takeuchi, Tatsushi Ruike, Ryo-ichi Nakamura, Ayumi Ihara, Tetsuyuki Ohashi, Kei-ichi Takata, Seisuke Kimura and Kengo Sakaguchi

Lĩnh vực: Applied Biological Science, Tokyo University of Science, Chiba, Japan

Nội dung tài liệu: Nghiên cứu này tập trung vào việc xác định đặc tính sinh hóa của một loại enzyme mới có tên là DmGEN, thuộc họ nuclease RAD2/XPG. DmGEN sở hữu hai vùng nuclease (miền N và miền I) tương tự như các nuclease XPG/class I. Tuy nhiên, nó có đặc điểm cấu trúc khác biệt, với hai miền nuclease nằm gần nhau như ở các nuclease FEN-1/class II và EXO-1/class III. Nghiên cứu đã xác định DmGEN có hoạt tính nuclease flap, đặc biệt ưu tiên cấu trúc flap bị chặn (blocked-flap) làm cơ chất. Enzyme này cũng có khả năng cắt chuỗi lagging của cấu trúc chạc sao chép mô hình. Các kết quả này cho thấy DmGEN đại diện cho một lớp mới (class IV) trong họ nuclease RAD2/XPG.

Mục lục chi tiết:

  • Keywords
  • Correspondence
  • Abbreviations
  • DmGEN shows a flap endonuclease activity, cleaving the blocked-flap structure and model replication fork
  • Drosophila melanogaster XPG-like endonuclease (DmGEN) is a new category of nuclease belonging to the RAD2/XPG family.
  • The RAD2/XPG family of nucleases, which have two conserved nuclease domains (the N domain and the I domain), are currently separated into three classes (XPG/class I, FEN-1/class II and EXO-1/class III) based on the types of nuclease activity and sequence homology.
  • DmGEN also has these conserved nuclease domains. The N and I domains of DmGEN were similar to those of XPG/class I.
  • The N domain of DmGEN showed 35.1, 25.0 and 10.9% homology (% identity) with the N domains of HsXPG, HsFEN-1 and HsEXO-1, respectively.
  • The I domain of DmGEN showed 44.2, 38.5 and 38.5% homology (% identity) with the I domains of HsXPG, HsFEN-1 and HsEXO-1, respectively.
  • The spacer region between the N and I domains is not required for nuclease activity, but contributes to substrate specificity.
  • DmGEN protein showed a relatively high degree of sequence homology with RAD2 nucleases, particularly XPG.
  • DmGEN had flap endonuclease activity, like FEN-1, but was not able to cleave the bubble structure, unlike XPG.
  • The flap endonuclease activity of DmGEN is more accurate and weaker than that of FEN-1.
  • DmGEN preferred a substrate in which the 5′-upstream of the flap is completely double-stranded.
  • DmGEN cleaved the lagging strand of the model replication fork with gaps.
  • DmGEN was localized in the nucleus throughout the 13 nuclear division cycles.
  • The biochemical properties of class IV nucleases are largely unknown in various animals and plants.
  • We suggest that DmGEN should be categorized in a new class (class IV) of the RAD2/XPG family.
  • Experimental procedures
  • Acknowledgements
  • References
  • Supplementary material