tailieunhanh - Báo cáo khoa học: DmGEN shows a flap endonuclease activity, cleaving the blocked-flap structure and model replication fork

Drosophila melanogasterXPG-like endonuclease (DmGEN) is a new cate-gory of nuclease belonging to the RAD2⁄XPG family. The DmGEN pro-tein has two nuclease domains (N and I domains) similar to XPG⁄class I nucleases; however, unlike class I nucleases, in DmGEN these two nuclease domains are positioned close to each other as in FEN-1⁄class II and EXO-1⁄ class III nucleases. | ễFEBS Journal DmGEN shows a flap endonuclease activity cleaving the blocked-flap structure and model replication fork Yoshihiro Kanai Gen Ishikawa Ryo Takeuchi Tatsushi Ruike Ryo-ichi Nakamura Ayumi Ihara Tetsuyuki Ohashi Kei-ichi Takata Seisuke Kimura and Kengo Sakaguchi Department of Applied BiologicalScience Tokyo University of Science Chiba Japan Keywords active-site mutant DmGEN novel flap endonuclease nuclease activity site-directed mutagenesis Correspondence K. Sakaguchi Department of Applied Biological Science Faculty of Science and Technology Tokyo University of Science 2641 Yamazaki Noda-shi Chiba-ken 278-8510 Japan Fax 81 4 7123 9767 Tel. 81 4 7124 1501 Ex. 3409 E-mail kengo@ Received 5 April 2007 revised 24 May 2007 accepted 7 June 2007 doi Drosophila melanogaster XPG-like endonuclease DmGEN is a new category of nuclease belonging to the RAD2 XPG family. The DmGEN protein has two nuclease domains N and I domains similar to XPG class I nucleases however unlike class I nucleases in DmGEN these two nuclease domains are positioned close to each other as in FEN-1 class II and EXO-1 class III nucleases. To confirm the properties of DmGEN we characterized the active-site mutant protein E143A E145A and found that DmGEN had flap endonuclease activity. DmGEN possessed weak nick-dependent 5 -3 exonuclease activity. Unlike XPG DmGEN could not incise the bubble structure. Interestingly based on characterization of flap endonuclease activity DmGEN preferred the blocked-flap structure as a substrate. This feature is distinctly different from FEN-1. Furthermore DmGEN cleaved the lagging strand of the model replication fork. Immunostaining revealed that DmGEN was present in the nucleus of actively proliferating Drosophila embryos. Thus our studies revealed that DmGEN belongs to a new class class IV of the RAD2. XPG nuclease family. The biochemical properties of DmGEN and its possible role are also discussed. DNA .

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