tailieunhanh - báo cáo khoa hoc : Analysis of subunit assembly and function of the Saccharomyces cerevisiae RNase H2 complex

RNase H2 ofSaccharomyces cerevisiaeconsists of three essential subunits (Rnh201, Rnh202 and Rnh203) and plays a critical role in the removal of RNA incorporated in duplex DNA. In the present study, we purified indi-vidual subunits and heterodimeric subcomplexes to examine the assembly and biochemical function of subunits of RNase H2in vitro. | IFEBS Journal Analysis of subunit assembly and function of the Saccharomyces cerevisiae RNase H2 complex Tuan Anh Nguyen Yon-Soo Tak Chul-Hwan Lee Young-Hoon Kang Il-Taeg Cho and Yeon-Soo Seo Center for DNA Replication and Genome Instability Department of BiologicalSciences Korea Advanced Institute of Science and Technology Daejeon South Korea Keywords Dna2 Fen1 genome stability Okazaki fragment processing PCNA RNase H2 Correspondence . Seo Center for DNA Replication and Genome Instability Department of BiologicalSciences Korea Advanced Institute of Science and Technology Daejeon 305-701 South Korea Fax 82 42 350 2610 Tel 82 42 350 2637 E-mail yeonsooseo@ Received 29 June 2011 revised 30 September 2011 accepted 3 October 2011 doi RNase H2 of Saccharomyces cerevisiae consists of three essential subunits Rnh201 Rnh202 and Rnh203 and plays a critical role in the removal of RNA incorporated in duplex DNA. In the present study we purified individual subunits and heterodimeric subcomplexes to examine the assembly and biochemical function of subunits of RNase H2 in vitro. Reconstitution experiments revealed that Rnh202 and Rnh203 first form a subcomplex followed by the recruitment of Rnh201 to complete complex formation. Rnh201 alone or in combination with Rnh203 showed neither substratebinding nor catalytic activity indicating that both activities of Rnh201 are latent until it becomes an integral part of the complex. However Rnh202 by itself showed substrate-binding activity. RNase H2 containing mutant Rnh202 defective in substrate binding had decreased substrate-binding activity indicating that Rnh202 contributes directly to substrate binding. Reconstitution of RNase H2 complexes with various mutant subunits allowed us to assess the influence of conserved amino acid residues in either Rnh201 or Rnh202 on substrate-binding and catalytic activities. We found that the substrate-binding activities of both Rnh201 and Rnh202 .