Analysis of subunit assembly and function of the Saccharomyces cerevisiae RNase H2 complex

Abstract

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 were critical for cleavage of the phosphodiester bond present between DNA and RNA in RNase H2 substrates. Structured digital abstractl RNH202 physically interacts with RNH201 by pull down (View interaction) l RNH201 binds to RNH203 by pull down (View interaction) l RNH202 binds to RNH203 by pull down (View interaction)

Ribonuclease H2 in health and disease

“…The eukaryotic homologue is the RNASEH2A subunit, and although it has no enzyme activity by itself, it forms an active heterotrimeric complex together with the accessory RNASEH2B and RNASEH2C subunits [12,14,24,25]. A high level of structural similarity between prokaryotic RNase HII and eukaryotic RNASEH2A was first shown when the crystal structure of the mouse enzyme was solved by Hollis and colleagues [26].…”

“…For example, a PIP-box [PCNA (proliferating-cell nuclear antigen)-interacting protein box] motif at the C-terminus of the RNASEH2B subunit guides the interaction between RNase H2 and PCNA [12] and its localization to replication foci [28]. Experiments with the S. cerevisiae proteins suggest that the B subunit may also have a role in substrate binding [25].…”

Transcript-RNA-templated DNA recombination and repair

“…To confirm that inactivation of RNase H1 and H2 allows for direct transcript RNA repair of a DSB in homologous DNA, we conducted a complementation test in the cis system using a vector expressing either a catalytically inactive mutant of RNH201, rnh201 -D39A 15 , or wild-type RNH201 . Results showed that when wild-type RNH201 was expressed from the plasmid in rnh1 rnh201 spt3 cells, there were no His + colonies following DSB induction ( Extended Data Fig.…”

A New Method, “Reverse Yeast Two-Hybrid Array” (RYTHA), Identifies Mutants that Dissociate the Physical Interaction Between Elg1 and Slx5

“…Rnh201 is the catalytic subunit of the RNase H2 complex, which protects genome integrity by removing RNA nucleotides incorporated into DNA during replication and/or Okazaki fragment synthesis (Nguyen et al 2011;Wahba et al 2011;Amon and Koshland 2016). Rnh201 plays a key role in DNA damage response and DNA replication processes (Allen-Soltero et al 2014).…”