PMC

RNase HI/1 and RNase HII/2 have distinct cleavage patterns on three different substrates. Three types of substrates are shown. (A) A single ribonucleotide in a duplex DNA that is cleaved by RNases HII/2 but not RNases HI/1 as noted by X. (B) Four consecutive ribonucleotides residues and (C) an RNA/DNA hybrid that is cleaved differently by the two classes of enzymes. Black arrows denote sites of cleavage by RNase HI/1 and red arrows represent hydrolysis by RNase HII/2.

(A) Comparison of amino acid sequences of human and S. cerevisiae RNase H2 subunits. Alignments are based on the consensus alignment of RNase H2 subunits from Crow et al. Reverse letters highlight homologies between the subunits. For RNases H2B and RNases H2C, a more generous highlighting is used and even then it is clear there is very little conservation between the human and yeast B and C subunits. (B) Schematic representation of the three components of human RNase H2. On top of each subunit are shown AGS-related mutations, and on the bottom the amino acids involved in catalysis, in RNASEH2A, and the PIP (PCNA Interacting Peptide) site of RNASEH2B.

RNase H1 organization in eukaryotes. (A) Two types of RNases H1 in eukaryotes are shown. The top “typical organization” is found in most RNases H1 from higher eukaryotes and consists of an MTS (Mitochondrial Targeting Sequence), HBD (Hybrid Binding Domain), CD (Connection Domain) and H-Domain (RNase H Domain). Not all “typical” RNases H1 have an MTS. Some, mainly fungal, RNases H1 have two HBDs as shown for S. cerevisiae. (B) Alignments of human (Hs), mouse (Mo), and three RNase H1 type proteins encoded in C. albicans (Ca1-3). The red bars represent the β-strands of human RNase H1, and the blue bars are the α-helices. The basic protrusion is shaded in grey as noted in the H-Domain alignment. (C) Alignment of the HBDs of S. cerevisiae RNase H1. Red circles in C and B are the partially conserved HBD amino acids discussed in the text.

Structural model. Interplay of HBD and H-Domain is orchestrated by the connection domain. (A) Co-crystal structure of human RNase H1 HBD and RNase H domain with RNA/DNA. Left and middle panels show HBD modeled either on the opposite side of RNase H domain (left) or immediately upstream (middle). The C-terminus of the HBD is connected to the N-terminus of the RNase H domain as indicated by dashed brown lines. The phosphate binding pocket is indicated as a “P” inside a grey circle. The middle panel also highlights the position of the basic protrusion. The right panel shows the RNA/DNA with the protein removed revealing the bend in the DNA marked by an arrow. (B) Processivity of RNase H1. In the model of processivity of RNase H1, the HBD anchors the enzyme to a hybrid permitting the RNase H domain to engage with several sites on a single RNA/DNA leading to multiple cleavages from a single binding event. In position A of the H-Domain, hydrolysis will occur near the HBD while in position B cleavage will be more distal. To achieve binding and cleavage at position C, the connection domain needs to be flexible to change the relative orientation of the HBD and H-Domains although the orientation with respect to the hybrid must remain the same.