Sharon J. Schultz

Ribonuclease H: properties, substrate specificity and roles in retroviral reverse transcription

Retroviral reverse transcriptases possess both a DNA polymerase and an RNase H activity. The linkage with the DNA polymerase activity endows the retroviral RNases H with unique properties not found in the cellular counterparts. In addition to the typical endonuclease activity on a DNA/RNA hybrid, cleavage by the retroviral enzymes is also directed by both DNA 3′ recessed and RNA 5′ recessed ends, and by certain nucleotide sequence preferences in the vicinity of the cleavage site. This spectrum of specificities enables retroviral RNases H to carry out a series of cleavage reactions during reverse transcription that degrade the viral RNA genome after minus‐strand synthesis, precisely generate the primer for the initiation of plus strands, facilitate the initiation of plus‐strand synthesis and remove both plus‐ and minus‐strand primers after they have been extended.

DNA relaxation by human topoisomerase I occurs in the closed clamp conformation of the protein

In cocrystal structures of human topoisomerase I and DNA, the enzyme is tightly clamped around the DNA helix. After cleavage and covalent attachment of the enzyme to the 3′ end at the nick, DNA relaxation requires rotation of the DNA helix downstream of the cleavage site. Models based on the cocrystal structure reveal that there is insufficient space in the protein for such DNA rotation without some deformation of the cap and linker regions of the enzyme. Alternatively, it is conceivable that the protein clamp opens to facilitate the rotation process. To distinguish between these two possibilities, we engineered two cysteines into the opposing loops of the “lips” region of the enzyme, which allowed us to lock the protein via a disulfide crosslink in the closed conformation around the DNA. Importantly, the rate of DNA relaxation when the enzyme was locked on the DNA was comparable to that observed in the absence of the disulfide crosslink. These results indicate that DNA relaxation likely proceeds without extensive opening of the enzyme clamp.

Sequence, Distance, and Accessibility Are Determinants of 5-End-directed Cleavages by Retroviral RNases H *

The RNase H activity of reverse transcriptase is essential for retroviral replication. RNA 5′-end-directed cleavages represent a form of RNase H activity that is carried out on RNA/DNA hybrids that contain a recessed RNA 5′-end. Previously, the distance from the RNA 5′-end has been considered the primary determinant for the location of these cleavages. Employing model hybrid substrates and the HIV-1 and Moloney murine leukemia virus reverse transcriptases, we demonstrate that cleavage sites correlate with specific sequences and that the distance from the RNA 5′-end determines the extent of cleavage. An alignment of sequences flanking multiple RNA 5′-end-directed cleavage sites reveals that both enzymes strongly prefer A or U at the +1 position and C or G at the –2 position, and additionally for HIV-1, A is disfavored at the –4 position. For both enzymes, 5′-end-directed cleavages occurred when sites were positioned between the 13th and 20th nucleotides from the RNA 5′-end, a distance termed the cleavage window. In examining the importance of accessibility to the RNA 5′-end, it was found that the extent of 5′-end-directed cleavages observed in substrates containing a free recessed RNA 5′-end was most comparable to substrates with a gap of two or three bases between the upstream and downstream RNAs. Together these finding demonstrate that the selection of 5′-end-directed cleavage sites by retroviral RNases H results from a combination of nucleotide sequence, permissible distance, and accessibility to the RNA 5′-end.

Analysis of Plus-strand Primer Selection, Removal, and Reutilization by Retroviral Reverse Transcriptases

The ability of reverse transcriptase to generate, extend, and remove the primer derived from the polypurine tract (PPT) is vital for reverse transcription, since this process determines one of the ends required for integration of the viral DNA. Based on the ability of the RNase H activity of Moloney murine leukemia virus reverse transcriptase to cleave a long RNA/DNA hybrid containing the PPT, it appears that cleavages that could generate the plus-strand primer can occur by an internal cleavage mechanism without any positioning by an RNA 5′-end, and such cleavages may serve to minimize cleavage events within the PPT itself. If the PPT were to be cleaved inappropriately just upstream of the normal plus-strand origin site, the resulting 3′-ends would not be extended by reverse transcriptase. Extension of the PPT primer by at least 2 nucleotides is sufficient for recognition and correct cleavage by RNase H at the RNA-DNA junction to remove the primer. Specific removal of the PPT primer after polymerase extension deviates from the general observation that primer removal occurs by cleavage one nucleotide away from the RNA-DNA junction and suggests that the same PPT specificity determinants responsible for generation of the PPT primer also direct PPT primer removal. Once the PPT primer has been extended and removed from the nascent plus-strand DNA, reinitiation at the resulting plus-strand primer terminus does not occur, providing a mechanism to prevent the repeated initiation of plus strands.

Cleavage Specificities of Moloney Murine Leukemia Virus RNase H Implicated in the Second Strand Transfer During Reverse Transcription

Reverse transcription of a retroviral RNA genome requires two template jumps to generate the linear double-stranded DNA required for integration. The RNase H activity of reverse transcriptase has several roles during this process. We have examined RNase H cleavages that define the maximal 3′ and 5′ ends of Moloney murine leukemia virus minus strand DNA prior to the second template jump. In both the endogenous reaction and on model substrates in vitro, RNase H cleaves the genomic RNA template between the second and third ribonucleotides 5′ of the U5/PBS junction, but other minor cleavages between 1 and 10 nucleotides 5′ of this junction are also observed. Similar experiments examining the specificity of RNase H for tRNA primer removal revealed that cleavage generally leaves a ribo A residue at the 5′ end of minus strand DNA. These observations suggest that three bases are typically duplicated on the ends of the minus strands, leading to an intermediate following the second jump which contains unpaired nucleotides. Model substrates mimicking the structure of this intermediate demonstrate that reverse transcriptase has little difficulty in utilizing such a branched structure for the initiation of displacement synthesis.

Specific Cleavages by RNase H Facilitate Initiation of Plus-Strand RNA Synthesis by Moloney Murine Leukemia Virus

ABSTRACT Successful generation, extension, and removal of the plus-strand primer is integral to reverse transcription. For Moloney murine leukemia virus, primer removal at the RNA/DNA junction leaves the 3′ terminus of the plus-strand primer abutting the downstream plus-strand DNA, but this 3′ terminus is not efficiently reutilized for another round of extension. The RNase H cleavage to create the plus-strand primer might similarly result in the 3′ terminus of this primer abutting downstream RNA, yet efficient initiation must occur to synthesize the plus-strand DNA. We hypothesized that displacement synthesis, RNase H activity, or both must participate to initiate plus-strand DNA synthesis. Using model hybrid substrates and RNase H-deficient reverse transcriptases, we found that displacement synthesis alone did not efficiently extend the plus-strand primer at a nick with downstream RNA. However, specific cleavage sites for RNase H were identified in the sequence immediately following the 3′ end of the plus-strand primer. During generation of the plus-strand primer, cleavage at these sites generated a gap. When representative gaps separated the 3′ terminus of the plus-strand primer from downstream RNA, primer extension significantly improved. The contribution of RNase H to the initiation of plus-strand DNA synthesis was confirmed by comparing the effects of downstream RNA versus DNA on plus-strand primer extension by wild-type reverse transcriptase. These data suggest a model in which efficient initiation of plus-strand synthesis requires the generation of a gap immediately following the plus-strand primer 3′ terminus.

Preferred Sequences within a Defined Cleavage Window Specify DNA 3′ End-directed Cleavages by Retroviral RNases H

The RNase H activity of reverse transcriptase carries out three types of cleavage termed internal, RNA 5′ end-directed, and DNA 3′ end-directed. Given the strong association between the polymerase domain of reverse transcriptase and a DNA 3′ primer terminus, we asked whether the distance from the primer terminus is paramount for positioning DNA 3′ end-directed cleavages or whether preferred sequences and/or a cleavage window are important as they are for RNA 5′ end-directed cleavages. Using the reverse transcriptases of human immunodeficiency virus, type 1 (HIV-1) and Moloney murine leukemia virus (M-MuLV), we determined the effects of sequence, distance, and substrate end structure on DNA 3′ end-directed cleavages. Utilizing sequence-matched substrates, our analyses showed that DNA 3′ end-directed cleavages share the same sequence preferences as RNA 5′ end-directed cleavages, but the sites must fall in a narrow window between the 15th and 20th nucleotides from the recessed end for HIV-1 reverse transcriptase and between the 17th and 20th nucleotides for M-MuLV. Substrates with an RNA 5′ end recessed by 1 (HIV-1) or 2–3 (M-MuLV) bases on a longer DNA could accommodate both types of end-directed cleavage, but further recession of the RNA 5′ end excluded DNA 3′ end-directed cleavages. For HIV-1 RNase H, the inclusion of the cognate dNTP enhanced DNA 3′ end-directed cleavages at the 17th and 18th nucleotides. These data demonstrate that all three modes of retroviral RNase H cleavage share sequence determinants that may be useful in designing assays to identify inhibitors of retroviral RNases H.