Identification of RNA binding motifs in the R2 retrotransposon-encoded reverse transcriptase.

R2 non-LTR retrotransposons insert at a specific site in the 28S rRNA genes of many animal phyla. R2 elements encode a single polypeptide with reverse transcriptase, endonuclease and nucleic acid binding domains. Integration involves separate cleavage of the two DNA strands at the target site and utilization of the released 3' ends to prime DNA synthesis. Critical to this integration is the ability of the protein to specifically bind 3' and 5' regions of the R2 RNA. In this report, alanine mutations in two conserved motifs N-terminal to the reverse transcriptase domain were generated and shown to result in proteins that retained the ability to cleave the first strand of the DNA target, to reverse transcribe RNA from an annealed primer and to displace annealed RNA when using DNA as a template. However, the mutant proteins had greatly reduced ability to bind 3' and 5' RNA in mobility shift assays, use the DNA target to prime reverse transcription and conduct second-strand DNA cleavage. These motifs thus appear to participate in all activities of the R2 protein known to require specific RNA binding. The similarity of these R2 RNA binding motifs to those of telomerase and group II introns is discussed.

Insights into the regulation of malate dehydrogenase: inhibitors, activators, and allosteric modulation by small molecules.

Cellular metabolism comprises a complex network of biochemical anabolic and catabolic processes that fuel the growth and survival of living organisms. The enzyme malate dehydrogenase (MDH) is most known for its role in oxidizing malate to oxaloacetate (OAA) in the last step of the tricarboxylic acid (TCA) cycle, but it also participates in the malate-aspartate shuttle in the mitochondria as well as the glyoxylate cycle in plants. These pathways and the specific reactions within them are dynamic and must be carefully calibrated to ensure a balance between nutrient/energy supply and demand. MDH structural and functional complexity requires a variety of regulatory mechanisms, including allosteric regulation, feedback, and competitive inhibition, which are often dependent on whether the enzyme is catalyzing its forward or reverse reaction. Given the role of MDH in central metabolism and its potential as a target for therapeutics in both cancer and infectious diseases, there is a need to better understand its regulation. The involvement of MDH in multiple pathways makes it challenging to identify which effectors are critical to its activity. Many of the in vitro experiments examining MDH regulation were done decades ago, and though allosteric sites have been proposed, none to date have been specifically mapped. This review aims to provide an overview of the current knowledge surrounding MDH regulation by its substrate, products, and other intermediates of the TCA cycle while highlighting all the gaps in our understanding of its regulatory mechanisms.

An Undergraduate Laboratory Exploring Mutational Mechanisms in Escherichia coli Based on the Luria-Delbrück Experiment.

Understanding the mechanism for DNA mutations is a key concept in most genetics and microbiology courses. In addition, understanding that most mutations occur prior to exposure to selection is an important yet often difficult concept for students to grasp. We developed an undergraduate laboratory activity on mutation mechanisms based on the classic experiment from Luria and Delbrück. The activity uses Escherichia coli as the model organism and the antibiotic streptomycin for selection. Students gain hands-on experience with an important experiment in genetics, and the laboratory contains an investigative component in having students calculate mutation rate for streptomycin resistance and in having the students design a follow-up experiment. E. coli has a knockout collection available, and we used a wild-type strain and a ΔmutS strain in the laboratory exercise. The ΔmutS strain is missing an enzyme in the mismatch repair pathway, and students calculate and compare the mutation rate and frequency for both the wild type and the knockout strain. Assessment of student learning showed that students had a significant gain in understanding of mutational mechanisms. An optional, additional experiment involving PCR and DNA sequencing of streptomycin-resistant mutants is also presented.

The diversity of retrotransposons and the properties of their reverse transcriptases.

A number of abundant mobile genetic elements called retrotransposons reverse transcribe RNA to generate DNA for insertion into eukaryotic genomes. Four major classes of retrotransposons are described here. First, the long-terminal-repeat (LTR) retrotransposons have similar structures and mechanisms to those of the vertebrate retroviruses. Genes that may enable these retrotransposons to leave a cell have been acquired by these elements in a number of animal and plant lineages. Second, the tyrosine recombinase retrotransposons are similar to the LTR retrotransposons except that they have substituted a recombinase for the integrase and recombine into the host chromosomes. Third, the non-LTR retrotransposons use a cleaved chromosomal target site generated by an encoded endonuclease to prime reverse transcription. Finally, the Penelope-like retrotransposons are not well understood but appear to also use cleaved DNA or the ends of chromosomes as primer for reverse transcription. Described in the second part of this review are the enzymatic properties of the reverse transcriptases (RTs) encoded by retrotransposons. The RTs of the LTR retrotransposons are highly divergent in sequence but have similar enzymatic activities to those of retroviruses. The RTs of the non-LTR retrotransposons have several unique properties reflecting their adaptation to a different mechanism of retrotransposition.

The reverse transcriptase encoded by the non-LTR retrotransposon R2 is as error-prone as that encoded by HIV-1.

Reverse transcriptases (RTs) encoded by a wide range of mobile retroelements have had a major impact on the structure and function of genomes. Among the most abundant elements in eukaryotes are the non long terminal repeat (LTR) retrotransposons. Here we compare the dNTP concentration requirements and error rates of the RT encoded by the non-LTR retrotransposon R2 of Bombyx mori with the well-characterized RTs of retroviruses. Surprisingly, R2 was found to have properties more similar to those of lentiviral RTs, such as human immunodeficiency virus type 1 (HIV-1), than to those of oncoretroviral RTs, such as murine leukemia virus. Like HIV-1 RT, R2 RT was able to synthesize DNA at low dNTP concentrations, suggesting that R2 is able to retrotranspose in nondividing cells. R2 RT also showed levels of misincorporation in biased dNTP pools and replication error rates in M13 lacZα forward mutation assays, similar to HIV-1 RT. Most of the R2 base substitutions in the forward mutation assay were caused by the misincorporation of dTMP. Analogous to HIV-1, the high error rate of R2 RT appears to be a result of its ability to extend mismatches once generated. We suggest that the low fidelity of R2 RT is a by-product of the flexibility of its active site/dNTP binding pocket required for the target-primed reverse transcription reaction used by R2 for retrotransposition. Finally, we discuss that in spite of the high R2 RT error rate, the long-term nucleotide substitution rate for R2 is not significantly above that associated with cellular DNA replication, based on the frequency of R2 retrotranspositions determined in natural populations.

Reduced dNTP binding affinity of 3TC-resistant M184I HIV-1 reverse transcriptase variants responsible for viral infection failure in macrophage.

We characterized HIV-1 reverse transcriptase (RT) variants either with or without the (-)-2',3'-deoxy-3'-thiacytidine-resistant M184I mutation isolated from a single HIV-1 infected patient. First, unlike variants with wild-type M184, M184I RT variants displayed significantly reduced DNA polymerase activity at low dNTP concentrations, which is indicative of reduced dNTP binding affinity. Second, the M184I variant displayed a approximately 10- to 13-fold reduction in dNTP binding affinity, compared with the Met-184 variant. However, the k(pol) values of these two RTs were similar. Third, unlike HIV-1 vectors with wild-type RT, the HIV-1 vector harboring M184I RT failed to transduce cell types containing low dNTP concentrations, such as human macrophage, likely due to the reduced DNA polymerization activity of the M184I RT under low cellular dNTP concentration conditions. Finally, we compared the binary complex structures of wild-type and M184I RTs. The Ile mutation at position 184 with a longer and more rigid beta-branched side chain, which was previously known to alter the RT-template interaction, also appears to deform the shape of the dNTP binding pocket. This can restrict ground state dNTP binding and lead to inefficient DNA synthesis particularly at low dNTP concentrations, ultimately contributing to viral replication failure in macrophage and instability in vivo of the M184I mutation.

DNA-directed DNA polymerase and strand displacement activity of the reverse transcriptase encoded by the R2 retrotransposon.

R2 elements are non-long terminal repeat (non-LTR) retrotransposons with a single open reading-frame encoding reverse transcriptase, DNA endonuclease and nucleic acid-binding domains. The elements are specialized for insertion into the 28 S rRNA genes of many animal phyla. The R2-encoded activities initiate retrotransposition by sequence-specific cleavage of the 28 S gene target site and the utilization of the released DNA 3' end to prime reverse transcription (target primed reverse transcription). The activity of the R2 polymerase on RNA templates has been shown to differ from retroviral reverse transcriptases (RTs) in a number of properties. We demonstrate that the R2-RT is capable of efficiently utilizing single-stranded DNA (ssDNA) as a template. The processivity of the enzyme on ssDNA templates is higher than its processivity on RNA templates. This finding suggests that R2-RT is also capable of synthesizing the second DNA strand during retrotransposition. However, R2-RT lacks the RNAse H activity that is typically used by retroviral and LTR-retrotransposon RTs to remove the RNA strand before the first DNA strand is used as template. Remarkably, R2-RT can displace RNA strands that are annealed to ssDNA templates with essentially no loss of processivity. Such strand displacement activity is highly unusual for a DNA polymerase. Thus the single R2 protein contains all the activities needed to make a double-stranded DNA product from an RNA transcript. Finally, during these studies we found an unexpected property of the highly sequence-specific R2 endonuclease domain. The endonuclease can non-specifically cleave ssDNA at a junction with double-stranded DNA. This activity suggests that second-strand cleavage of the target site may not be sequence specific, but rather is specified by a single-stranded region generated when the first DNA strand is used to prime reverse transcription.

Modification of human immunodeficiency virus type 1 reverse transcriptase to target cells with elevated cellular dNTP concentrations.

Retroviruses and DNA viruses utilize cellular dNTPs as substrates for their DNA polymerases during viral replication in infected cells. However, because of S phase-dependent dNTP biosynthesis, the availability of cellular dNTPs significantly varies among cell types (e.g. dividing versus nondividing cells and normal versus tumor cells). Here we tested whether alterations in the dNTP utilization efficiency and dNTP binding affinity of viral DNA polymerases can switch viral infection specificity to cell types with different dNTP concentrations. We employed an HIV-1 reverse transcriptase (RT) mutant (Q151N), which is catalytically active only at high dNTP concentrations because of its reduced dNTP binding affinity. Indeed, the modified HIV-1 vector harboring the Q151N mutant RT preferentially transduced tumor cells containing higher cellular dNTP concentrations than primary cells (e.g. human lung fibroblasts (HLFs) and human keratinocytes). Although the wild type HIV-1 vector transduced both HLFs and tumor cells, the Q151N vector failed to transduce HLFs and keratinocytes but efficiently transduced tumor cells. Pretreatment of HLFs with deoxynucleosides, which increase cellular dNTP pools, enabled the mutant vector to transduce HLFs, suggesting that the transduction failure of the RT mutant vector to primary cells is because of inefficient reverse transcription in low cellular dNTP environments. We also observed that the Q151N vector expressing herpes simplex virus-thymidine kinase renders tumor cells sensitive to gancyclovir. This study validates a novel strategy in which modifications of viral DNA polymerases in various vector systems allow the delivery of target genes exclusively to tumor cells exploiting elevated cellular dNTP concentration as a tumor cell-specific host factor.

Kinetic evidence for interaction of human immunodeficiency virus type 1 reverse transcriptase with the 3'-OH of the incoming dTTP substrate.

Two previously identified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants, Q151N and V148I, are known to have reduced dNTP binding affinity but possess wild-type chemical catalysis rates. Structural modeling based on the crystal structure of the HIV-1 RT ternary complex with dTTP proposes that Q151N loses the interaction with the 3'-OH of the incoming dTTP and that V148I disrupts positioning of Q151 for this interaction. On the basis of this, we predicted that while wild-type (WT) HIV-1 RT would have decreased binding affinity to dTTP analogues lacking 3'-OH, compared to dTTP, the Q151N and V148I RT mutants should have decreased but similar affinity to both dTTP and dTTP analogues. Pre-steady-state kinetics on WT RT showed 14- and 53-fold higher K(d) values for the 3'-OH lacking ddTTP and acyTTP, compared to dTTP. In contrast, the Q151N and V148I mutants, which were predicted to have lost H-bonding interaction with the 3'-OH of dTTP, showed higher but similar K(d) values for dTTP, ddTTP, and acyTTP. Interestingly, the Q151N and V148I RTs bound to AZTTP approximately 12 and 18 times more tightly than to dTTP, respectively. Our structure modeling suggests that these RT mutants can interact with the azido moiety of AZTTP, which is 1.4 A longer than the 3'-OH of dTTP. The kinetic data presented in this report demonstrate the functional role of the Q151 residue in HIV-1 RT interaction with dTTP and its analogues containing chemical modifications at the 3'-C of the sugar moiety.

Macrophage tropism of HIV-1 depends on efficient cellular dNTP utilization by reverse transcriptase.

Retroviruses utilize cellular dNTPs to perform proviral DNA synthesis in infected host cells. Unlike oncoretroviruses, which replicate in dividing cells, lentiviruses, such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus, are capable of efficiently replicating in non-dividing cells (terminally differentiated macrophages) as well as dividing cells (i.e. activated CD4+ T cells). In general, non-dividing cells are likely to have low cellular dNTP content compared with dividing cells. Here, by employing a novel assay for cellular dNTP content, we determined the dNTP concentrations in two HIV-1 target cells, macrophages and activated CD4+ T cells. We found that human macrophages contained 130-250-fold lower dNTP concentrations than activated human CD4+ T cells. Biochemical analysis revealed that, unlike oncoretroviral reverse transcriptases (RTs), lentiviral RTs efficiently synthesize DNA even in the presence of the low dNTP concentrations equivalent to those found in macrophages. In keeping with this observation, HIV-1 vectors containing mutant HIV-1 RTs, which kinetically mimic oncoretroviral RTs, failed to transduce human macrophages despite retaining normal infectivity for activated CD4+ T cells and other dividing cells. These results suggest that the ability of HIV-1 to infect macrophages, which is essential to establishing the early pathogenesis of HIV-1 infection, depends, at least in part, on enzymatic adaptation of HIV-1 RT to efficiently catalyze DNA synthesis in limited cellular dNTP substrate environments.