Actin and myosin contribute to mammalian mitochondrial DNA maintenance.

Mitochondrial DNA maintenance and segregation are dependent on the actin cytoskeleton in budding yeast. We found two cytoskeletal proteins among six proteins tightly associated with rat liver mitochondrial DNA: non-muscle myosin heavy chain IIA and ß-actin. In human cells, transient gene silencing of MYH9 (encoding non-muscle myosin heavy chain IIA), or the closely related MYH10 gene (encoding non-muscle myosin heavy chain IIB), altered the topology and increased the copy number of mitochondrial DNA; and the latter effect was enhanced when both genes were targeted simultaneously. In contrast, genetic ablation of non-muscle myosin IIB was associated with a 60% decrease in mitochondrial DNA copy number in mouse embryonic fibroblasts, compared to control cells. Gene silencing of ß-actin also affected mitochondrial DNA copy number and organization. Protease-protection experiments and iodixanol gradient analysis suggest some ß-actin and non-muscle myosin heavy chain IIA reside within human mitochondria and confirm that they are associated with mitochondrial DNA. Collectively, these results strongly implicate the actomyosin cytoskeleton in mammalian mitochondrial DNA maintenance.

Quantitative regulation of class switch recombination by switch region transcription.

The isotype specificity of immunoglobulin (Ig) class switching is regulated by a cytokine which induces transcription of a specific switch (S) region, giving rise to so-called germline transcripts. Although previous studies have demonstrated that germline transcription of an S region is required for class switch recombination (CSR) of that particular S region, it has not been shown whether the level of S region transcription affects the efficiency of CSR. We addressed this question by using an artificial DNA construct containing a constitutively transcribed mu switch (Smu) region and an alpha switch (Salpha) region driven by a tetracycline-responsive promoter. The construct was introduced into a switch-inducible B lymphoma line and the quantitative correlation between Salpha region transcription and class switching efficiency was evaluated. The level of Salpha transcription was linearly correlated with CSR efficiency, reaching a plateau at saturation. On the other hand, we failed to obtain the evidence to support involvement of either RNA-DNA heteroduplex or trans germline transcripts in CSR. Taken together, it is likely that S region transcription and/or transcript processing in situ may be required for CSR. We propose that because of the unusual properties of S region DNA, transcription induces the DNA to transiently be single stranded, permitting secondary structure(s) to form. Such structures may be recognition targets of a putative class switch recombinase.

HIV-1 reverse transcriptase interaction with model RNA-DNA duplexes.

HIV-1 reverse transcriptase (HIV-1 RT) is a multifunctional enzyme responsible for converting viral RNA into preintegrative DNA during the early stages of viral infection. DNA polymerase and RNase H activities are required, and several conformationally distinct primer-templates must be accommodated by the enzyme during the process. Parameters of interaction between model substrates (ligands) and HIV-1 RT (wild type p66/p51 and the RNase H-deficient mutant p66(E478Q)/p51) (analytes) were estimated by surface plasmon resonance at 25 degrees C, pH 8.0. Binding of RT to the ligands is specific and can be analyzed using a conventional 1:1 binding algorithm. RNA-DNA hybrids with 5'-template overhangs of 6 and 12 nucleotides bind to RT approximately one order of magnitude stronger than the corresponding 36-mer with blunt ends due to slower dissociation. Immobilization of the latter through either the 5'-end of RNA or DNA strand does not change the equilibrium constant (K(D)) for wild-type RT but the values of kinetic constants of association and dissociation differ significantly. For the p66(E478Q)/p51 enzyme, orientation effects are notable even altering the K(D) value. Binding of the p66(E478Q)/p51 to any RNA-DNA hybrids is slightly stronger compared with wild type. Data can be interpreted in terms of the mechanism of reverse transcription.

Sorting of mannose 6-phosphate receptors mediated by the GGAs.

The delivery of soluble hydrolases to lysosomes is mediated by the cation-independent and cation-dependent mannose 6-phosphate receptors. The cytosolic tails of both receptors contain acidic-cluster-dileucine signals that direct sorting from the trans-Golgi network to the endosomal-lysosomal system. We found that these signals bind to the VHS domain of the Golgi-localized, gamma-ear-containing, ARF-binding proteins (GGAs). The receptors and the GGAs left the trans-Golgi network on the same tubulo-vesicular carriers. A dominant-negative GGA mutant blocked exit of the receptors from the trans-Golgi network. Thus, the GGAs appear to mediate sorting of the mannose 6-phosphate receptors at the trans-Golgi network.

Signal-binding specificity of the mu4 subunit of the adaptor protein complex AP-4.

The medium (mu) chains of the adaptor protein (AP) complexes AP-1, AP-2, and AP-3 recognize distinct subsets of tyrosine-based (YXXphi) sorting signals found within the cytoplasmic domains of integral membrane proteins. Here, we describe the signal-binding specificity and affinity of the medium subunit mu4 of the recently described adaptor protein complex AP-4. To elucidate the determinants of specificity, we screened a two-hybrid combinatorial peptide library using mu4 as a selector protein. Statistical analyses of the results revealed that mu4 prefers aspartic acid at position Y+1, proline or arginine at Y+2, and phenylalanine at Y-1 and Y+3 (phi). In addition, we examined the interaction of mu4 with naturally occurring YXXphi signals by both two-hybrid and in vitro binding analyses. These experiments showed that mu4 recognized the tyrosine signal from the human lysosomal protein LAMP-2, HTGYEQF. Using surface plasmon resonance measurements, we determined the apparent dissociation constant for the mu4-YXXphi interaction to be in the micromolar range. To gain insight into a possible role of AP-4 in intracellular trafficking, we constructed a Tac chimera bearing a mu4-specific YXXphi signal. This chimera was targeted to the endosomal-lysosomal system without being internalized from the plasma membrane.

The absence of ribonuclease H1 or H2 alters the sensitivity of Saccharomyces cerevisiae to hydroxyurea, caffeine and ethyl methanesulphonate: implications for roles of RNases H in DNA replication and repair.

BACKGROUND: RNA of RNA-DNA hybrids can be degraded by ribonucleases H present in all organisms including the eukaryote Saccharomyces cerevisiae. Determination of the number and roles of the RNases H in eukaryotes is quite feasible in S. cerevisiae. RESULTS: Two S. cerevisiae RNases H, related to Escherichia coli RNase HI and HII, are not required for growth under normal conditions, yet, compared with wild-type cells, a double-deletion strain has an increased sensitivity to hydroxyurea (HU) and is hypersensitive to caffeine and ethyl methanesulphonate (EMS). In the absence of RNase H1, RNase H2 activity increases, and cells are sensitive to EMS but not HU and are more tolerant of caffeine; the latter requires RNase H2 activity. Cells missing only RNase H2 exhibit increased sensitive to HU and EMS but not caffeine CONCLUSIONS: Mutant phenotypes infer that some RNA-DNA hybrids are recognized by both RNases H1 and H2, while other hybrids appear to be recognized only by RNase H2. Undegraded RNA-DNA hybrids have an effect when DNA synthesis is impaired, DNA damage occurs or the cell cycle is perturbed by exposure to caffeine suggesting a role in DNA replication/repair that can be either beneficial or detrimental to cell viability.

Isolation of RNase H genes that are essential for growth of Bacillus subtilis 168.

Two genes encoding functional RNase H (EC 3.1.26.4) were isolated from a gram-positive bacterium, Bacillus subtilis 168. Two DNA clones exhibiting RNase H activities both in vivo and in vitro were obtained from a B. subtilis DNA library. One (28.2 kDa) revealed high similarity to Escherichia coli RNase HII, encoded by the rnhB gene. The other (33.9 kDa) was designated rnhC and encodes B. subtilis RNase HIII. The B. subtilis genome has an rnhA homologue, the product of which has not yet shown RNase H activity. Analyses of all three B. subtilis genes revealed that rnhB and rnhC cannot be simultaneously inactivated. This observation indicated that in B. subtilis both the rnhB and rnhC products are involved in certain essential cellular processes that are different from those suggested by E. coli rnh mutation studies. Sequence conservation between the rnhB and rnhC genes implies that both originated from a single ancestral RNase H gene. The roles of bacterial RNase H may be indicated by the single rnhC homologue in the small genome of Mycoplasma species.

Identification of the genes encoding Mn2+-dependent RNase HII and Mg2+-dependent RNase HIII from Bacillus subtilis: classification of RNases H into three families.

Database searches indicated that the genome of Bacillus subtilis contains three different genes encoding RNase H homologues. The ypdQ gene encodes an RNase HI homologue with 132 amino acid residues, whereas the rnh and ysgB genes encode RNase HII homologues with 255 and 313 amino acid residues, respectively. RNases HI and HII show no significant sequence similarity. These genes were individually expressed in Escherichia coli; the recombinant proteins were purified, and their enzymatic properties were compared with those of E. coli RNases HI and HII. We found that the ypdQ gene product showed no RNase H activity. The 2.2 kb pair genomic DNA containing this gene did not suppress the RNase H deficiency of an E. coli rnhA mutant, indicating that this gene product shows no RNase H activity in vivo as well. In contrast, the rnh (rnhB) gene product (RNase HII) showed a preference for Mn2+, as did E. coli RNase HII, whereas the ysgB (rnhC) gene product (RNase HIII) exhibited a Mg2+-dependent RNase H activity. Oligomeric substrates digested with these enzymes indicate similar recognition of these substrates by B. subtilis and E. coli RNases HII. Likewise, B. subtilis RNase HIII and E. coli RNase HI have generated similar products. These results suggest that B. subtilis RNases HII and HIII may be functionally similar to E. coli RNases HII and HI, respectively. We propose that Mn2+-dependent RNase HII is universally present in various organisms and Mg2+-dependent RNase HIII, which may have evolved from RNase HII, functions as a substitute for RNase HI.

Cloning, expression, and mapping of ribonucleases H of human and mouse related to bacterial RNase HI.

We identified two human sequences and one mouse sequence in the database of expressed sequence tags that are highly homologous to the N-terminal sequence of eukaryotic RNases H1. The cDNAs for human RNASEH1 and mouse Rnaseh1 were obtained, their nucleotide sequences determined, and the proteins expressed in Escherichia coli and partially purified. Both proteins have RNase H activity in vitro and they bind to dsRNA and RNA-DNA hybrids through the N-terminal conserved motif present in eukaryotic RNases H1. The RNASEH1 gene is expressed in all human tissues at similar levels, indicating that RNase H1 may be a housekeeping protein. The human RNASEH1 and mouse Rnaseh1 cDNAs were used to isolate BAC genomic clones that were used as probes for fluorescence in situ hybridization. The human gene was localized to chromosome 17p11.2 and the mouse gene to a nonsyntenic region on chromosome 12A3. The chromosomal location and possible disease association of the human RNASEH1 gene are discussed.

A common 40 amino acid motif in eukaryotic RNases H1 and caulimovirus ORF VI proteins binds to duplex RNAs.

Eukaryotic RNases H from Saccharomyces cerevisiae , Schizosaccharomyces pombe and Crithidia fasciculata , unlike the related Escherichia coli RNase HI, contain a non-RNase H domain with a common motif. Previously we showed that S.cerevisiae RNase H1 binds to duplex RNAs (either RNA-DNA hybrids or double-stranded RNA) through a region related to the double-stranded RNA binding motif. A very similar amino acid sequence is present in caulimovirus ORF VI proteins. The hallmark of the RNase H/caulimovirus nucleic acid binding motif is a stretch of 40 amino acids with 11 highly conserved residues, seven of which are aromatic. Point mutations, insertions and deletions indicated that integrity of the motif is important for binding. However, additional amino acids are required because a minimal peptide containing the motif was disordered in solution and failed to bind to duplex RNAs, whereas a longer protein bound well. Schizosaccharomyces pombe RNase H1 also bound to duplex RNAs, as did proteins in which the S.cerevisiae RNase H1 binding motif was replaced by either the C.fasciculata or by the cauliflower mosaic virus ORF VI sequence. The similarity between the RNase H and the caulimovirus domain suggest a common interaction with duplex RNAs of these two different groups of proteins.

Kinetic and stoichiometric analysis for the binding of Escherichia coli ribonuclease HI to RNA-DNA hybrids using surface plasmon resonance.

To understand how ribonucleases H recognize RNA-DNA hybrid substrates, we analyzed kinetic parameters of binding of Escherichia coli RNase HI to RNA-DNA hybrids ranging in length from 18 to 36 base pairs (bp) using surface plasmon resonance (BIAcoreTM). The kon and koff values for the binding of the enzyme to the 36-bp substrate were 1.5 x 10(6) M-1 s-1 and 3.2 x 10(-2) s-1, respectively. Similar values were obtained with the shorter substrates. Using uncleavable 2'-O-methylated RNA-DNA substrates, values for kon and koff were 2.1 x 10(5) M-1 s-1 and 1.3 x 10(-1) s-1 in the absence of Mg2+ that were further reduced in the presence of Mg2+ to 7.4 x 10(3) M-1 s-1 and 2.6 x 10(-2) s-1. Kinetic parameters similar to the wild-type enzyme were obtained using an active-site mutant enzyme, Asp134 replaced by Ala, whereas a greatly reduced on-rate was observed for another inactive mutant enzyme, in which the basic protrusion is eliminated, thereby distinguishing between poor catalysis and inability to bind to the substrate. Stoichiometric analyses of RNase HI binding to substrates of 18, 24, 30, and 36 bp are consistent with previous reports suggesting that RNase HI binds to 9-10 bp of RNA-DNA hybrid.

The isolated RNase H domain of murine leukemia virus reverse transcriptase. Retention of activity with concomitant loss of specificity.

Retroviral RNases H are similar in sequence and structure to Escherichia coli RNase HI and yet have differences in substrate specificities, metal ion requirements, and specific activities. Separation of reverse transcriptase (RT) into polymerase and RNase H domains yields an active RNase H from murine leukemia virus (MuLV) but an inactive human immunodeficiency virus (HIV) RNase H. The "handle region" present in E. coli RNase HI but absent in HIV RNase H contributes to the binding to its substrate and when inserted into HIV RNase H results in an active enzyme retaining some degree of specificity. Here, we show MuLV protein containing the C-terminal 175 amino acids with its own handle region or that of E. coli RNase HI has the same specific activity as the RNase H of RT, retains a preference for Mn2+ as the cation required for activity, and has association rate (KA) 10% that of E. coli RNase HI. However, with model substrates, specificities for removal of the tRNAPro primer and polypurine tract stability are lost, indicating specificity of RNase H of MuLV requires the remainder of the RT. Differences in KA, while significant, appear insufficient to account for the differences in specific activities of the bacterial and viral RNases H.

Antiretroviral effect of a gag-RNase HI fusion gene.

We have previously shown that a molecule consisting of a fusion of a Ca(2+)-dependent nuclease (from Staphylococcus aureus) to a retroviral coat protein specifies a potent antiviral specific for that retrovirus. Genes specifying such fusion proteins can be delivered to virus-susceptible cells, providing an antiviral gene therapy aimed at limiting virus spread. We report here the results of experiments to vary the nuclease moiety of such fusion proteins. We found that one nuclease. Serratia marcescens nuclease, was extremely toxic to host cells and hence not likely to be useful for therapeutic purposes. A second nuclease, Escherichia coli RNase Hl was found to be nontoxic and highly effective against a murine leukemia virus when it was fused to the leukemia virus coat protein. The fusion protein was enzymatically active and stably expressed, without apparent toxicity to host cells. Reduction in infectious virus output was as high as 97-99%. These studies provide a model system for the development of gene therapeutic agents aimed at combating retroviral infections in vivo.

Ribonuclease H renaturation gel assay using a fluorescent-labeled substrate.

Ribonucleases H (RNases H) are enzymes that specifically degrade the RNA of RNA-DNA hybrids. These enzymes are involved in DNA replication, reverse transcription (RT) and antisense oligodeoxyribonucleotide-mediated arrest of translation. One of the most valuable tools for assaying RNase H activity is the renaturation gel assay with which such activities can be detected using purified protein preparations or crude extracts. Radioactive substrates [32P labeled poly(rA)-poly(dT) hybrid] are commonly used with exposure of the gel to X-ray film; this is possible at any time without disturbing the renaturation-degradation process. Here, we describe a method using fluorescent-labeled substrates. RNA-DNA substrates are synthesized by first transcribing DNA with T7 RNA polymerase using Bodipy-TR-14-UTP and the four normal nucleoside triphosphates. The run-off transcript is annealed to a short oligomeric DNA complementary to the 3'-end of the transcript, and the DNA portion of the hybrid is formed by RT. This RNA-DNA is added to the polyacrylamide mixture before polymerization, and SDS-PAGE is performed as usual. After various periods of renaturation, the gel is scanned to detect fluorescent substrate using the red-excited laser of a fluorescence scanner. This fluorescence method has all of the advantages of using radio-labeled substrates and none of its disadvantages, and the sensitivities of the two methods are comparable. In addition, we show that the sensitivity of this procedure can be increased if damaging chemicals remaining in the gel after polymerization are eliminated by simultaneous electrophoresis of the RNase H and a protein with higher mobility.

Escherichia coli RNase HI inhibits murine leukaemia virus reverse transcription in vitro and yeast retrotransposon Ty1 transposition in vivo.

BACKGROUND: Reverse transcription, which converts an RNA genome into double-stranded DNA, requires both the polymerase and RNase H activities of reverse transcriptase (RT). In vitro, poorly processive RT dissociates from partially copied RNA-DNA hybrids, that are usually extended by a second RT molecule. Despite similar structures, RNase HI of Escherichia coli can degrade RNA-DNA hybrids that are resistant to RNase H of RT. E. coli RNase HI is used to determine the accessibility to and requirement for RNA-DNA hybrids in reverse transcription in vivo and in vitro. RESULTS: In the presence of E. coli RNase HI, reverse transcription yields incomplete cDNA molecules due to degradation of RNA-DNA hybrids. Delivery of E. coli RNase HI to Ty1 particles via fusion to the capsid protein can reduce retrotransposition by more than 99%, also indicating inhibition of DNA synthesis in vivo. CONCLUSION: Inhibition of both reverse transcription in vitro and retrotransposition in vivo by E. coli RNase HI indicates that the poor processivity of RT exposes RNA-DNA hybrids critical for reverse transcription to degradation. Targeting a cellular RNase H to HIV may help define the site(s) of RNA-DNA hybrids that are susceptible to nonretroviral RNase H and may be useful for gene therapy to inhibit retroviral replication.

Cloning, sequence analysis, overproduction in Escherichia coli and enzymatic characterization of the RNase HI from Mycobacterium smegmatis.

Activity gel analysis of cell extracts from slow- and fast-growing mycobacteria confirmed the presence of several RNase H activities in both classes of organism. The rnhA gene from Mycobacterium smegmatis (Ms) was subsequently cloned using an internal gene segment probe [Mizrahi et al., Gene 136 (1993) 287-290]. The gene encodes a polypeptide of 159 amino acids that shares 50% identity with the RNase HI from Escherichia coli (Ec). However, unlike its counterparts from Gram- bacteria, Ms rnhA does not form an overlapping divergent transcriptional unit with dnaQ (encoding the epsilon (proofreading) subunit of DNA polymerase III). Ms RNase HI was overproduced in Ec as an enzymatically active maltose-binding protein (MBP) fusion protein which cleaved the RNA strand of an RNA.DNA hybrid with a similar site selectivity to that of its Ec homologue.

The non-RNase H domain of Saccharomyces cerevisiae RNase H1 binds double-stranded RNA: magnesium modulates the switch between double-stranded RNA binding and RNase H activity.

Eukaryotic ribonucleases H of known sequence are composed of an RNase H domain similar in size and sequence to that of Escherichia coli RNase HI and additional domains of unknown function. The RNase H1 of Saccharomyces cerevisiae has such an RNase H domain at its C-terminus. Here we show that the N-terminal non-RNase H portion of the yeast RNase H1 binds tightly to double-stranded RNA (dsRNA) and RNA-DNA hybrids even in the absence of the RNase H domain. Two copies of a sequence with limited similarity to the dsRNA-binding motif are present in this N-terminus. When the first of these sequences is altered, the protein no longer binds tightly to dsRNA and exhibits an increase in RNase H activity. Unlike other dsRNA-binding proteins, increasing the Mg2+ concentration from 0.5 mM to 5 mM inhibits binding of RNase H1 to dsRNA; yet a protein missing the RNase H domain binds strongly to dsRNA even at the higher Mg2+ concentration. These results suggest that binding to dsRNA and RNase H activity are mutually exclusive, and the Mg2+ concentration is critical for switching between the activities. Changes in the Mg2+ concentration or proteolytic severing of the dsRNA-binding domain could alter the activity or location of the RNase H and may govern access of the enzyme to the substrate. Sequences similar to the dsRNA-binding motif are present in other eukaryotic RNases H and the transactivating protein of cauliflower mosaic virus, suggesting that these proteins may also bind to dsRNA.

Defects in primer-template binding, processive DNA synthesis, and RNase H activity associated with chimeric reverse transcriptases having the murine leukemia virus polymerase domain joined to Escherichia coli RNase H.

The RNase H domain of murine leukemia virus (MuLV) reverse transcriptase (RT) was replaced with Escherichia coli RNase H, and the effect on RNase H activity and processive DNA synthesis was studied, using RNA-DNA hybrids containing sequences from the MuLV polypurine tract (PPT). Two chimeric RTs, having the entire polymerase domain or all but the last 19 amino acids, were expressed. In both cases, these RTs made multiple cuts in PPT-containing substrates, whereas wild-type cleavages occurred primarily at sites consistent with the distance between the polymerase and RNase H active sites. Primer extension assays performed with the chimeric RTs, an RNase H-minus RT, and wild-type showed that the presence of a wild-type viral RNase H domain facilitates processive DNA synthesis. When wild-type RT was bound to primer-template, two retarded bands could be detected in band-shift assays. In the absence of primer extension, a high proportion of enzyme-bound primer-template was associated with the faster-migrating band, whereas with DNA synthesis, more of the bound radioactivity was in the super-shifted complex. This suggests that the super-shifted complex contains the active form of RT. The mutant RTs were deficient in formation of this complex, but the chimeric RTs were somewhat less defective than the RNase H-minus mutant. Our results demonstrate that in the wild-type enzyme, the RNase H domain is required to stabilize the interaction between RT and primer-template.