The attachment of minicircles to kinetoplast DNA networks during replication

Kinetoplast DNA (kDNA), the trypanosomatid mitochondrial DNA, is a network containing several thousand interlocked minicircles. During kDNA synthesis, minicircles dissociate from the network, and after replication their progeny reattach to the network periphery. Using electron microscopy autoradiography, we found that newly synthesized 3H-labeled minicircles, after short labeling periods, are concentrated in two peripheral zones on opposite sides of the network. These must be minicircle attachment sites, adjacent to the two diametrically opposed complexes of replication proteins observed previously. From the pattern of radiolabeling during longer pulses, we reached the unexpected conclusion that minicircle attachment around the entire network periphery may be due to a relative movement of the kinetoplast and the two complexes. The kinetoplast probably rotates between two fixed complexes.     

Further purification and characterization of a multienzyme complex for DNA synthesis in human cells

The 21 S complex of enzymes for DNA synthesis in the combined low salt nuclear extract-post microsomal supernatant from HeLa cells [Malkas et al. (1990) Biochemistry 29:6362-6374] was purified by poly (ethylene glycol) precipitation, Q-Sepharose chromatography, Mono Q Fast Protein Liquid Chromatography (FPLC), and velocity gradient centrifugation. The procedure gives purified enzyme complex at a yield of 45%. The 21 S enzyme complex remains intact and functional in the replication of simian virus 40 DNA throughout the purification. Sedimentation analysis showed that the 21 S enzyme complex exists in the crude HeLa cell extract and that simian virus 40 in vitro DNA replication activity in the cell extract resides exclusively with the 21 S complex. The results of enzyme and immunological analysis indicate that DNA polymerase alpha-primase, a 3',5' exonuclease, DNA ligase I, RNase H, and topoisomerase I are associated with the purified enzyme complex. Denaturing polyacrylamide gel electrophoresis of the purified enzyme complex showed the presence of about 30 polypeptides in the size range of 300 to 15 kDa. Immunofluorescent imaging analysis, with antibodies to DNA polymerase alpha,beta and DNA ligase I, showed that polymerase alpha and DNA ligase I are localized to granular-like foci within the nucleus during S-phase. In contrast, DNA polymerase beta, which is not associated with the 21 S complex, is diffusely distributed throughout the nucleoplasm.     

In vivo determination of replication origins of human mitochondrial DNA by ligation-mediated polymerase chain reaction

A large part of replication is aborted in human mitochondria, the result being a D-loop. As few attempts have been made to distinguish free 5' ends of true replicate from those of abortive ones, we examined the 5' ends of true replicate of human mitochondrial DNA at one nucleotide resolution in vivo by making use of ligation-mediated polymerase chain reaction. The distribution and relative amounts of origins of the true replicate are exactly the same as those of total newly synthesized heavy strands, which means that the abortion of replication is independent of 5' ends. Treatment of DNA with RNase H frees 5' ends on both heavy and light strands. This is the first in vivo evidence for covalently attached primer RNA to nascent strand in human mitochondrial DNA.     

A nested-PCR-based schizodeme method for identifying Leishmania kinetoplast minicircle classes directly from clinical samples and its application to the study of the epidemiology of Leishmania tropica in Pakistan

A nested PCR was developed to amplify the variable region of the kinetoplast minicircles of all Leishmania species which infect mammals. Each Leishmania parasite contains approximately 10,000 kinetoplast DNA minicircles, which are unequally distributed among approximately 10 minicircle classes. The PCR primers were designed to bind within the 120-bp conserved region which is common to all minicircle classes; the remaining approximately 600 bp of each minicircle is highly conserved within each minicircle class but highly divergent between classes. The nested PCR generated a strong signal from a minimum of 0.1 fg of Leishmania DNA. Restriction digests of the amplicons from the highest dilutions suggested that minicircles from only a limited number of minicircle classes had acted as template in the reaction. One PCR product was directly sequenced and found to be derived from only one minicircle class. Since the primers amplify all minicircle classes, this indicated that as little as 1/10 of one Leishmania parasite was present in the PCR template. This demonstrated that the nested PCR achieved very nearly the maximum theoretically possible sensitivity and is therefore a potentially useful method for diagnosis. The nested PCR was tested for sensitivity on 20 samples from patients from the Timargara refugee camp, Pakistan. Samples were collected by scraping out a small amount of tissue with a scalpel from an incision at the edge of the lesion; the tissue was smeared on one microscope slide and placed in a tube of 4 M guanidine thiocyanate, in which the sample was stable for at least 1 month. DNA for PCR was prepared by being bound to silica in the presence of 6 M guanidine thiocyanate; washed in guanidine thiocyanate, ethanol, and acetone; and eluted with 10 mM Tris-HCl. PCR products of the size expected for Leishmania tropica were obtained from 15 of the 20 samples in at least one of three replicate reactions. The negative samples were from lesions that had been treated with glucantime or were over 6 months old, in which parasites are frequently scanty. This test is now in routine use for the detection and identification of Leishmania parasites in our clinical laboratory. Fingerprints produced by restriction digests of the PCR products were defined as complex or simple. There were no reproducible differences between the complex restriction patterns of the kinetoplast DNA of any of the parasites from Timargara camp with HaeIII and HpaII. The simple fingerprints were very variable and were interpreted as being the product of PCR on a limited subset of minicircle classes, and consequently, it was thought that the variation was determined by the particular minicircle classes that had been represented in the template. The homogeneity of the complex fingerprints suggests that the present epidemic of cutaneous leishmaniasis in Timargara camp may be due to the spread of a single clone of L. tropica.     

Uridylate-containing RNA sequences determine specificity for binding and polyadenylation by the catalytic subunit of vaccinia virus poly(A) polymerase

VP55, the catalytic subunit of vaccinia virus poly(A) polymerase, has the remarkable property of adding 30-35 adenylates to RNA 3' ends in a rapid processive burst before an abrupt transition to slow, non-processive adenylate addition. Here, we demonstrate that this property results from the affinity of the enzyme for uridylate residues within the 3' 31-40 nt of the RNA primer. At physiological salt concentrations, both polyadenylation and stable VP55 binding required the presence of multiple uridylates within a 31-40 nt length of RNA, though specific RNA sequences were not necessary. Even DNA in which the deoxythymidylate residues were replaced with ribouridylates, could be polyadenylated in a processive manner. Both the unmethylated pyrimidine ring and a 2'-OH on the associated sugar are features of ribouridylates that are important for priming. The abrupt termination of processive polyadenylation was attributed to translocation of VP55 along the nascent poly(A) tail, which lacks uridylates for stable binding. As evidence for translocation and interaction with newly synthesized RNA, other homopolymer tails were synthesized by VP55 in the presence of Mn2+, which relaxes its donor nucleotide specificity. Only during poly(U) tail synthesis did processive nucleotide addition fail to terminate.     

Synthesis of polyribonucleotide chains from the 3'-hydroxyl terminus of oligodeoxynucleotides by Escherichia coli primase

Escherichia coli primase synthesizes RNA primers on DNA templates for the initiation of DNA replication. The sole known activity of primase is to catalyze synthesis of short RNA chains de novo. We now report a novel activity of primase, namely that it can synthesize RNA from the 3'-hydroxyl terminus of a pre-existing oligodeoxynucleotide. The oligonucleotide-primed synthesis of RNA by primase occurs in both of the G4oric-specific priming system and the dnaB protein associated general priming system. This priming reaction of primase is verified by a number of biochemical methods, including inhibition by modified 3'-phosphate of oligonucleotides and deoxyribonuclease I and ribonuclease H cleavages. We also show that the primed RNA is an effective primer for the synthesis of DNA chain by E. coli DNA polymerase III holoenzyme. The significance of this finding to primases generating multimeric length RNA is discussed.     

Structure of the Escherichia coli primase/single-strand DNA-binding protein/phage G4oric complex required for primer RNA synthesis

Escherichia coli primase/SSB/single-stranded phage G4oric is a simple system to study how primase interacts with DNA template to synthesize primer RNA for initiation of DNA replication. By a strategy of deletion analysis and antisense oligonucleotide protection on small single-stranded G4oric fragments, we have identified the DNA sequences required for binding primase and the critical location of single-strand DNA-binding (SSB) protein. Together with the previous data, we have defined the structure of the primase/SSB/G4oric priming complex. Two SSB tetramers bind to the G4oric secondary structure, which dictates the spacing of 3' and 5' bound adjacent SSB tetramers and leaves SSB-free regions on both sides of the stem-loop structure. Two primase molecules then bind separately to specific DNA sequences in the 3' and 5' SSB-free G4oric regions. Binding of the 3' SSB tetramer, upstream of the primer RNA initiation site, is also necessary for priming. The generation of a primase-recognition target by SSB phasing at DNA hairpin structures may be applicable to the binding of initiator proteins in other single-stranded DNA priming systems. Novel techniques used in this study include antisense oligonucleotide protection and RNA synthesis on an SSB-melted, double-stranded DNA template.     

In vivo addition of poly(A) tail and AU-rich sequences to the 3' terminus of the Sindbis virus RNA genome: a novel 3'-end repair pathway

Alphaviruses are mosquito-transmitted RNA viruses that cause important diseases in both humans and livestock. Sindbis virus (SIN), the type species of the alphavirus genus, carries a 11.7-kb positive-sense RNA genome which is capped at its 5' end and polyadenylated at its 3' end. The 3' nontranslated region (3'NTR) of the SIN genome carries many AU-rich motifs, including a 19-nucleotide (nt) conserved element (3'CSE) and a poly(A) tail. This 3'CSE and the adjoining poly(A) tail are believed to regulate the synthesis of negative-sense RNA and genome replication in vivo. We have recently demonstrated that the SIN genome lacking the poly(A) tail was infectious and that de novo polyadenylation could occur in vivo (K. R. Hill, M. Hajjou, J. Hu, and R. Raju, J. Virol. 71:2693-2704, 1997). Here, we demonstrate that the 3'-terminal 29-nt region of the SIN genome carries a signal for possible cytoplasmic polyadenylation. To further investigate the polyadenylation signals within the 3'NTR, we generated a battery of mutant genomes with mutations in the 3'NTR and tested their ability to generate infectious virus and undergo 3' polyadenylation in vivo. Engineered SIN genomes with terminal deletions within the 19-nt 3'CSE were infectious and regained their poly(A) tail. Also, a SIN genome carrying the poly(A) tail but lacking a part or the entire 19-nt 3'CSE was also infectious. Sequence analysis of viruses generated from these engineered SIN genomes demonstrated the addition of a variety of AU-rich sequence motifs just adjacent to the poly(A) tail. The addition of AU-rich motifs to the mutant SIN genomes appears to require the presence of a significant portion of the 3'NTR. These results indicate the ability of alphavirus RNAs to undergo 3' repair and the existence of a pathway for the addition of AU-rich sequences and a poly(A) tail to their 3' end in the infected host cell. Most importantly, these results indicate the ability of alphavirus replication machinery to use a multitude of AU-rich RNA sequences abutted by a poly(A) motif as promoters for negative-sense RNA synthesis and genome replication in vivo. The possible roles of cytoplasmic polyadenylation machinery, terminal transferase-like enzymes, and the viral polymerase in the terminal repair processes are discussed.