Complementary Strand Replication Research Articles

The recombination mediator RAD51D promotes geminiviral infection

To study a possible role for homologous recombination in geminivirus replication, we challenged Arabidopsis recombination gene knockouts by Euphorbia yellow mosaic virus infection. Our results show that the RAD51 paralog RAD51D, rather than RAD51 itself, promotes viral replication at early stages of infection. Blot hybridization analyses of replicative intermediates using one- and two-dimensional gels and deep sequencing point to an unexpected facet of recombination-dependent replication, the repair by single-strand annealing (SSA) during complementary strand replication. A significant decrease of both intramolecular, yielding defective DNAs and intermolecular recombinant molecules between the two geminiviral DNA components (A, B) were observed in the absence of RAD51D. By contrast, DNA A and B reacted differentially with the generation of inversions. A model to implicate single-strand annealing recombination in geminiviral recombination-dependent replication is proposed.

The contribution of translesion synthesis polymerases on geminiviral replication

Geminiviruses multiply primarily in the plant phloem, but never in meristems. Their Rep protein can activate DNA synthesis in differentiated cells. However, when their single-stranded DNA is injected into the phloem by insects, no Rep is present for inducing initial complementary strand replication. Considering a contribution of translesion synthesis (TLS) polymerases in plants, four of them (Polη, Polζ, Polκ, Rev1) are highly and constitutively expressed in differentiated tissues like the phloem. Two geminiviruses (Euphorbia yellow mosaic virus, Cleome leaf crumple virus), inoculated either biolistically or by whiteflies, replicated in Arabidopsis thaliana mutant lines of these genes to the same extent as in wild type plants. Comparative deep sequencing of geminiviral DNAs, however, showed a high exchange rate (10−4–10−3) similar to the phylogenetic variation described before and a significant difference in nucleotide substation rates if Polη and Polζ were absent, with a differential response to the viral DNA components.

Rad54 is not essential for any geminiviral replication mode in planta.

The circular single-stranded DNA of phytopathogenic geminiviruses is propagated by three modes: complementary strand replication (CSR), rolling circle replication (RCR) and recombination-dependent replication (RDR), which need host plant factors to be carried out. In addition to necessary host polymerases, proteins of the homologous recombination repair pathway may be considered essential, since geminiviruses are particularly prone to recombination. Among several others, Rad54 was suggested to be necessary for the RCR of Mungbean yellow mosaic India virus. This enzyme is a double-stranded DNA-dependent ATPase and chromatin remodeller and was found to bind and modulate the viral replication-initiator protein in vitro and in Saccharomyces cerevisiae. In contrast to the previous report, we scrutinized the requirement of Rad54 in planta for two distinct fully infectious geminiviruses with respect to the three replication modes. Euphorbia yellow mosaic virus and Cleome leaf crumple virus were inoculated into Rad54-deficient and wildtype Arabidopsis thaliana plant lines to compare the occurrence of viral DNA forms. Replication intermediates were displayed in the time course of infection by one and two-dimensional agarose gel electrophoresis and Southern hybridization. The experiments showed that Rad54 was neither essential for CSR, RCR nor RDR, and it had no significant influence on virus titers during systemic infection.

Replicative intermediates of porcine circovirus in animal tissue cultured cells or in bacteria undergoing copy-release replication

Porcine circovirus (PCV) has been assumed to replicate its genome via the rolling-circle replication (RCR) mechanism because it encodes a Rep protein that contains several amino acid motifs commonly found in other RCR biological systems. Two proteins, Rep and Rep', are essential for PCV DNA replication in mammalian cells. In this work, replicative intermediates of PCV-infected porcine kidney (PK15) cells or copy-release of PCV genomes from a head-to-tail tandem construct (without Rep') in Escherichia coli were examined. In PK15 cells, replicative intermediates consistent with complementary-strand replication which converts single-stranded circular genome to double-stranded supercoiled DNA and RCR which generates single-stranded plus strand progeny genome were observed. To a lesser extent, intermediates suggestive of recombination-dependent replication were also detected. In Escherichia coli, copy release of the single-stranded circular PCV genome with conversion to a supercoiled molecule by complementary-strand synthesis was observed. However, replicative intermediates indicative of RCR were not detected.

Replicative intermediates of maize streak virus found during leaf development

Geminiviruses of the genera Begomovirus and Curtovirus utilize three replication modes: complementary-strand replication (CSR), rolling-circle replication (RCR) and recombination-dependent replication (RDR). Using two-dimensional gel electrophoresis, we now show for the first time that maize streak virus (MSV), the type member of the most divergent geminivirus genus, Mastrevirus, does the same. Although mastreviruses have fewer regulatory genes than other geminiviruses and uniquely express their replication-associated protein (Rep) from a spliced transcript, the replicative intermediates of CSR, RCR and RDR could be detected unequivocally within infected maize tissues. All replicative intermediates accumulated early and, to varying degrees, were already present in the shoot apex and leaves at different maturation stages. Relative to other replicative intermediates, those associated with RCR increased in prevalence during leaf maturation. Interestingly, in addition to RCR-associated DNA forms seen in other geminiviruses, MSV also apparently uses dimeric open circular DNA as a template for RCR.

Replicative intermediates of Tomato leaf curl virus and its satellite DNAs

Several plant geminiviruses have been shown recently to utilize both rolling-circle replication (RCR) and recombination-dependent replication (RDR) strategies. A highly specific binding of the viral replication-associated protein (Rep) to its cognate DNA is essential for initiation of viral DNA replication and for the recognition of DNA components of the bipartite geminiviruses of the Begomovirus genus. We have extended the replication analysis to the monopartite Australian Tomato leaf curl virus (ToLCV), its Rep binding deficient mutants, and the satellite DNAs it supports. Analyses of viral DNA by two-dimensional agarose gel electrophoresis after fractionation by single-stranded (ss) DNA-selective cellulose chromatography revealed that DNA intermediates of ToLCV and its mutant were identical. Both RCR and RDR intermediates were identified. New ToLCV DNA forms were observed and characterized as subgenomic topoisomers, heterogeneous open circular double-stranded (ds) DNA, and degradation products. A 1350-nt DNA β satellite associated with the unrelated Cotton leaf curl Multan virus (CLCuMV) was supported by ToLCV and produced intermediates of both RCR and RDR, suggesting that replication strategies of satellites are determined by the helper virus. Replicative intermediates of the 682 nt ToLCV satellite DNA could not be resolved; however, concatemers of up to octamer were detected, together with a field of hybridizing material suggestive of complementary strand replication on heterogeneous circular ssDNA templates.

Nucleotide sequence of the genome of the filamentous bacteriophage I2-2: module evolution of the filamentous phage genome.

The nucleotide sequence of the circular single-stranded genome of the filamentous Escherichia coli phage I2-2 has been determined and compared with those of the filamentous E. coli phages Ff(M13, fl, or fd) and IKe. The I2-2 DNA sequence comprises 6744 nucleotides; 139 nucleotides less than that of the N- and I2-plasmid-specific phage IKe, and 337 (336) nucleotides more than that of the F-plasmid-specific phage Ff. Nucleotide sequence comparisons have indicated that I2-2, IKe, and Ff have a similar genetic organization, and that the genomes of I2-2 and IKe are evolutionarily more closely related than those of I2-2 and Ff. The studies have further demonstrated that the I2-2 genome is a composite replicon, composed of only two-thirds of the ancestral genome of IKe. Only a contiguous I2-2 DNA sequence of 4615 nucleotides encompassing not only the coat protein and phage assembly genes, but also the signal required for efficient phage morphogenesis, was found to be significantly homologous to sequences in the genomes of IKe and Ff. No homology was observed between the consecutive DNA sequence that contains the origins for viral and complementary strand replication and the replication genes. Although other explanations cannot be ruled out, our data strongly suggest that the ancestor filamentous phage genome of phages I2-2 and IKe has exchanged its replication module during evolution with that of another replicon, e.g., a plasmid that also replicates via the so-called rolling circle mechanism.

Pulse-label analysis and mapping of the two terminal regions of asynchronous complementary strand replication of mitochondrial DNA in transformed hamster cells

In vivo pulse-chase radioactive labeling studies were performed to localize within the physical map of C 13 B 4 hamster mtDNA † † Abbreviations used: mtDNA, mitochondrial DNA; for all other abbreviations, see accompanying paper (Nass, 1980). the two terminal regions of heavy and light complementary strand synthesis. These terminal segments have been defined operationally as that region on the H- and L-strand that is synthesized last. mtDNA of monolayer cultures was pulsed with [ 3H]thymidine for a minimum period of 10 minutes, which is about one-tenth of one round of mtDNA synthesis, followed by chase periods of up to 120 minutes. The properties of the labeled closed circular replicative intermediates E-mtDNA, C-mtDNA and D-mtDNA were analyzed in CsCl PrI 2 gradients and in neutral sucrose velocity and alkaline CsCl gradients. Both terminally labeled α and β daughter molecules were found to pass through the E-mtDNA stage. Sensitivity of C 13 B 4 mtDNA to alkali and ribonuclease A indicated the presence of covalently linked ribonucleotides. The distribution (specific activity) of pulse-chase radioactivity relative to uniform label was followed in electrophoretically separated HpaII + HinIII and HpaI restriction fragments (freed of 7 S initiation sequences) and corrected for thymine content. The strand specificity of the pulse-label was determined by hybridization of restriction fragments with H- and L-strands of mtDNA. The kinetic data agree precisely with electron microscopic determinations of H- and L-strand origins at respective genome positions of 0 and 67 ± 3, which are located on HpaII + HindIII fragments 9 and 6, respectively (Nass, 1980). The two terminal regions are within the predicted genome sector between about 67 and 100/0 map units; the highest terminal pulse-chase radioactivity extends within 5 to 15% of the genome's length behind each origin. The kinetics of early labeling events were found to differ at the two termini. The evidence indicates that the majority of L-strand initiation/termination sites are in the region near map position 67 ± 3, and confirms the highly asynchronous replication mechanism of this DNA.