The minimal replicator element of the Kaposi's sarcoma-associated herpesvirus terminal repeat supports replication in a semiconservative and cell-cycle-dependent manner

Kaposi's sarcoma-associated herpesvirus (KSHV) persists as episomes in infected cells by circularizing at the terminal repeats (TRs). The KSHV episome carries multiple reiterated copies of the terminal repeat, and each copy is capable of supporting replication. Expression of the latency-associated nuclear antigen (LANA) is critical for the replication of TR-containing plasmids. A 32-bp sequence upstream of LANA binding site 1 (LBS1), referred to as RE (replication element), along with LANA binding sites 1 and 2 (RE-LBS1/2), is sufficient to support replication (J. Hu and R. Renne, J. Virol. 79:2637-2642, 2005). In this report we demonstrate that the minimal replicator element (RE-LBS1/2) replicates in synchrony with the host cellular DNA, and only once, in a cell-cycle-dependent manner. Overexpression of the mammalian replication inhibitor geminin blocked replication of the plasmid containing the minimal replicator element, confirming the involvement of the host cellular replication control mechanism, and prevented rereplication of the plasmid in the same cell cycle. Overexpression of Cdt1 also rescued the replicative ability of the RE-LBS1/2-containing plasmids. A chromatin immunoprecipitation assay performed using anti-origin recognition complex 2 (alpha-ORC2) and alpha-LANA antibodies from cells transfected with RE-LBS1/2, RE-LBS1, LBS1, or RE showed the association of ORC2 with the RE region. Expression of LANA increased the number of copies of chromatin-bound DNA of replication elements, suggesting that LANA is important for the recruitment of ORCs and may contribute to the stabilization of the replication protein complexes at the RE site.     

Mutational analysis of the 18-base-pair inverted repeat element at the bovine papillomavirus origin of replication: identification of critical sequences for E1 binding and in vivo replication.

Replication of bovine papillomavirus requires two viral proteins, E1 and E2-TA. Previously we demonstrated that sequences within an imperfect 18-bp inverted repeat (IR) element were sufficient to confer specific binding of the E1 protein to the origin region (S. E. Holt, G. Schuller, and V. G. Wilson, J. Virol. 68:1094-1102, 1994). To identify critical nucleotides for E1 binding and origin function, a series of individual point mutations was constructed at each nucleotide position in the 18-bp IR. Binding of E1 to these point mutations established that both the position of the mutation and the specific nucleotide change were important for the E1-DNA interaction. Equivalent mutations from each half of the IR exhibited similar binding, suggesting that the halves were functionally symmetric for E1 interactions. Each of these mutations was evaluated also for origin function in vivo by a transient-replication assay. No single point mutation eliminated replication capacity completely, though many mutants were severely impaired, demonstrating an important functional contribution for the E1 binding site. Furthermore, E1 binding was not sufficient for replication, as several origin mutants bound E1 well in vitro but replicated poorly in vivo. This suggests that certain nucleotides within the 18-bp IR may be involved in postbinding events necessary for replication initiation. The results with the point mutations suggest that E1-E1 interactions are important for stable complex formation and also indicate that there is some flexibility with regard to formation of a functional E1 replication complex at the origin.     

Amplicons on human chromosome 11q are located in the early/late-switch regions of replication timing

Amplicons are frequently found in human tumor genomes, but the mechanism of their generation is still poorly understood. We previously measured the replication timing of the genes along the entire length of human chromosomes 11q and 21q and found that many "disease-related" genes are located in timing-transition regions. In this study, further scrutiny of the updated replication-timing map of human chromosome 11q revealed that both amplicons on human chromosomal bands 11q13 and 11q22 are located in the early/late-switch regions of replication timing in two human cell lines (THP-1 and Jurkat). Moreover, examination of synteny in the human and mouse genomes revealed that synteny breakage in both genomes occurred primarily at the early/late-switch regions of replication timing that we had identified. In conclusion, we found that the early/late-switch regions of replication timing coincided with "unstable" regions of the genome.     

The DNA unwinding element in a yeast replication origin functions independently of easily unwound sequences present elsewhere on a plasmid.

We have previously identified a DNA unwinding element (DUE) in autonomously replicating sequences (ARSs) and demonstrated a correlation between single-strand-specific nuclease hypersensitivity of the DUE and ARS-mediated plasmid replication in yeast. The DUE in the H4 ARS is the most easily unwound sequence in a supercoiled DNA molecule, in the context of the Ylp5 plasmid. To determine whether sequences which are more readily unwound than the ARS can influence replication activity, we have inserted such sequences, called 'torsional sinks', into the plasmids at a site distal to the ARS. We show that the torsional sink sequences effect reduction or elimination of the nuclease hypersensitivity of a variety of H4 ARS derivatives. However, we detect no difference in the in vivo replication activity of an individual ARS plasmid with or without a torsional sink. Thus, the function of the DUE in a yeast replication origin is unaffected by easily unwound sequences present elsewhere on the same plasmid.     

Solution structure and metal-ion binding of the P4 element from bacterial RNase P RNA

We determined the solution structure of two 27-nt RNA hairpins and their complexes with cobalt(III)-hexammine (Co(NH3)3+(6)) by NMR spectroscopy. The RNA hairpins used in this study are the P4 region from Escherichia coli RNase P RNA and a C-to-U mutant that confers altered divalent metal-ion specificity (Ca2+ replaces Mg2+) for catalytic activity of this ribozyme. Co(NH3)3+(6) is a useful spectroscopic probe for Mg(H2O)2+(6)-binding sites because both complexes have octahedral symmetry and have similar radii. The thermodynamics of binding to both RNA hairpins was studied using chemical shift changes upon titration with Mg2+, Ca2+, and Co(NH3)3+(6). We found that the equilibrium binding constants for each of the metal ions was essentially unchanged when the P4 model RNA hairpin was mutated, although the NMR structures show that the RNA hairpins adopt different conformations. In the C-to-U mutant a C.G base pair is replaced by U.G, and the conserved bulged uridine in the P4 wild-type stem shifts in the 3' direction by 1 nt. Intermolecular NOE cross-peaks between Co(NH3)3+(6) and RNA protons were used to locate the site of Co(NH3)3+(6) binding to both RNA hairpins. The metal ion binds in the major groove near a bulge loop, but is shifted 5' by more than 1 bp in the mutant. The change of the metal-ion binding site provides a possible explanation for changes in catalytic activity of the mutant RNase P in the presence of Ca2+.     

DNA binding specificity of the bovine papillomavirus E1 protein is determined by sequences contained within an 18-base-pair inverted repeat element at the origin of replication.

Bovine papillomavirus type 1 (BPV-1) DNA replicates episomally and requires two virally expressed proteins, E1 and E2, for this process. Both proteins bind to the BPV-1 genome in the region that functions as the origin of replication. The binding sequences for the E2 protein have been characterized previously, but little is known about critical sequence requirements for E1 binding. Using a bacterially expressed E1 fusion protein, we examined binding of the BPV-1 E1 protein to the origin region. E1 strongly protected a 28-bp segment of the origin (nucleotides 7932 to 15) from both DNase I and exonuclease III digestion. Additional exonuclease III protection was observed beyond the core region on both the 5' and 3' sides, suggesting that E1 interacted with more distal sequences as well. Within the 28-bp protected core, there were two overlapping imperfect inverted repeats (IR), one of 27 bp and one of 18 bp. We show that sequences within the smaller, 18-bp IR element were sufficient for specific recognition of DNA by E1 and that additional BPV-1 sequences beyond the 18-bp IR element did not significantly increase origin binding by E1 protein. While the 18-bp IR element contained sequences sufficient for specific binding by E1, E1 did not form a stable complex with just the isolated 18-bp element. Formation of a detectable E1-DNA complex required that the 18-bp IR be flanked by additional DNA sequences. Furthermore, binding of E1 to DNA containing the 18-bp IR increased as a function of overall increasing fragment length. We conclude that E1-DNA interactions outside the boundaries of the 18-bp IR are important for thermodynamic stabilization of the E1-DNA complex. However, since the flanking sequences need not be derived from BPV-1, these distal E1-DNA interactions are not sequence specific. Comparison of the 18-bp IR from BPV-1 with the corresponding region from other papillomaviruses revealed a symmetric conserved consensus sequence, T-RY--TTAA--RY-A, that may reflect the specific nucleotides critical for E1-DNA recognition.     

Dependence of timing of mitotic events on the rate of protein synthesis and DNA replication in sea urchin early cleavages

Abstract. To understand what processes affect the cell-cycle timing of mitotic events in early cleavage cycles of sea urchin embryos, a study was made on the effects of (a) reducing protein synthesis with emetine and (b) DNA replication with aphidi-colin, on the timing of nuclear envelope breakdown, anaphase onset and cytokinesis. When protein synthesis was slightly inhibited by administration of emetine, the delay in the mitotic events increased, with an increase in the delay in accumulation of proteins up to the levels to which cells must synthesize the proteins to execute the cleavage. This indicated that protein synthesis affects the timing of mitotic events. The delay in cleavage cycles caused by a slight inhibition of DNA replication with aphidicolin was in proportion to the concentration of aphidicolin administered, suggesting that DNA replication also affects the timing of mitotic events. Furthermore, it was confirmed that accumulation of the proteins to the levels required for execution of the first cleavage precedes completion of DNA replication as a requirement for execution of the first cleavage. These results imply the existence of process(es) affected by protein synthesis that are included in a feedback control system which prevents the initiation of mitosis until after the completion of DNA replication; it is the characteristic of a cell-cycle control system that has been predicted theoretically.     

A new expression system for mammalian cells based on putative replicator sequences of the mouse and a truncated thymidine kinase gene

We have constructed a new expression vector for mammalian cells. The vector contains a truncated tk gene for amplification under selective conditions, a sequence putatively supporting the replication of plasmid DNA in eukaryotic cells (murine autonomously replicating sequence) and an expression cassette for the cDNA to be studied. As a model cDNA we have used that of human tissue-type plasminogen activator (t-PA). Analysis of Hirt supernatants and chromosomal DNA from L cells, prepared six weeks after isolation of the clones indicated a 50- to 500-fold amplification of the expression construct in the cells. Concomitantly, the expression of t-PA was dramatically increased. Our data are consistent with episomal persistence of the expression construct, with a head-to-tail mode of integration into the mouse genome and with coexistence of both episomal plasmids and head-to-tail integrates. In tk-deficient cell lines other then L-cells, such as mouse mastocytoma or rat hepatoma cells, a strong selection against the persistence of the expression construct was noted. After long-term propagation of the L-cells under selective conditions the expression of the indicator gene continually decreases, but finally a constant plateau level of expression is established. Expression could be restored to the original level by blocking more efficiently the de novo synthesis of nucleosides.     

Binding sites for the herpes simplex virus immediate-early protein ICP4 impose an increased dependence on viral DNA replication on simple model promoters located in the viral genome.

We examined the ability of binding sites for the herpes simplex virus immediate-early protein ICP4 to alter the regulation of closely linked promoters by placing strong ICP4 binding sites upstream or downstream of simple TATA promoters in the intact viral genome. We found that binding sites strongly reduced the levels of expression at early times postinfection and that this effect was partially overcome after the onset of viral DNA replication. These data confirm that DNA-bound ICP4 can inhibit the activity of a closely linked promoter and raise the possibility that ICP4 binding sites contribute to temporal regulation during infection.     

Asymmetric replication in vitro from a human sequence element is dependent on adeno-associated virus Rep protein.

The DNA of human parvovirus adeno-associated virus type 2 (AAV) integrates preferentially into a defined region of human chromosome 19. Southern blots of genomic DNA from latently infected cell lines revealed that the provirus was not simply inserted into the cellular DNA. Both the proviral and adjoining cellular DNA organization indicated that integration occurred by a complex, coordinated process involving limited DNA replication and rearrangements. However, the mechanism for targeted integration has remained obscure. The two larger nonstructural proteins (Rep68 and Rep78) of AAV bind to a sequence element that is present in both the integration locus (P1) and the AAV inverted terminal repeat. This binding may be important for targeted integration. To investigate the mechanism of targeted integration, we tested the cloned integration site subfragment in a cell-free replication assay in the presence or absence of recombinant Rep proteins. Extensive, asymmetric replication of linear or open-circular template DNA was dependent on the presence of P1 sequence and Rep protein. The activities of Rep on the cloned P1 element are analogous to activities on the AAV inverted terminal repeat. Replication apparently initiates from a 3'-OH generated by the sequence-specific nicking activity of Rep. This results in a covalent attachment between Rep and the 5'-thymidine of the nick. The complexity of proviral structures can be explained by the participation of limited DNA replication facilitated by Rep during integration.