In the Simian Virus 40 In Vitro Replication System, Start Site Selection by the Polymerase α-Primase Complex Is Not Significantly Altered by Changes in the Concentration of Ribonucleotides

The simian virus 40 (SV40) in vitro replication system was previously used to demonstrate that the human polymerase (Pol) alpha-primase complex preferentially initiates DNA synthesis at pyrimidine-rich trinucleotide sequences. However, it has been reported that under certain conditions, nucleoside triphosphate (NTP) concentrations play a critical role in determining where eukaryotic primase initiates synthesis. Therefore, we have examined whether increased NTP concentrations alter the template locations at which SV40 replication is initiated. Our studies demonstrate that elevated ribonucleotide concentrations do not significantly alter which template sequences serve as initiation sites. Of considerable interest, the sequences that serve as initiation sites in the SV40 system are similar to those that serve as initiation sites for prokaryotic primases. It is also demonstrated that regardless of the concentration of ribonucleotides present in the reactions, DNA synthesis initiated outside of the core origin. These studies provide additional evidence that the Pol alpha-primase complex can initiate DNA synthesis only after a considerable amount of single-stranded DNA is generated.     

The functional role of a DNA primase in chloroplast DNA replication in Chlamydomonas reinhardtii.

A complementation experiment was developed to identify the protein component that is essential for the in vitro replication of a cloned template containing a chloroplast DNA replication origin of Chlamydomonas reinhardtii. Using this method, we have identified a DNA primase activity that copurified with DNA polymerase from the crude protein mixture. The primase catalyzed the synthesis of short RNA primers on single-stranded DNA templates. Among the synthetic templates, the order of preference was poly(dA), poly(dT), and poly(dC). The primer size range for these templates was 11-18, 5-12, and 3-11 nucleotides, respectively. On a single-stranded template containing the chloroplast DNA replication origin, the primer length range reached 19 to 27 nucleotides, indicating a better processtivity. Several initiation sites were mapped on both strands of the cloned replication origin. Some preferential initiation sites were located on A tracks spaced at one helical turn apart within the bending locus. Primase improved the template specificity of the in vitro DNA replication system and enhanced the incorporation of radioactive dATP into the supercoiled template containing the core sequences of the chloroplast DNA replication origin.     

Initiation of RNA-primed DNA synthesis in vitro by DNA polymerase alpha-primase.

The initiation of new DNA strands at origins of replication in animal cells requires de novo synthesis of RNA primers by primase and subsequent elongation from RNA primers by DNA polymerase alpha. To study the specificity of primer site selection by the DNA polymerase alpha-primase complex (pol alpha-primase), a natural DNA template containing a site for replication initiation was constructed. Two single-stranded DNA (ssDNA) molecules were hybridized to each other generating a duplex DNA molecule with an open helix replication 'bubble' to serve as an initiation zone. Pol alpha-primase recognizes the open helix region and initiates RNA-primed DNA synthesis at four specific sites that are rich in pyrimidine nucleotides. The priming site positioned nearest the ssDNA-dsDNA junction in the replication 'bubble' template is the preferred site for initiation. Using a 40 base oligonucleotide template containing the sequence of the preferred priming site, primase synthesizes RNA primers of 9 and 10 nt in length with the sequence 5'-(G)GAAGAAAGC-3'. These studies demonstrate that pol alpha-primase selects specific nucleotide sequences for RNA primer formation and suggest that the open helix structure of the replication 'bubble' directs pol alpha-primase to initiate RNA primer synthesis near the ssDNA-dsDNA junction.     

Simian virus 40 DNA replication: functional organization of regulatory elements.

The efficiency of simian virus 40 (SV40) DNA replication is dependent on the structural organization of the regulatory region. The enhancing effect of the G + C-rich 21-base-pair (bp) repeats on SV40 DNA replication is position and dose dependent and to some extent orientation dependent. The inverted orientation is about 50% as effective as the normal orientation of the 21-bp repeat region. Movement of the 21-bp repeat region 180 or 370 bp upstream of the ori sequence abolishes its enhancing effect, whereas no replication is detected if the 21-bp repeat region is placed downstream of the ori sequence. The dose-dependent enhancement of the 21-bp repeat of SV40 DNA replication as first described in single transfection by Bergsma et al. (D. J. Bergsma, D. M. Olive, S. W. Hartzell, and K. N. Subramanian, Proc. Natl. Acad. Sci. USA 79:381-385, 1982) is dramatically amplified in mixed transfection. In the presence of the 21-bp repeat region, the 72-bp repeat region can enhance SV40 DNA replication. In the presence of the 21-bp repeats and a competitive environment, the 72-bp repeat region exhibits a cis-acting inhibitory effect on SV40 DNA replication.     

Role of replication and CpG methylation in fragile X syndrome CGG deletions in primate cells

Instability of the fragile X CGG repeat involves both maternally derived expansions and deletions in the gametes of full-mutation males. It has also been suggested that the absence of aberrant CpG methylation may enhance repeat deletions through an unknown process. The effect of CGG tract length, DNA replication direction, location of replication initiation, and CpG methylation upon CGG stability were investigated using an SV40 primate replication system. Replication-dependant deletions with 53 CGG repeats were observed when replication was initiated proximal to the repeat, with CGG as the lagging-strand template. When we initiated replication further from the repeat, while maintaining CGG as the lagging-strand template or using CCG as the lagging-strand template, significant instability was not observed. CpG methylation of the unstable template stabilized the repeat, decreasing both the frequency and the magnitude of deletion events. Furthermore, CpG methylation slowed the efficiency of replication for all templates. Interestingly, replication forks displayed no evidence of a block at the CGG repeat tract, regardless of replication direction or CpG methylation status. Templates with 20 CGG repeats were stable under all circumstances. These results reveal that CGG deletions occur during replication and are sensitive to replication-fork dynamics, tract length, and CpG methylation.     

Sequence requirements for protein-primed DNA replication of bacteriophage PRD1

In vitro studies have demonstrated that linear duplex, protein-free DNA molecules containing an inverted terminal repeat (ITR) sequence of the PRD1 genome at one end can undergo replication by a protein-primed mechanism. No DNA replication was observed when the ITR sequence was deleted or was not exposed at the terminus of the template DNA. We have determined the minimal origin of replication by analyzing the template activity of various deletion derivatives. Our results showed that the terminal 20 base-pairs of ITR are required for efficient in vitro DNA replication. We have found that, within the minimal replication origin region, there are complementary sequences. A site-specific mutagenesis analysis showed that most of the point mutations in the complementary sequences markedly reduced the template activity. The analyses of the results obtained with synthetic oligonucleotides have revealed that the specificity of the replication origin is strand specific and even on a single-stranded template a particular DNA sequence including a 3'-terminal C residue is required for the initiation of PRD1 DNA replication in vitro.     

Use of simian virus 40 replication to amplify Epstein-Barr virus shuttle vectors in human cells.

We have increased the copy number of Epstein-Barr virus vectors that also carry the origin of replication of simian virus 40 (SV40) by providing a transient dose of SV40 T antigen. T antigen was supplied in trans by transfection of a nonreplicating plasmid which expresses T antigen into cells carrying Epstein-Barr virus-SV40 vectors. A significant increase in vector copy number occurred over the next few days. We also observed a high frequency of intramolecular recombination when the vector carried a repeat segment in direct orientation, but not when the repeat was in inverted orientation or absent. Furthermore, by following the mutation frequency for a marker on the vector after induction of SV40 replication, it was determined that SV40 replication generates a detectable increase in the deletion frequency but no measurable increase in the frequency of point mutations.     

Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase.

Studies of simian virus 40 (SV40) DNA replication in a reconstituted cell-free system have established that T antigen and two cellular replication proteins, replication protein A (RP-A) and DNA polymerase alpha-primase complex, are necessary and sufficient for initiation of DNA synthesis on duplex templates containing the SV40 origin of DNA replication. To better understand the mechanism of initiation of DNA synthesis, we analyzed the functional interactions of T antigen, RP-A, and DNA polymerase alpha-primase on model single-stranded DNA templates. Purified DNA polymerase alpha-primase was capable of initiating DNA synthesis de novo on unprimed single-stranded DNA templates. This reaction involved the synthesis of a short oligoribonucleotide primer which was then extended into a DNA chain. We observed that the synthesis of ribonucleotide primers by DNA polymerase alpha-primase is dramatically stimulated by SV40 T antigen. The presence of T antigen also increased the average length of the DNA product synthesized on primed and unprimed single-stranded DNA templates. These stimulatory effects of T antigen required direct contact with DNA polymerase alpha-primase complex and were most marked at low template and polymerase concentrations. We also observed that the single-stranded DNA binding protein, RP-A, strongly inhibits the primase activity of DNA polymerase alpha-primase, probably by blocking access of the enzyme to the template. T antigen partially reversed the inhibition caused by RP-A. Our data support a model in which DNA priming is mediated by a complex between T antigen and DNA polymerase alpha-primase with the template, while RP-A acts to suppress nonspecific priming events.     

Sequence-specific functions of the early palindrome domain within the SV40 core origin of replication.

The early palindrome domain within the SV40 core origin of replication is essential for the initiation of replication. Studies with single point mutants in this region suggested that the early palindrome domain does not function as a cruciform structure, but may be involved in the initiation of SV40 DNA replication in a sequence-specific manner. Two mutants, base-substituted at a primase initiation site nucleotide 5214, showed dramatic decreases in DNA replication in monkey cells. Despite earlier reports to the contrary, disruption of the cruciform configuration or polypyrimidine tract does not invariably lead to lack of replication function, as some mutants unable to form this structure replicate normally. Gel retention assays and DNase I footprinting with the nuclear proteins of monkey cells showed that the 5'GAGGC3' pentanucleotide repeats on either side of early palindrome domain interact with monkey nuclear protein. The early palindrome domain may affect the interaction of SV40 DNA with nuclear protein, and participate in SV40 DNA replication.     

SV40 deletion mutants lacking the 21-bp repeated sequences are viable, but have noncomplementable deficiencies.

We have constructed deletion mutants of simian virus 40 (SV40) lacking the two tandemly repeated copies or all three copies of the 21-bp repeated sequence located in the origin region. The mutants were viable, but had lower infectivities compared to the wild type. The mutant lacking two copies of the 21-bp repeat grew fairly well indicating that the one copy of the 21-bp repeat it contains is adequate. The other mutant lacking all the three copies of the 21-bp repeat was also viable but grew poorly. The viability of this mutant suggests that the upstream 72-bp repeated sequence compensates, though only partially, for the absence of the 21-bp repeat. The growth deficiencies of the deletion mutants could not be overcome by complementation with temperature-sensitive helper mutants providing either the early or the late functions of the virus, suggesting that the deficiencies lie in both early and late gene expression and/or in replication.     

Replication of simian virus 40 origin-containing DNA during infection with a recombinant Autographa californica multiple nuclear polyhedrosis virus expressing large T antigen.

Autographica californica multiple nuclear polyhedrosis virus (AcMNPV) has been shown to encode many of the enzymes involved in the replication of its own DNA. Although the AcMNPV genome contains multiple sets of reiterated sequences that are thought to function as origins of DNA replication, no initiator protein has yet been identified in the set of viral replication enzymes. In this study, the ability of a heterologous origin initiator system to promote DNA replication in AcMNPV-infected cells was examined. A recombinant AcMNPV that expressed the simian virus 40 (SV40) large T antigen was surprisingly found to induce the efficient replication of a transfected plasmid containing an SV40 origin. This replication was subsequently found to involve three essential components: (i) T antigen, since replication of SV40 origin-containing plasmids was not induced by wild-type AcMNPV which did not express this protein; (ii) an intact SV40 core origin, since deletion of specific functional motifs within the origin resulted in a loss of replicative abilities; and (iii) one or more AcMNPV-encoded proteins, since viral superinfection was required for plasmid amplification. Characterization of the replicated DNA revealed that it existed as a high-molecular-weight concatemer and underwent significant levels of homologous recombination between inverted repeat sequences. These properties were consistent with an AcMNPV-directed mode of DNA synthesis rather than that of SV40 and suggested that T antigen-SV40 origin complexes may be capable of initiating DNA replication reactions that can be completed by AcMNPV-encoded enzymes.     

The origin of bidirectional DNA replication in polyoma virus.

The nucleotide locations of RNA-p-DNA covalent linkages in polyoma virus (PyV) replicating DNA were mapped in the region containing the genetically required origin of DNA replication (ori). These linkages mark the initiation sites for RNA-primed DNA synthesis. A clear transition was identified between the presence of these linkages (discontinuous DNA synthesis) and their absence (continuous DNA synthesis) on each strand of ori. This demonstrated that PyV DNA replication, like simian virus 40 (SV40), is semi-discontinuous, and thus revealed the location of the origin of bidirectional DNA replication (OBR). The transition site on the template encoding PyV late mRNA occurred at the junction of ori-core and T-antigen binding site A. This was essentially the same site as previously observed in SV40 (Hay and DePamphilis, 1982). However, in contrast to SV40, the transition site on the template encoding PyV early mRNA was displaced towards the late gene side of ori. This resulted in a 16 nucleotide gap within ori in which no RNA-p-DNA linkages were observed on either strand. A model for the initiation of PyV DNA replication is presented.