The sequence of the RNA primer and the DNA template influence the initiation of plus-strand DNA synthesis in hepatitis B virus

For hepadnaviruses, the RNA primer for plus-strand DNA synthesis is generated by the final RNase H cleavage of the pregenomic RNA at an 11 nt sequence called DR1 during the synthesis of minus-strand DNA. This RNA primer initiates synthesis at one of two distinct sites on the minus-strand DNA template, resulting in two different end products; duplex linear DNA or relaxed circular DNA. Duplex linear DNA is made when initiation of synthesis occurs at DR1. Relaxed circular DNA, the major product, is made when the RNA primer translocates to the sequence complementary to DR1, called DR2 before initiation of DNA synthesis. We studied the mechanism that determines the site of the final RNase H cleavage in hepatitis B virus (HBV). We showed that the sites of the final RNase H cleavage are always a fixed number of nucleotides from the 5' end of the pregenomic RNA. This finding is similar to what was found previously for duck hepatitis B virus (DHBV), and suggests that all hepadnaviruses use a similar mechanism. Also, we studied the role of complementarity between the RNA primer and the acceptor site at DR2 in HBV. By increasing the complementarity, we were able to increase the level of priming at DR2 over that seen in the wild-type virus. This finding suggests that the level of initiation of plus-strand DNA synthesis at DR2 is sub-maximal for wild-type HBV. Finally, we studied the role of the sequence at the 5' end of the RNA primer that is outside of the DR sequence. We found that substitutions or insertions in this region affected the level of priming at DR1 and DR2.     

Purine analog substitution of the HIV-1 polypurine tract primer defines regions controlling initiation of plus-strand DNA synthesis

Despite extensive study, the mechanism by which retroviral reverse transciptases (RTs) specifically utilize polypurine tract (PPT) RNA for initiation of plus-strand DNA synthesis remains unclear. Three sequence motifs within or adjacent to the purine-rich elements are highly conserved, namely, a rU:dA tract region immediately 5′ to the PPT, an rA:dT-rich sequence constituting the upstream portion of the PPT and a downstream rG:dC tract. Using an in vitro HIV-1 model system, we determined that the former two elements define the 5′ terminus of the (+)-strand primer, whereas the rG:dC tract serves as the primary determinant of initiation specificity. Subsequent analysis demonstrated that G→A or A→G substitution at PPT positions −2, −4 and +1 (relative to the scissile phosphate) substantially reduces (+)-strand priming. We explored this observation further using PPT substrates substituted with a variety of nucleoside analogs [inosine (I), purine riboside (PR), 2-aminopurine (2-AP), 2,6-diaminopurine (2,6-DAP), isoguanine (iG)], or one of the naturally occurring bases at these positions. Our results demonstrate that for PPT positions −2 or +1, substituting position 2 of the purine was an important determinant of cleavage specificity. In addition, cleavage specificity was greatly affected by substituting −4G with an analog containing a 6-NH2 moiety.     

Mechanism of initiation of in vitro DNA synthesis by the immunopurified complex between yeast DNA polymerase I and DNA primase

The immunopurified yeast DNA-polymerase-I--DNA-primase complex synthesizes oligo(rA) and oligo(rG) molecules that are used as primer for replication of poly(dT) and poly(dC). Neither initiation nor DNA synthesis is observed with poly(dA) and poly(dI). Nitrocellulose-filter binding shows that the enzyme complex binds to deoxypyrimidine polymers, but not to deoxypurine polymers. Although the yeast complex initiates DNA synthesis on deoxypyrimidine homopolymers, it prefers to elongate pre-existing primer molecules rather than to initiate de novo DNA replication. The size of the oligo(rA) and oligo(rG) primer molecules has been determined by urea/polyacrylamide gel electrophoresis: longer oligoribonucleotides are synthesized when their utilization is prevented by omitting dNTP. An oligodeoxythymidylate template with a chain length as short as five residues can support oligo(rA) synthesis catalyzed by the yeast DNA-polymerase--DNA-primase complex and the size of the oligoribonucleotide products synthesized with oligodeoxythymidylate of differing chain length has also been determined. The mechanistic properties of the DNA-polymerase--DNA-primase complexes, purified from different eukaryotic organisms, appear to be very similar. The possible biological implication of the studies on the mechanism and specificity of initiation of DNA synthesis in a well-defined model template system has been discussed.     

Acetylcholine-induced initiation of thymic lymphoblast DNA synthesis and proliferation

Low (5 × 10⁻⁹ M to 10⁻⁷ M) acetylcholine concentrations cause a calcium-independent stimulation of the initiation of DNA synthesis and proliferation of lymphoblasts which are part of rat thymocyte populations suspended in vitro. A much higher (5 × 10⁻⁵ M) acetylcholine concentration also stimulates lymphoblast DNA synthesis and proliferation, but this action is calcium-dependent. This proliferogenic response to acetylcholine is however not clearly mediated by either cyclic GMP or cyclic AMP.     

DNA synthesis in polyoma virus infection. II. Relationship between viral DNA replication and initiation of cellular DNA replicons

The rate of synthesis of cellular DNA is stimulated in stationary phase mouse embryo cells infected with polyoma virus. Nascent cellular DNA strands pulselabeled with [³H]thymidine in the presence of replicating viral DNA are smaller, by an average of 2·1 × 10⁷ daltons, than DNA made under similar conditions in uninfected cells. Previous work (Cheevers et al., 1972) has indicated that this observation is the consequence of activation in infected cells of cellular DNA initiation sites not in operation during a similar pulse-labeling interval in uninfected cells. Similar results were obtained using cells infected with the temperature-sensitive Ts-a mutant of polyoma at 32 °C, which permits both the induction of cellular DNA synthesis and replication of viral DNA. However, at a temperature of 39 °C, which permits only the induction of cellular DNA replication in Ts-a-infected cells, the size of newly synthesized DNA is not different from that of uninfected cells. Similarly, in rat embryo cells abortively infected with polyoma (wild-type), stimulation of cellular DNA synthesis occurs but viral DNA replication is restricted, and no difference is apparent in the size of newly formed DNA as compared to uninfected cells. These results are interpreted to mean that in productively infected cells, polyoma DNA and some regions of the host genome may be co-ordinately replicated.     

Transcriptional role in DNA replication: degradation of RNA primer during DNA synthesis

DNA synthesis catalyzed in vitro by E. coli DNA polymeraseI in the presence of single stranded fd DNA or poly (dT) as template is stimulated by RNA primers. When poly(dT) fully or partially saturated with polyriboadenylic acid strands is used as template - primer, DNA synthesis proceeds with concomitant degradation of the ribostrands to 5′-adenosine monophosphate. The fragment of DNA polymerase lacking the 5′→3′ exonuclease shows comparable RNA primer dependency but reduced efficiency for the degradation of the RNA primer from the 5′-end.     

Inhibition of initiation of DNA synthesis by aminoglycoside antibiotics

In a $\text{dna}\text{C}{}^{\mathrm{ts}}$ mutant of $\text{E. coli}$, the reinitiation of DNA synthesis, which occurred by the shift of the culture from a restrictive temperature to a permissive temperature, was markedly prevented by habakacin, dibekacin, kanamycin, and gentamicin. On the contrary, chloramphenicol did not inhibit the reinitiation synthesis for 30 min. In a parallel experiment, leucine uptake into protein was profoundly blocked by chloramphenicol, but only slightly by habekacin. Habekacin did not significantly affect DNA elongation of the cells at a restrictive temperature. We propose that inhibition of initiation of replication by aminoglycoside antibiotics is related to their lethality.     

N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis

The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5′-2-deoxyribose-5-phosphate lyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polβ, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or an 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polβ preferentially generated −1 or −2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of −2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of −1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase within the non-homologous end joining pathway.     

Modular architecture of the bacteriophage T7 primase couples RNA primer synthesis to DNA synthesis

DNA primases are template-dependent RNA polymerases that synthesize oligoribonucleotide primers that can be extended by DNA polymerase. The bacterial primases consist of zinc binding and RNA polymerase domains that polymerize ribonucleotides at templating sequences of single-stranded DNA. We report a crystal structure of bacteriophage T7 primase that reveals its two domains and the presence of two Mg(2+) ions bound to the active site. NMR and biochemical data show that the two domains remain separated until the primase binds to DNA and nucleotide. The zinc binding domain alone can stimulate primer extension by T7 DNA polymerase. These findings suggest that the zinc binding domain couples primer synthesis with primer utilization by securing the DNA template in the primase active site and then delivering the primed DNA template to DNA polymerase. The modular architecture of the primase and a similar mechanism of priming DNA synthesis are likely to apply broadly to prokaryotic primases.