A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of 'third generation' instruments that will sequence a diploid mammalian genome for ∼$1,000 in ∼24 h.

/pdf/the-potential-and-challenges-of-nanopore-sequencing-51qxscif85.pdf

The Potential and Challenges of Nanopore Sequencing

A 3.5 angstrom resolution electron density map of the HIV-1 reverse transcriptase heterodimer complexed with nevirapine, a drug with potential for treatment of AIDS, reveals an asymmetric dimer. The polymerase (pol) domain of the 66-kilodalton subunit has a large cleft analogous to that of the Klenow fragment of Escherichia coli DNA polymerase I. However, the 51-kilodalton subunit of identical sequence has no such cleft because the four subdomains of the pol domain occupy completely different relative positions. Two of the four pol subdomains appear to be structurally related to subdomains of the Klenow fragment, including one containing the catalytic site. The subdomain that appears likely to bind the template strand at the pol active site has a different structure in the two polymerases. Duplex A-form RNA-DNA hybrid can be model-built into the cleft that runs between the ribonuclease H and pol active sites. Nevirapine is almost completely buried in a pocket near but not overlapping with the pol active site. Residues whose mutation results in drug resistance have been approximately located.

Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor.

http://www.jbc.org/content/267/1/166.full.pdf

8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G----T and A----C substitutions.

We have determined the DNA sequence of the long unique region (UL) in the genome of herpes simplex virus type 1 (HSV-1) strain 17. The UL sequence contained 107,943 residues and had a base composition of 66.9% G + C. Together with our previous work, this completes the sequence of HSV-1 DNA, giving a total genome length of 152,260 residues of base composition 68.3% G + C. Genes in the UL region were located by the use of published mapping analyses, transcript structures and sequence data, and by examination of DNA sequence characteristics. Fifty-six genes were identified, accounting for most of the sequence. Some 28 of these are at present of unknown function. The gene layout for UL was found to be very similar to that for the corresponding part of the genome of varicella-zoster virus, the only other completely sequenced alphaherpesvirus, and the amino acid sequences of equivalent proteins showed a range of similarities. In the whole genome of HSV-1 we now recognize 72 genes which encode 70 distinct proteins.

The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1.

RNA viruses exploit all known mechanisms of genetic variation to ensure their survival. Distinctive features of RNA virus replication include high mutation rates, high yields, and short replication times. As a consequence, RNA viruses replicate as complex and dynamic mutant swarms, called viral quasispecies. Mutation rates at defined genomic sites are affected by the nucleotide sequence context on the template molecule as well as by environmental factors. In vitro hypermutation reactions offer a means to explore the functional sequence space of nucleic acids and proteins. The evolution of a viral quasispecies is extremely dependent on the population size of the virus that is involved in the infections. Repeated bottleneck events lead to average fitness losses, with viruses that harbor unusual, deleterious mutations. In contrast, large population passages result in rapid fitness gains, much larger than those so far scored for cellular organisms. Fitness gains in one environment often lead to fitness losses in an alternative environment. An important challenge in RNA virus evolution research is the assignment of phenotypic traits to specific mutations. Different constellations of mutations may be associated with a similar biological behavior. In addition, recent evidence suggests the existence of critical thresholds for the expression of phenotypic traits. Epidemiological as well as functional and structural studies suggest that RNA viruses can tolerate restricted types and numbers of mutations during any specific time point during their evolution. Viruses occupy only a tiny portion of their potential sequence space. Such limited tolerance to mutations may open new avenues for combating viral infections.

RNA virus mutations and fitness for survival

https://www.annualreviews.org/doi/pdf/10.1146/annurev.bi.63.070194.004021

Function and structure relationships in DNA polymerases.

Site-directed mutagenesis of the large fragment of DNA polymerase I (Klenow fragment) yielded two mutant proteins lacking 3',5'-exonuclease activity but having normal polymerase activity. Crystallographic analysis of the mutant proteins showed that neither had any alteration in protein structure other than the expected changes at the mutation sites. These results confirmed the presumed location of the exonuclease active site on the small domain of Klenow fragment and its physical separation from the polymerase active site. An anomalous scattering difference Fourier of a complex of the wild-type enzyme with divalent manganese ion and deoxythymidine monophosphate showed that the exonuclease active site has binding sites for two divalent metal ions. The properties of the mutant proteins suggest that one metal ion plays a role in substrate binding while the other is involved in catalysis of the exonuclease reaction.

https://science.sciencemag.org/content/sci/240/4849/199.full.pdf

Genetic and crystallographic studies of the 3',5'-exonucleolytic site of DNA polymerase I

On careful examination of existing kinetic data for correct and incorrect dNTP incorporations by a variety of DNA polymerases, it is apparent that these enzymes resist a unified description. Instead, the picture that emerges is a rather complex one: for most polymerases, there is evidence for a noncovalent step preceding phosphoryl transfer, but there are less reliable data for determining whether the noncovalent step or phosphoryl transfer is rate-limiting during misincorporation. Although the structural conservation in the polymerase superfamily is probably reflected in a common set of intermediates along the reaction pathway, the energetics of these species vary even when closely related polymerases are compared. Consequently, some polymerases apparently show more discrimination between correctly paired and mispaired dNTPs in the binding step, and polymerases may differ in terms of which step of the reaction is rate-limiting in correct and incorrect insertion reactions. Because of the higher energy barrier in the misincorporation reaction, at least some of the intermediates both before and after the rate-limiting step in the misincorporation pathway will have higher energies than the corresponding intermediates in correct incorporation; consequently, these steps can serve as kinetic checkpoints.

DNA polymerase fidelity: kinetics, structure, and checkpoints.

https://science.sciencemag.org/content/sci/266/5193/2022.full.pdf

Catherine M. Joyce

Papers

Function and structure relationships in DNA polymerases.

Genetic and crystallographic studies of the 3',5'-exonucleolytic site of DNA polymerase I

DNA polymerase fidelity: kinetics, structure, and checkpoints.

A unified polymerase mechanism for nonhomologous DNA and RNA polymerases

Identification of residues critical for the polymerase activity of the Klenow fragment of DNA polymerase I from Escherichia coli.