Rapid incorporation kinetics and improved fidelity of a novel class of 3'-OH unblocked reversible terminators.

Recent developments of unique nucleotide probes have expanded our understanding of DNA polymerase function, providing many benefits to techniques involving next-generation sequencing (NGS) technologies. The cyclic reversible termination (CRT) method depends on efficient base-selective incorporation of reversible terminators by DNA polymerases. Most terminators are designed with 3'-O-blocking groups but are incorporated with low efficiency and fidelity. We have developed a novel class of 3'-OH unblocked nucleotides, called Lightning Terminators™, which have a terminating 2-nitrobenzyl moiety attached to hydroxymethylated nucleobases. A key structural feature of this photocleavable group displays a 'molecular tuning' effect with respect to single-base termination and improved nucleotide fidelity. Using Therminator DNA polymerase, we demonstrate that these 3'-OH unblocked terminators exhibit superior enzymatic performance compared to two other reversible terminators, 3'-O-amino-TTP and 3'-O-azidomethyl-TTP. Lightning Terminators show maximum incorporation rates (k(pol)) that range from 35 to 45 nt/s, comparable to the fastest NGS chemistries, yet with catalytic efficiencies (k(pol)/K(D)) comparable to natural nucleotides. Pre-steady-state kinetic studies of thymidine analogs revealed that the major determinant for improved nucleotide selectivity is a significant reduction in k(pol) by >1000-fold over TTP misincorporation. These studies highlight the importance of structure-function relationships of modified nucleotides in dictating polymerase performance.

Improved nucleotide selectivity and termination of 3'-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates.

We describe a novel 3'-OH unblocked reversible terminator with the potential to improve accuracy and read-lengths in next-generation sequencing (NGS) technologies. This terminator is based on 5-hydroxymethyl-2'-deoxyuridine triphosphate (HOMedUTP), a hypermodified nucleotide found naturally in the genomes of numerous bacteriophages and lower eukaryotes. A series of 5-(2-nitrobenzyloxy)methyl-dUTP analogs (dU.I-dU.V) were synthesized based on our previous work with photochemically cleavable terminators. These 2-nitrobenzyl alkylated HOMedUTP analogs were characterized with respect to incorporation, single-base termination, nucleotide selectivity and photochemical cleavage properties. Substitution at the α-methylene carbon of 2-nitrobenzyl with alkyl groups of increasing size was discovered as a key structural feature that provided for the molecular tuning of enzymatic properties such as single-base termination and improved nucleotide selectivity over that of natural nucleotides. 5-[(S)-α-tert-Butyl-2-nitrobenzyloxy]methyl-dUTP (dU.V) was identified as an efficient reversible terminator, whereby, sequencing feasibility was demonstrated in a cyclic reversible termination (CRT) experiment using a homopolymer repeat of ten complementary template bases without detectable UV damage during photochemical cleavage steps. These results validate our overall strategy of creating 3'-OH unblocked reversible terminator reagents that, upon photochemical cleavage, transform back into a natural state. Modified nucleotides based on 5-hydroxymethyl-pyrimidines and 7-deaza-7-hydroxymethyl-purines lay the foundation for development of a complete set of four reversible terminators for application in NGS technologies.

Competition of Escherichia coli DNA polymerases I, II and III with DNA Pol IV in stressed cells.

Escherichia coli has five DNA polymerases, one of which, the low-fidelity Pol IV or DinB, is required for stress-induced mutagenesis in the well-studied Lac frameshift-reversion assay. Although normally present at approximately 200 molecules per cell, Pol IV is recruited to acts of DNA double-strand-break repair, and causes mutagenesis, only when at least two cellular stress responses are activated: the SOS DNA-damage response, which upregulates DinB approximately 10-fold, and the RpoS-controlled general-stress response, which upregulates Pol IV about 2-fold. DNA Pol III was also implicated but its role in mutagenesis was unclear. We sought in vivo evidence on the presence and interactions of multiple DNA polymerases during stress-induced mutagenesis. Using multiply mutant strains, we provide evidence of competition of DNA Pols I, II and III with Pol IV, implying that they are all present at sites of stress-induced mutagenesis. Previous data indicate that Pol V is also present. We show that the interactions of Pols I, II and III with Pol IV result neither from, first, induction of the SOS response when particular DNA polymerases are removed, nor second, from proofreading of DNA Pol IV errors by the editing functions of Pol I or Pol III. Third, we provide evidence that Pol III itself does not assist with but rather inhibits Pol IV-dependent mutagenesis. The data support the remaining hypothesis that during the acts of DNA double-strand-break (DSB) repair, shown previously to underlie stress-induced mutagenesis in the Lac system, there is competition of DNA polymerases I, II and III with DNA Pol IV for action at the primer terminus. Up-regulation of Pol IV, and possibly other stress-response-controlled factor(s), tilt the competition in favor of error-prone Pol IV at the expense of more accurate polymerases, thus producing stress-induced mutations. This mutagenesis assay reveals the DNA polymerases operating in DSB repair during stress and also provides a sensitive indicator for DNA polymerase competition and choice in vivo.

Single-strand-specific exonucleases prevent frameshift mutagenesis by suppressing SOS induction and the action of DinB/DNA polymerase IV in growing cells.

Escherichia coli strains carrying null alleles of genes encoding single-strand-specific exonucleases ExoI and ExoVII display elevated frameshift mutation rates but not base substitution mutation rates. We characterized increased spontaneous frameshift mutation in ExoI- ExoVII- cells and report that some of this effect requires RecA, an inducible SOS DNA damage response, and the low-fidelity, SOS-induced DNA polymerase DinB/PolIV, which makes frameshift mutations preferentially. We also find that SOS is induced in ExoI- ExoVII- cells. The data imply a role for the single-stranded exonucleases in guarding the genome against mutagenesis by removing excess single-stranded DNA that, if left, leads to SOS induction and PolIV-dependent mutagenesis. Previous results implicated PolIV in E. coli mutagenesis specifically during starvation or antibiotic stresses. Our data imply that PolIV can also promote mutation in growing cells under genome stress due to excess single-stranded DNA.

Roles of E. coli double-strand-break-repair proteins in stress-induced mutation.

Special mechanisms of mutation are induced during growth-limiting stress and can generate adaptive mutations that permit growth. These mechanisms may provide improved models for mutagenesis in antibiotic resistance, evolution of pathogens, cancer progression and chemotherapy resistance. Stress-induced reversion of an Escherichia coli episomal lac frameshift allele specifically requires DNA double-strand-break-repair (DSBR) proteins, the SOS DNA-damage response and its error-prone DNA polymerase, DinB. We distinguished two possible roles for the DSBR proteins. Each might act solely upstream of SOS, to create single-strand DNA that induces SOS. This could upregulate DinB and enhance mutation globally. Or any or all of them might function other than or in addition to SOS promotion, for example, directly in error-prone DSBR. We report that in cells with SOS genes derepressed constitutively, RecA, RuvA, RuvB, RuvC, RecF, and TraI remain required for stress-induced mutation, demonstrating that these proteins act other than via SOS induction. RecA and TraI also act by promoting SOS. These and additional results with hyper-mutating recD and recG mutants support roles for these proteins via error-prone DSBR. Such mechanisms could localize stress-induced mutagenesis to small genomic regions, a potentially important strategy for adaptive evolution, both for reducing additional deleterious mutations in rare adaptive mutants and for concerted evolution of genes.

Modeling variation in tumors in vivo.

Transgenic mice that allow mutant cells to be visualized in situ were used to study variation in tumors. These mice carry the G11 placental alkaline phosphatase (PLAP) transgene, a mutant allele rendered incapable of producing its enzyme product by a frameshift caused by insertion of a tract of G:C base pairs in a coding region. Spontaneous deletion of one G:C base pair from this tract restores gene function, and cells with PLAP activity can be detected histochemically. To study tumors, the G11 PLAP transgene was introduced into the polyoma virus middle T antigen mammary tumor model. Tumors in these mice exhibited up to 300 times more PLAP+ cells than normal tissues. PLAP+ cells were located throughout each tumor. Many of the PLAP+ cells were singlets, but clusters also were common, with one cluster containing >30,000 cells. Comparison of these data to simulations produced by computer models suggested that multiple factors were involved in generating mutant cells in tumors. Although genetic instability appeared to have occurred in most tumors, large clusters were much more common than expected based on instability alone.

Adaptive mutation and amplification in Escherichia coli: two pathways of genome adaptation under stress.

The neo-Darwinists suggested that evolution is constant and gradual, and thus that genetic changes that drive evolution should be too. However, more recent understanding of phenomena called adaptive mutation in microbes indicates that mutation rates can be elevated in response to stress, producing beneficial and other mutations. We review evidence that, in Escherichia coli, two separate mechanisms of stress-induced genetic change occur that revert a lac frameshift allele allowing growth on lactose medium. First, compensatory frameshift ("point") mutations occur by a mechanism that includes DNA double-strand breaks and (we have suggested) their error-prone repair. Point mutation requires induction of the RpoS-dependent general stress response, and the SOS DNA damage response leading to upregulation of the error-prone DNA polymerase DinB (Pol IV), and occurs during a transient limitation of post-replicative mismatch repair activity. A second mechanism, adaptive amplification, entails amplification of the leaky lac allele to 20-50 tandem repeats. These provide sufficient beta-galactosidase activity for growth, thereby apparently deflecting cells from the point mutation pathway. Unlike point mutation, amplification neither occurs in hypermutating cells nor requires SOS or DinB, but like point mutation, amplification requires the RpoS-dependent stress response. Similar processes are being found in other bacterial systems and yeast. Stress-induced genetic changes may underlie much of microbial evolution, pathogenesis and antibiotic resistance, and also cancer formation, progression and drug resistance.

Visualization of mosaicism in tissues of normal and mismatch-repair-deficient mice carrying a microsatellite-containing transgene.

To determine the frequency of mutation in different cell types of mammals, transgenic mice that allow mutant cells to be visualized in situ were used. These mice carry a defective allele of the human placental alkaline phosphatase (PLAP) gene. The allele does not produce enzyme because the reading frame is shifted by an insertion of 7 G:C basepairs. The insertion is adjacent to four existing G:C basepairs, so the allele has a tract of 11Gs. The G11 PLAP allele was studied in wildtype mice and in mice deficient in mismatch-repair (MMR) due to lack of either Pms2 or Mlh1. PLAP(+) cells were counted in brain, heart, kidney, and liver. In wildtype mice, there was an average of between 5 and 30 PLAP(+) events per million cells. No cells with alkaline phosphatase activity were detected in tissues from mice lacking the PLAP gene. In MMR-deficient mice, the number of PLAP(+) allele was increased by at least three-order of magnitude in brain, heart and kidney, but <10-fold in liver. These data show that MMR is vital to maintaining repeat stability in brain, heart and kidney cells. The reason for the different results in the liver is not clear. Cells in the liver were shown to be capable of expressing of PLAP enzyme and PLAP mRNA was present in this organ.