Polymerase recognition of synthetic oligodeoxyribonucleotides incorporating degenerate pyrimidine and purine bases

A universal base that is capable of substituting for any of the four natural bases in DNA would be of great utility in both mutagenesis and recombinant DNA experiments. This paper describes the properties of oligonucleotides incorporating two degenerate bases, the pyrimidine base 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one and the purine base N6-methoxy-2,6-diaminopurine, designated P and K, respectively. An equimolar mixture of the analogues P and K (called M) acts, in primers, as a universal base. The thermal stability of oligonucleotide duplexes were only slightly reduced when natural bases were replaced by P or K. Templates containing the modified bases were copied by Taq polymerase; P behaved as thymine in 60% of copying events and as cytosine in 40%, whereas K behaved as if it were guanine (13%) or adenine (87%). The dUTPase gene of Caenorhabditis elegans, which we have found to contain three nonidentical homologous repeats, was used as a model system to test the use of these bases in primers for DNA synthesis. A pair of oligodeoxyribonucleotides, each 20 residues long and containing an equimolar mixture of P and K at six positions, primed with high specificity both T7 DNA polymerase in sequencing reactions and Taq polymerase in PCRs; no nonspecific amplification was obtained on genomic DNA of C. elegans. Use of P and K can significantly reduce the complexity of degenerate oligonucleotide mixtures, and when used together, P and K can act as a universal base.

Extensive purification of a putative RNA polymerase I holoenzyme from plants that accurately initiates rRNA gene transcription in vitro

RNA polymerase I (pol I) is a nuclear enzyme whose function is to transcribe the duplicated genes encoding the precursor of the three largest ribosomal RNAs. We report a cell-free system from broccoli (Brassica oleracea) inflorescence that supports promoter-dependent RNA pol I transcription in vitro. The transcription system was purified extensively by DEAE-Sepharose, Biorex 70, Sephacryl S300, and Mono Q chromatography. Activities required for pre-rRNA transcription copurified with the polymerase on all four columns, suggesting their association as a complex. Purified fractions programmed transcription initiation from the in vivo start site and utilized the same core promoter sequences required in vivo. The complex was not dissociated in 800 mM KCl and had a molecular mass of nearly 2 MDa based on gel filtration chromatography. The most highly purified fractions contain ≈30 polypeptides, two of which were identified immunologically as RNA polymerase subunits. These data suggest that the occurrence of a holoenzyme complex is probably not unique to the pol II system but may be a general feature of eukaryotic nuclear polymerases.

Inactivation of DNA proofreading obviates the need for SOS induction in frameshift mutagenesis

Translesion synthesis at replication-blocking lesions requires the induction of proteins that are controlled by the SOS system in Escherichia coli. Of the proteins identified so far, UmuD′, UmuC, and RecA* were shown to facilitate replication across UV-light-induced lesions, yielding both error-free and mutagenic translesion-synthesis products. Similar to UV lesions, N-2-acetylaminofluorene (AAF), a chemical carcinogen that forms covalent adducts at the C8 position of guanine residues, is a strong replication-blocking lesion. Frameshift mutations are induced efficiently by AAF adducts when located within short repetitive sequences in a two-step mechanism; AAF adducts incorporate a cytosine across from the lesion and then form a primer-template misaligned intermediate that, upon elongation, yields frameshift mutations. Recently, we have shown that although elongation from the nonslipped intermediate depends on functional umuDC+ gene products, elongation from the slipped intermediate is umuDC+-independent but requires another, as yet biochemically uncharacterized, SOS function. We now show that in DNA Polymerase III-proofreading mutant strains (dnaQ49 and mutD5 strains), elongation from the slipped intermediate is highly efficient in the absence of SOS induction—in contrast to elongation from the nonslipped intermediate, which still requires UmuDC functions.

Coordinate Regulation of G- and C Strand Length during New Telomere Synthesis

We have used the ciliate Euplotes to study the role of DNA polymerase in telomeric C strand synthesis. Euplotes provides a unique opportunity to study C strand synthesis without the complication of simultaneous DNA replication because millions of new telomeres are made at a stage in the life cycle when no general DNA replication takes place. Previously we showed that the C-strands of newly synthesized telomeres have a precisely controlled length while the G-strands are more heterogeneous. This finding suggested that, although synthesis of the G-strand (by telomerase) is the first step in telomere addition, a major regulatory step occurs during subsequent C strand synthesis. We have now examined whether G- and C strand synthesis might be regulated coordinately rather than by two independent mechanisms. We accomplished this by determining what happens to G- and C strand length if C strand synthesis is partially inhibited by aphidicolin. Aphidicolin treatment caused a general lengthening of the G-strands and a large increase in C strand heterogeneity. This concomitant change in both the G- and C strand length indicates that synthesis of the two strands is coordinated. Since aphidicolin is a very specific inhibitor of DNA polα and polδ, our results suggest that this coordinate length regulation is mediated by DNA polymerase.

Nonlinearity in genetic decoding: Homologous DNA replicase genes use alternatives of transcriptional slippage or translational frameshifting

The τ and γ subunits of DNA polymerase III are both encoded by a single gene in Escherichia coli and Thermus thermophilus. γ is two-thirds the size of τ and shares virtually all its amino acid sequence with τ. E. coli and T. thermophilus have evolved very different mechanisms for setting the approximate 1:1 ratio between τ and γ. Both mechanisms put ribosomes into alternate reading frames so that stop codons in the new frame serve to make the smaller γ protein. In E. coli, ≈50% of initiating ribosomes translate the dnaX mRNA conventionally to give τ, but the other 50% shift into the −1 reading frame at a specific site (A AAA AAG) in the mRNA to produce γ. In T. thermophilus ribosomal frameshifting is not required: the dnaX mRNA is a heterogeneous population of molecules with different numbers of A residues arising from transcriptional slippage on a run of nine T residues in the DNA template. Translation of the subpopulation containing nine As (or +/− multiples of three As) yields τ. The rest of the population of mRNAs (containing nine +/− nonmultiples of three As) puts ribosomes into the alternate reading frames to produce the γ protein(s). It is surprising that two rather similar dnaX sequences in E. coli and T. thermophilus lead to very different mechanisms of expression.

Immunoassays with rolling circle DNA amplification: A versatile platform for ultrasensitive antigen detection

We describe an adaptation of the rolling circle amplification (RCA) reporter system for the detection of protein Ags, termed “immunoRCA.” In immunoRCA, an oligonucleotide primer is covalently attached to an Ab; thus, in the presence of circular DNA, DNA polymerase, and nucleotides, amplification results in a long DNA molecule containing hundreds of copies of the circular DNA sequence that remain attached to the Ab and that can be detected in a variety of ways. Using immunoRCA, analytes were detected at sensitivities exceeding those of conventional enzyme immunoassays in ELISA and microparticle formats. The signal amplification afforded by immunoRCA also enabled immunoassays to be carried out in microspot and microarray formats with exquisite sensitivity. When Ags are present at concentrations down to fM levels, specifically bound Abs can be scored by counting discrete fluorescent signals arising from individual Ag–Ab complexes. Multiplex immunoRCA also was demonstrated by accurately quantifying Ags mixed in different ratios in a two-color, single-molecule-counting assay on a glass slide. ImmunoRCA thus combines high sensitivity and a very wide dynamic range with an unprecedented capability for single molecule detection. This Ag-detection method is of general applicability and is extendable to multiplexed immunoassays that employ a battery of different Abs, each labeled with a unique oligonucleotide primer, that can be discriminated by a color-coded visualization system. ImmunoRCA-profiling based on the simultaneous quantitation of multiple Ags should expand the power of immunoassays by exploiting the increased information content of ratio-based expression analysis.

Efficient translesion replication in the absence of Escherichia coli Umu proteins and 3′–5′ exonuclease proofreading function

Translesion replication (TR) past a cyclobutane pyrimidine dimer in Escherichia coli normally requires the UmuD′2C complex, RecA protein, and DNA polymerase III holoenzyme (pol III). However, we find that efficient TR can occur in the absence of the Umu proteins if the 3′–5′ exonuclease proofreading activity of the pol III ɛ-subunit also is disabled. TR was measured in isogenic uvrA6 ΔumuDC strains carrying the dominant negative dnaQ allele, mutD5, or ΔdnaQ spq-2 mutations by transfecting them with single-stranded M13-based vectors containing a specifically located cis-syn T–T dimer. As expected, little TR was observed in the ΔumuDC dnaQ+ strain. Surprisingly, 26% TR occurred in UV-irradiated ΔumuDC mutD5 cells, one-half the frequency found in a uvrA6 umuDC+mutD5 strain. lexA3 (Ind−) derivatives of the strains showed that this TR was contingent on two inducible functions, one LexA-dependent, responsible for ≈70% of the TR, and another LexA-independent, responsible for the remaining ≈30%. Curiously, the ΔumuDC ΔdnaQ spq-2 strain exhibited only the LexA-independent level of TR. The cause of this result appears to be the spq-2 allele, a dnaE mutation required for viability in ΔdnaQ strains, since introduction of spq-2 into the ΔumuDC mutD5 strain also reduces the frequency of TR to the LexA-independent level. The molecular mechanism responsible for the LexA-independent TR is unknown but may be related to the UVM phenomenon [Palejwala, V. A., Wang, G. E., Murphy, H. S. & Humayun, M. Z. (1995) J. Bacteriol. 177, 6041–6048]. LexA-dependent TR does not result from the induction of pol II, since TR in the ΔumuDC mutD5 strain is unchanged by introduction of a ΔpolB mutation.

Enzymatic processing of DNA containing tandem dihydrouracil by endonucleases III and VIII

Endonuclease III from Escherichia coli, yeast (yNtg1p and yNtg2p) and human and E.coli endonuclease VIII have a wide substrate specificity, and recognize oxidation products of both thymine and cytosine. DNA containing single dihydrouracil (DHU) and tandem DHU lesions were used as substrates for these repair enzymes. It was found that yNtg1p prefers DHU/G and exhibits much weaker enzymatic activity towards DNA containing a DHU/A pair. However, yNtg2p, E.coli and human endonuclease III and E.coli endonuclease VIII activities were much less sensitive to the base opposite the lesion. Although these enzymes efficiently recognize single DHU lesions, they have limited capacity for completely removing this damaged base when DHU is present on duplex DNA as a tandem pair. Both E.coli endonuclease III and yeast yNtg1p are able to remove only one DHU in DNA containing tandem lesions, leaving behind a single DHU at either the 3′- or 5′-terminus of the cleaved fragment. On the other hand, yeast yNtg2p can remove DHU remaining on the 5′-terminus of the 3′ cleaved fragment, but is unable to remove DHU remaining on the 3′-terminus of the cleaved 5′ fragment. In contrast, both human endonuclease III and E.coli endonuclease VIII can remove DHU remaining on the 3′-terminus of a cleaved 5′ fragment, but are unable to remove DHU remaining on the 5′-terminus of a cleaved 3′ fragment. Tandem lesions are known to be generated by ionizing radiation and agents that generate reactive oxygen species. The fact that these repair glycosylases have only a limited ability to remove the DHU remaining at the terminus suggests that participation of other repair enzymes is required for the complete removal of tandem lesions before repair synthesis can be efficiently performed by DNA polymerase.

The transcript release factor PTRF augments ribosomal gene transcription by facilitating reinitiation of RNA polymerase I

Termination of murine rDNA transcription by RNA polymerase I (Pol I) requires pausing of Pol I by terminator-bound TTF-I (transcription termination factor for Pol I), followed by dissociation of the ternary complex by PTRF (Pol I and transcript release factor). To examine the functional correlation between transcription termination and initiation, we have compared transcription on terminator-containing and terminator-less rDNA templates. We demonstrate that terminated RNA molecules are more efficiently synthesized than run-off transcripts, indicating that termination facilitates reinitiation. Transcriptional enhancement is observed in multiple- but not single-round transcription assays measuring either promoter-dependent or promoter-independent Pol I transcription. Increased synthesis of terminated transcripts is observed in crude extracts but not in a PTRF-free reconstituted transcription system, indicating that PTRF-mediated release of pre-rRNA is responsible for transcriptional enhancement. Consistent with PTRF serving an important role in modulating the efficiency of rRNA synthesis, PTRF exhibits pronounced charge heterogeneity, is phosphorylated at multiple sites and fractionates into transcriptionally active and inactive forms. The results suggest that regulation of PTRF activity may be an as yet unrecognized means to control the efficiency of ribosomal RNA synthesis.

Directed evolution of polymerase function by compartmentalized self-replication

We describe compartmentalized self-replication (CSR), a strategy for the directed evolution of enzymes, especially polymerases. CSR is based on a simple feedback loop consisting of a polymerase that replicates only its own encoding gene. Compartmentalization serves to isolate individual self-replication reactions from each other. In such a system, adaptive gains directly (and proportionally) translate into genetic amplification of the encoding gene. CSR has applications in the evolution of polymerases with novel and useful properties. By using three cycles of CSR, we obtained variants of Taq DNA polymerase with 11-fold higher thermostability than the wild-type enzyme or with a >130-fold increased resistance to the potent inhibitor heparin. Insertion of an extra stage into the CSR cycle before the polymerase reaction allows its application to enzymes other than polymerases. We show that nucleoside diphosphate kinase and Taq polymerase can form such a cooperative CSR cycle based on reciprocal catalysis, whereby nucleoside diphosphate kinase produces the substrates required for the replication of its own gene. We also find that in CSR the polymerase genes themselves evolve toward more efficient replication. Thus, polymerase genes and their encoded polypeptides cooperate to maximize postselection copy number. CSR should prove useful for the directed evolution of enzymes, particularly DNA or RNA polymerases, as well as for the design and study of in vitro self-replicating systems mimicking prebiotic evolution and viral replication.

A type IB topoisomerase with DNA repair activities

Previously we have characterized type IB DNA topoisomerase V (topo V) in the hyperthermophile Methanopyrus kandleri. The enzyme has a powerful topoisomerase activity and is abundant in M. kandleri. Here we report two characterizations of topo V. First, we found that its N-terminal domain has sequence homology with both eukaryotic type IB topoisomerases and the integrase family of tyrosine recombinases. The C-terminal part of the sequence includes 12 repeats, each repeat consisting of two similar but distinct helix-hairpin-helix motifs; the same arrangement is seen in recombination protein RuvA and mammalian DNA polymerase β. Second, on the basis of sequence homology between topo V and polymerase β, we predict and demonstrate that topo V possesses apurinic/apyrimidinic (AP) site-processing activities that are important in base excision DNA repair: (i) it incises the phosphodiester backbone at the AP site, and (ii) at the AP endonuclease cleaved AP site, it removes the 5′ 2-deoxyribose 5-phosphate moiety so that a single-nucleotide gap with a 3′-hydroxyl and 5′-phosphate can be filled by a DNA polymerase. Topo V is thus the prototype for a new subfamily of type IB topoisomerases and is the first example of a topoisomerase with associated DNA repair activities.

Evidence that replication fork components catalyze establishment of cohesion between sister chromatids

Accurate chromosome segregation requires that replicated sister chromatids are held together until anaphase, when their “cohesion” is dissolved, and they are pulled to opposite spindle poles by microtubules. Establishment of new cohesion between sister chromatids in the next cell cycle is coincident with replication fork passage. Emerging evidence suggests that this temporal coupling is not just a coincident timing of independent events, but rather that the establishment of cohesion is likely to involve the active participation of replication-related activities. These include PCNA, a processivity clamp for some DNA polymerases, Trf4/Pol σ (formerly Trf4/Polκ), a novel and essential DNA polymerase, and a modified Replication Factor C clamp–loader complex. Here we describe recent advances in how cohesion establishment is linked to replication, highlight important unanswered questions in this new field, and describe a “polymerase switch” model for how cohesion establishment is coupled to replication fork progression. Building the bridges between newly synthesized sister chromatids appears to be a fundamental but previously unrecognized function of the eukaryotic replication machinery.

Tuning DNA “strings”: Modulating the rate of DNA replication with mechanical tension

Recent experiments have measured the rate of replication of DNA catalyzed by a single enzyme moving along a stretched template strand. The dependence on tension was interpreted as evidence that T7 and related DNA polymerases convert two (n = 2) or more single-stranded template bases to double helix geometry in the polymerization site during each catalytic cycle. However, we find structural data on the T7 enzyme–template complex indicate n = 1. We also present a model for the “tuning” of replication rate by mechanical tension. This model considers only local interactions in the neighborhood of the enzyme, unlike previous models that use stretching curves for the entire polymer chain. Our results, with n = 1, reconcile force-dependent replication rate studies with structural data on DNA polymerase complexes.

Somatic mosaicism in Wiskott–Aldrich syndrome suggests in vivo reversion by a DNA slippage mechanism

Somatic mosaicism caused by in vivo reversion of inherited mutations has been described in several human genetic disorders. Back mutations resulting in restoration of wild-type sequences and second-site mutations leading to compensatory changes have been shown in mosaic individuals. In most cases, however, the precise genetic mechanisms underlying the reversion events have remained unclear, except for the few instances where crossing over or gene conversion have been demonstrated. Here, we report a patient affected with Wiskott–Aldrich syndrome (WAS) caused by a 6-bp insertion (ACGAGG) in the WAS protein gene, which abrogates protein expression. Somatic mosaicism was documented in this patient whose majority of T lymphocytes expressed nearly normal levels of WAS protein. These lymphocytes were found to lack the deleterious mutation and showed a selective growth advantage in vivo. Analysis of the sequence surrounding the mutation site showed that the 6-bp insertion followed a tandem repeat of the same six nucleotides. These findings strongly suggest that DNA polymerase slippage was the cause of the original germ-line insertion mutation in this family and that the same mechanism was responsible for its deletion in one of the propositus T cell progenitors, thus leading to reversion mosaicism.

Requirement of proliferating cell nuclear antigen in RAD6-dependent postreplicational DNA repair.

The proliferating cell nuclear antigen (PCNA) acts as a processivity factor for replicative DNA polymerases and is essential for DNA replication. In vitro studies have suggested a role for PCNA-in the repair synthesis step of nucleotide excision repair, and PCNA interacts with the cyclin-dependent kinase inhibitor p21. However, because of the lack of genetic evidence, it is not clear which of the DNA repair processes are in fact affected by PCNA in vivo. Here, we describe a PCNA mutation, pol30-46, that confers ultraviolet (UV) sensitivity but has no effect on growth or cell cycle progression, and the mutant pcna interacts normally with DNA polymerase delta and epsilon. Genetic studies indicate that the pol30-46 mutation is specifically defective in RAD6-dependent postreplicational repair of UV damaged DNA, and this mutation impairs the error-free mode of bypass repair. These results implicate a role for PCNA as an intermediary between DNA replication and postreplicational DNA repair.