Slippage synthesis of simple sequence DNA.

The analysis of slippage synthesis of simple sequence DNA in vitro sheds some light on the question of how simple sequences arise in vivo. We show that it is possible to synthesize all types of repetitious di- and trinucleotide motifs starting from short primers and a polymerase in vitro. The rate of this synthesis depends on a sequence specific slippage rate, but is independent of the length of the fragments being synthesized. This indicates that only the ends of the DNA fragments are involved in determining this rate and that slippage is accordingly a short range effect. Slippage synthesis occurs also on a fixed template where only one strand is free to move, a situation which resembles chromosome replication in vivo. It seems therefore likely that slippage during replication is the cause of the observed length polymorphism of simple sequence stretches between individuals of a population.     

Analysis of slipped sequences in EST projects

Slippage is an important sequencing problem that can occur in EST projects. However, very few studies have addressed this. We propose three new methods to detect slippage artifacts: arithmetic mean method, geometric mean method, and echo coverage method. Each method is simple and has two different strategies for processing sequences: suffix and subsequence. Using the 291,689 EST sequences produced in the SUCEST project, we performed comparative tests between our proposed methods and the SUCEST method. The subsequence strategy is better than the suffix strategy, because it is not anchored at the end of the sequence, so it is more flexible to find slippage at the beginning of the EST. In a comparison with the SUCEST method, the advantage of our methods is that they do not discard the majority of the sequences marked as slippage, but instead only remove the slipped artifact from the sequence. Based on our tests the echo coverage method with subsequence strategy shows the best compromise between slippage detection and ease of calibration.     

The determination of the sequences present in the shadow bands of a dinucleotide repeat PCR.

A Polymerase Chain Reaction (PCR) of a DNA sequence containing a CA repeat produces a main band but also several shadow bands that differ by 2 base pairs below the main band. In the experiments described in this paper, these shadow bands were excised from a DNA sequencing gel and directly sequenced. It was found that the sequence in the CA repeat was ambiguous. However, the sequence 5' and 3' to the CA repeat was clear and unambiguous. It is proposed that the shadow bands are generated by 2 base pair random deletions in the CA repeat region. During this process the sequence becomes 'scrambled' only in the CA repeat region. The shadow bands were shown to occur during the PCR since the genomic DNA template did not contain the shadow bands. It is probable that the shadow bands arise by slippage during the PCR. It is predicted that a thermostable DNA polymerase with a high processivity would greatly reduce the occurrence of shadow bands.     

Base stacking and even/odd behavior of hairpin loops in DNA triplet repeat slippage and expansion with DNA polymerase

Repetitions of CAG or CTG triplets in DNA can form intrastrand hairpin loops with combinations of normal and mismatched base pairs that easily rearrange. Such loops may promote primer-template slippage in DNA replication or repair to give triplet-repeat expansions like those associated with neurodegenerative diseases. Using self-priming sequences (e.g. (CAG)(16)(CTG)(4)), we resolve all hairpin loops formed and measure their slippage and expansion rates with DNA polymerase at 37 degrees C. Comparing CAG/CTG loop structures with GAC/GTC structures, having similar hydrogen bonding but different base stacking, we find that CAG, CTG, and GTC triplets predominantly form even-membered loops that slip in steps of two triplets, whereas GAC triplets favor odd-numbered loops. Slippage rates decline as hairpin stability increases, supporting the idea that slippage initiates more easily in less stable regions. Loop stabilities (in low salt) increase in the order GTC < CAG < GAC < CTG, while slippage rates decrease in the order GTC > CAG approximately GAC > CTG. Loops of GTC compared with CTG melt 9 degrees C lower and slip 6-fold faster. We interpret results in terms of base stacking, by relating melting temperature to standard enthalpy changes for doublets of base pairs and mispairs, considering enthalpy-entropy compensation.     

Sequence composition and context effects on the generation and repair of frameshift intermediates in mononucleotide runs in Saccharomyces cerevisiae

DNA polymerase slippage occurs frequently in tracts of a tandemly repeated nucleotide, and such slippage events can be genetically detected as frameshift mutations. In long mononucleotide runs, most frameshift intermediates are repaired by the postreplicative mismatch repair (MMR) machinery, rather than by the exonucleolytic proofreading activity of DNA polymerase. Although mononucleotide runs are hotspots for polymerase slippage events, it is not known whether the composition of a run and the surrounding context affect the frequency of slippage or the efficiency of MMR. To address these issues, 10-nucleotide (10N) runs were inserted into the yeast LYS2 gene to create +1 frameshift alleles. Slippage events within these runs were detected as Lys(+) revertants. 10G or 10C runs were found to be more unstable than 10A or 10T runs, but neither the frequency of polymerase slippage nor the overall efficiency of MMR was greatly influenced by sequence context. Although complete elimination of MMR activity (msh2 mutants) affected all runs similarly, analyses of reversion rates in msh3 and msh6 mutants revealed distinct specificities of the yeast Msh2p-Msh3p and Msh2p-Msh6p mismatch binding complexes in the repair of frameshift intermediates in different sequence contexts.     

Nucleotide analogs facilitate base conversion with 3' mismatch primers

We compared the efficiency of PCR amplification using primers containing either a nucleotide analog or a mismatch at the 3' base. To determine the distribution of bases inserted opposite eight different analogs, 3' analog primers were used to amplify four different templates. The products from the reactions with the highest amplification efficiency were sequenced.Analogs allowing efficient amplification followed by insertion of a new base at that position are herein termed 'convertides'. The three convertides with the highest amplification efficiency were used to convert sequences containing C, T, G and A bases into products containing the respective three remaining bases. Nine templates were used to generate conversion products, as well as non-conversion control products with no base change. We compared the ability of natural bases to convert specific sites with and without a preconversion step using nucleotide analog primers. Conversion products were identified by a ligation detection reaction using primers specific for the converted sequence. We found that conversions resulting in transitions were easier to accomplish than transversions and that sequence context influences conversion. Specifically, primer slippage appears to be an important mechanism for producing artifacts via polymerase extension of a 3' base or analog transiently base paired to neighboring bases of the template. Nucleotide analogs could often reduce conversion artifacts and increase the yield of the expected product. While new analogs are needed to reliably achieve transversions, the current set have proven effective for creating transition conversions.     

Fold-back structures at the distal end influence DNA slippage at the proximal end during mononucleotide repeat expansions.

Polymerase slippage during DNA synthesis by the Klenow fragment of DNA polymerase across A, C, G and T repeats (30 bases) has been studied. Within minutes, duplexes that contain only repeats (30 bp) expand dramatically to several hundred base pairs long. Rate comparisons in a repeat duplex when one strand was expanded as against that when both strands were expanded suggest a model of migrating hairpin loops which in the latter case coalesce into a duplex. Moreover, slippage (at the proximal or 3'-end) is subject to positive and negative effects from the 5'-end (distal) of the same strand. Growing T and G strands generate T.A:T and G-G:C motif fold-back structures at the distal end that hamper slippage at the proximal end. On the other hand, growing tails at the distal end upon annealing with excess complementary template accentuates proximal slippage several-fold.     

DNA Sequence Effects on Single Base Deletions Arising during DNA Polymerization in Vitro by Escherichia Coli Klenow Fragment Polymerase

Most single base deletions detected after DNA polymerization in vitro directed by either Escherichia coli DNA polymerase I or its Klenow fragment are opposite Pu in the template. The most frequent study, were previously found to be associated with the consensus template context 5'-PyTPu-3'. In this study, the predictive power of the consensus sequence on single base deletion frequencies was directly tested by parallel comparison of mutations arising in four related DNAs differing by a single base. G, a deletion hotspot within the template context 5'-TTGA-3', was substituted by each of the 3 other bases. Previous studies had shown that deletions opposite the G were frequent but that deletions opposite its neighboring A were never detected. Based on the predictions of the consensus, the substitution of T for G should produce frequent deletions opposite the neighboring A due to its new 5'-TTTA-3' template context. This prediction was fulfilled; no deletions of this A were detected in the other templates. The consensus further predicted that deletions opposite template C would be lower than those opposite either A or G at the same site and this prediction was also fulfilled. The C substitution also produced a new hotspot for 1 bp deletions 14 bp away. The new hotspot depends on quasi-palindromic misalignment of the newly synthesized DNA strand during polymerization; accurate, but ectopically templated synthesis is responsible for this mutagenesis. Mutations templated by quasi-palindromic misalignments have previously been recognized when they produced complex sequence changes; here we show that this mechanism can produce frequent single base deletions. The unique stimulation of misalignment mutagenesis by the C substitution in the template is consistent with the singular ability of C at that site to contribute to extended complementary pairing during the DNA misalignment that precedes mutagenesis.     

UmuD2 Inhibits a Non-covalent Step during DinB-mediated Template Slippage on Homopolymeric Nucleotide Runs

Escherichia coli DinB (DNA polymerase IV) possesses an enzyme architecture resulting in specialized lesion bypass function and the potential for creating −1 frameshifts in homopolymeric nucleotide runs. We have previously shown that the mutagenic potential of DinB is regulated by the DNA damage response protein UmuD₂. In the current study, we employ a pre-steady-state fluorescence approach to gain a mechanistic understanding of DinB regulation by UmuD₂. Our results suggest that DinB, like its mammalian and archaeal orthologs, uses a template slippage mechanism to create single base deletions on homopolymeric runs. With 2-aminopurine as a fluorescent reporter in the DNA substrate, the template slippage reaction results in a prechemistry fluorescence change that is inhibited by UmuD₂. We propose a model in which DNA templates containing homopolymeric nucleotide runs, when bound to DinB, are in an equilibrium between non-slipped and slipped conformations. UmuD₂, when bound to DinB, displaces the equilibrium in favor of the non-slipped conformation, thereby preventing frameshifting and potentially enhancing DinB activity on non-slipped substrates.     

In vitro replication slippage by DNA polymerases from thermophilic organisms

Replication slippage of DNA polymerases is a potential source of spontaneous genetic rearrangements in prokaryotic and eukaryotic cells. Here we show that different thermostable DNA polymerases undergo replication slippage in vitro, during single-round replication of a single-stranded DNA template carrying a hairpin structure. Low-fidelity polymerases, such as Thermus aquaticus (Taq), high-fidelity polymerases, such as Pyrococcus furiosus (Pfu) and a highly thermostable polymerase from Pyrococcus abyssi (Pyra exo(-)) undergo slippage. Thermococcus litoralis DNA polymerase (Vent) is also able to slip; however, slippage can be inhibited when its strand-displacement activity is induced. Moreover, DNA polymerases that have a constitutive strand-displacement activity, such as Bacillus stearothermophilus DNA polymerase (Bst), do not slip. Polymerases that slip during single-round replication generate hairpin deletions during PCR amplification, with the exception of Vent polymerase because its strand-displacement activity is induced under these conditions. We show that these hairpin deletions occurring during PCR are due to replication slippage, and not to a previously proposed process involving polymerization across the hairpin base.     

Strand asymmetry of +1 frameshift mutagenesis at a homopolymeric run by DNA polymerase III holoenzyme of Escherichia coli

We have recently shown that single-base frameshifts were predominant among mutations induced within the rpsL target sequence upon oriC plasmid DNA replication in vitro. We found that the occurrence of +1 frameshifts at a run of 6 residues of dA/dT could be increased proportionally by increasing the concentration of dATP present in the in vitro replication. Using single-stranded circular DNA containing either the coding sequence of the rpsL gene or its complementary sequence, the +1 frameshift mutagenesis by DNA polymerase III holoenzyme of Escherichia coli was extensively examined. A(6) --> A(7) frameshifts occurred 30 to 90 times more frequently during DNA synthesis with the noncoding sequence (dT tract) template than with the coding sequence (dA tract). Excess dATP enhanced the occurrence of +1 frameshifts during DNA synthesis with the dT tract template, but no other dNTPs showed such an effect. In the presence of 0.1 mM dATP, the A(6) --> A(7) mutagenesis with the dT tract template was not inhibited by 1.5 mM dCTP, which is complementary to the residue immediately upstream of the dT tract. These results strongly suggested that the A(6) --> A(7) frameshift mutagenesis possesses an asymmetric strand nature and that slippage errors leading to the +1 frameshift are made during chain elongation within the tract rather than by misincorporation of nucleotides opposite residues next to the tract.     

Plastome mutator-induced alterations arise in Oenothera chloroplast DNA through template slippage.

The plastome mutator of Oenothera hookeri strain Johansen causes deletions and duplications at target sites defined by direct repeats in the plastid genome. Previous studies characterized the mutations long after they had occurred and could not discriminate between the possibilities that the plastome mutator acted through unequal homologous recombination or template slippage. From the known hotspots, the rRNA spacer in the large inverted repeat was chosen for this study because it contains both direct and indirect repeats. Identical deletions were recovered from independently derived plants; the altered regions were always flanked by direct repeats. The regions in which the deletions occurred have the potential to form secondary structures that would stabilize the intervening sequence. Of the two affected regions, the one with the stronger potential secondary structure was altered more frequently. Because no duplication products or inversions were recovered, it is proposed that the plastome mutator acts through template slippage rather than through a recombination mechanism.     

Fourier and wavelet transform analysis, a tool for visualizing regular patterns in DNA sequences

A correlation function that compares each base in a DNA sequence to its various neighbours and which is subsequently processed by Fourier and wavelet transforms has been developed. The procedure has been applied to sequences from the human chromosome 22, to nef genes from various HIV clones and to myosin heavy chain DNA. It permits to readily visualize regular features in DNA which are related to the stability of heteroduplexes formed upon strand slippage.     

Frameshift mutagenesis by eucaryotic DNA polymerases in vitro

The frequency and specificity of frameshift errors produced during a single round of in vitro DNA synthesis by DNA polymerases-alpha, -beta, and -gamma (pol-alpha, -beta, and -gamma, respectively) have been determined. DNA polymerase-beta is the least accurate enzyme, producing frameshift errors at an average frequency of one error for each 1,000-3,000 nucleotides polymerized, a frequency similar to its average base substitution accuracy. DNA polymerase-alpha is approximately 10-fold more accurate, producing frameshifts at an average frequency of one error for every 10,000-30,000 nucleotides polymerized, a frequency which is about 2- to 6-fold lower than the average pol-alpha base substitution accuracy. DNA polymerase-gamma is highly accurate, producing on the average less than one frameshift error for every 200,000-400,000 nucleotides polymerized. This represents a more than 10-fold higher fidelity than for base substitutions. Among the collection of sequenced frameshifts produced by DNA polymerases-alpha and beta, both common features and distinct specificities are apparent. These specificities suggest a major role for eucaryotic DNA polymerases in modulating frameshift fidelity. Possible mechanisms for production of frameshifts are discussed in relation to the observed biases. One of these models has been experimentally supported using site-directed mutagenesis to change the primary DNA sequence of the template. Alteration of a pol-beta frameshift hotspot sequence TTTT to CTCT reduced the frequency of pol-beta-dependent minus-one-base errors at this site by more than 30-fold, suggesting that more than 97% of the errors at the TTTT run involve a slippage mechanism.