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, 37 (5), 1682-9

DNA Models of Trinucleotide Frameshift Deletions: The Formation of Loops and Bulges at the Primer-Template Junction

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DNA Models of Trinucleotide Frameshift Deletions: The Formation of Loops and Bulges at the Primer-Template Junction

Walter A Baase et al. Nucleic Acids Res.

Abstract

Although mechanisms of single-nucleotide residue deletion have been investigated, processes involved in the loss of longer nucleotide sequences during DNA replication are poorly understood. Previous reports have shown that in vitro replication of a 3'-TGC TGC template sequence can result in the deletion of one 3'-TGC. We have used low-energy circular dichroism (CD) and fluorescence spectroscopy to investigate the conformations and stabilities of DNA models of the replication intermediates that may be implicated in this frameshift. Pyrrolocytosine or 2-aminopurine residues, site-specifically substituted for cytosine or adenine in the vicinity of extruded base sequences, were used as spectroscopic probes to examine local DNA conformations. An equilibrium mixture of four hybridization conformations was observed when template bases looped-out as a bulge, i.e. a structure flanked on both sides by duplex DNA. In contrast, a single-loop structure with an unusual unstacked DNA conformation at its downstream edge was observed when the extruded bases were positioned at the primer-template junction, showing that misalignments can be modified by neighboring DNA secondary structure. These results must be taken into account in considering the genetic and biochemical mechanisms of frameshift mutagenesis in polymerase-driven DNA replication.

Figures

Figure 1.
Figure 1.
Oligonucleotide constructs and nomenclature. (a) gT indicates the template strand used to construct duplex molecules with GC bps flanking the 3 nt loop at positions [−3,−2,−1] (c) aT indicates the template strand used to construct duplex molecules with AT bps flanking the [−3,−2,−1] loop sequence. The positions of the probe residues 2-AP (Ap) or PC (PC) are indicated in the name of the template strand in square brackets. Potential looped out sequences of the T strand are underlined. (b) and (d) are primer strands (P) that are partially complementary to the gT strands (gP) or to the aT strands (aP). Primer strands of different lengths are designated by the position of the 3′-OH terminus. The numbering scheme at the top of the Figure refers to both strands. gC and aC are fully complementary to gT and aT, respectively.
Figure 2.
Figure 2.
Fluorescence changes observed during loop formation. Fluorescence intensities of 1 μM duplex molecules formed with template DNA T and various length primer strands, gP (see Figure 1 for nomenclature). Fl is the fluorescence intensity of the duplex molecule relative to the signal of the ss T strand. The signal of the fully duplex molecule without extrahelical bases is shown at the right of each panel. gTβ (circles); gT (squares). Template sequences had a single PC residue at position [−4] (a) or position [1] (b).
Figure 3.
Figure 3.
Fluorescence of duplex oligonucleotides with GC bps flanking the loop sequence. Fluorescence signals of 1 μM duplex molecules with primer strands of various lengths, gP, were normalized to the intensities of the corresponding gP-5 and gP-4 complexes (see text). (a) gTα[1] (circles), gTβ[1] (squares) and gTγ[1] (triangles). (b) gT[1] (circles) and gT[−1,1] (squares). See Figure 1 for nomenclature. In order to show the self-quenching of adjacent PC residues in ss template oligonucleotide gT[−1,1] (see text), signals of gT[−1,1] complexes are normalized relative to the intensity of gT[−1]/gP-5 and gT[−1]/gP-4.
Figure 4.
Figure 4.
Fluorescence of duplex oligonucleotides with AT bps flanking the loop sequence. The sequences of the template strands aTα[1] (open circles) and aTβ[1] (open squares) are identical except for the loop (Figure 1). Closed circles are Fl signals from complexes of gTα[1] with the aP primers. Fluorescence intensities of 1 μM duplex molecules with various length primer strands, aP, have been normalized to the signals of the aP-5 and aP-4 complexes.
Figure 5.
Figure 5.
Spectroscopic characterization of bulge and P/T loop DNA constructs. (a,b) ss template DNA (black); duplex DNA with oligonucleotide gC as the complementary strand (blue); bulge DNA with complementary strand gP6 (red). The yellow lines are the best fits of the bulge CD spectra (see text). (c,d) duplex DNA with complementary oligonucleotide gP-5 (yellow), gP-4 (black), gP1 (red) or gP2 (blue). Oligonucleotide concentrations were 13 μM. Template strands have either a PC residue at position [−4], gT[−4] (a,c) or a PC dimer at positions [−1,1], gT[–1,1] (b,d). Oligonucleotide sequences and numbering schemes are shown in Figure 1. CD spectra and fluorescence intensities (inserts) are plotted per mole PC residue. Fl intensity is in arbitrary units that are the same in all Inserts.
Figure 6.
Figure 6.
Hybridization schemes of the gT/gP2 construct. Upper strand is gP2; lower strand represents either gT[−4] or gT[−1,1], depending on the position(s) of the PC residue (X) The different base pairing schemes are named at the left by the positions of the looped-out bases in the template strand. {0} is a possible alignment in which the two 3′ terminal bases of the primer are not base-paired with the template strand.

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References

    1. Streisinger G, Okada Y, Emrich J, Newton J, Tsugita A, Terzaghi E, Inouye M. Frameshift mutations and the genetic code. Cold Spring Harb. Symp. Quant. Biol. 1966;31:77–84. - PubMed
    1. Kunkel TA, Soni A. Mutagenesis by transient misalignment. J. Biol. Chem. 1988;263:14784–14789. - PubMed
    1. Bebenek K, Kunkel TA. Frameshift errors initiated by nucleotide misincorporation. Proc. Natl Acad. Sci. USA. 1990;87:4946–4950. - PMC - PubMed
    1. Tippin B, Kobayashi S, Bertram JG, Goodman MF. To slip or skip, visualizing frameshift mutation dynamics for error-prone DNA polymerases. J. Biol. Chem. 2004;279:45360–45368. - PubMed
    1. Garcia-Diaz M, Bebenek K, Krahn JM, Pedersen LC, Kunkel TA. Structural analysis of strand misalignment during DNA synthesis by a human DNA polymerase lambda. Cell. 2006;124:331–342. - PubMed

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