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. 2005 Mar 14;33(5):1604-17.
doi: 10.1093/nar/gki307. Print 2005.

Structural roles of CTG repeats in slippage expansion during DNA replication

Lai Man Chi  1 , Sik Lok Lam
Affiliations

Structural roles of CTG repeats in slippage expansion during DNA replication

Lai Man Chi et al. Nucleic Acids Res. .

Abstract

CTG triplet repeat sequences have been found to form slipped-strand structures leading to self-expansion during DNA replication. The lengthening of these repeats causes the onset of neurodegenerative diseases, such as myotonic dystrophy. In this study, electrophoretic and NMR spectroscopic studies have been carried out to investigate the length and the structural roles of CTG repeats in affecting the hairpin formation propensity. Direct NMR evidence has been successfully obtained the first time to support the presence of three types of hairpin structures in sequences containing 1-10 CTG repeats. The first type contains no intra-loop hydrogen bond and occurs when the number of repeats is less than four. The second type has a 4 nt TGCT-loop and occurs in sequences with even number of repeats. The third type contains a 3 nt CTG-loop and occurs in sequences with odd number of repeats. Although stabilizing interactions have been identified between CTG repeats in both the second and third types of hairpins, the structural differences observed account for the higher hairpin formation propensity in sequences containing even number of CTG repeats. The results of this study confirm the hairpin loop structures and explain how slippage occurs during DNA replication.

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Figures

Figure 1
Figure 1
(A) Imino and methyl protons (in bold) in Watson–Crick base pair and T·T mismatch. Owing to the asymmetric arrangement of the T·T mismatches, two T·T pairing modes are present. Rapid transition between the two pairing modes is possible. (B) Numbering scheme of (CTG)n. Nucleotides in gray represent the extended stem regions that promote hairpin formation.
Figure 2
Figure 2
NMR determined and mfold predicted structures of (CTG)n.
Figure 3
Figure 3
Non-denaturing gel of (CTG)n at (A) 10 μM and (B) 1 mM. (C) Hairpin populations of (CTG)n determined at 10 μM (black) and 1 mM (gray). The populations were calculated from the average of four trials and the uncertainties were determined from the standard deviations of the average values.
Figure 4
Figure 4
(A) Imino and (B) methyl regions of (CTG)n with n = 1–3. The label TR(m·n) refers to the assignment of TRm·TRn.
Figure 5
Figure 5
(A) Imino and (B) methyl regions of (CTG)3, (CTG)3 after slow cool treatment and extended (CTG)3. By comparing the G8 and T9 imino intensities and the methyl intensities of the hairpin and duplex conformers, the duplex population was found to increase in the slow cool state but decrease in the extended (CTG)3 sequence.
Figure 6
Figure 6
Imino regions of (CTG)4, (CTG)4 after slow cool treatment, extended (CTG)4 and extended TR1C-(CTG)4. The extended (CTG)4 sample contains four additional G–C base pairs at the stem terminals. The extension of the stem region promotes the hairpin population and thus a decrease in the duplex population. The imino peaks at 10.53 and 10.76 p.p.m. were found to decrease accordingly, confirming that these two peaks belong to the duplex conformer. In the extended TR1C-(CTG)4 sample, TR1 was substituted by a cytosine nucleotide TR1C, the imino region shows no signal at 10.53 and 10.60 p.p.m. As no TR1C·TR4 pairing was expected in both the hairpin and duplex conformers, the absence of signal at these chemical shifts confirms the assignments of TR1·TR4 in the (CTG)4 duplex and hairpin, respectively.
Figure 7
Figure 7
(A) Imino and (B) methyl regions of (CTG)n with n = 4, 6, 8 and 10.
Figure 8
Figure 8
(A) Imino and (B) methyl regions of (CTG)5, extended (CTG)5, extended (CTG)5 after slow cool treatment and extended TR2C-(CTG)5.
Figure 9
Figure 9
(A) Imino and (B) methyl regions of (CTG)5, (CTG)7 and (CTG)9.
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References

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