Strand Switching During Synthesis of dATTCT dAgaat Dna Repeats Can Result in Complex Expansion Mutations

Primer-template misalignment can occur forward or backward along the same template strand, resulting in duplications and deletions, respectively;

however, misalignment within a palindromic or quasi-palindromic sequence can also occur on a different template strand. This can occur in an intermolecular fashion within a single replication fork from the leading to the lagging strand (or from the lagging to the leading strand) or between two different chromosomes (Fig. 3). Strand switching can also occur in an intramolecular fashion when the nascent strand snaps back on itself, forming

Fig. 3 Intermolecular strand switching can occur within quasi-palindromic repeats forming perfect inverted repeats. A quasi-palindromic sequence, including d(CXG)n, d(CCTG)n • d(CAGG)n, and d(ATTCT)n • d(AGAAT)n repeats can form various degrees of mispaired hairpin structures in one or both strands. The self-complementary base-pairing potential can lead to an intramolecular or an intermolecular strand switch. For the intramolecular strand switch, during leading-strand synthesis the nascent strand can unpair (step B) and form a mispaired hairpin region (denoted by the shaded region of the helix) (step C). Continued synthesis down the hairpin can lead to the formation of a perfect inverted repeat (denoted by the thicker line) (step D). For the intermolecular strand switch, following unwinding (step E), the 3' end of the nascent strand pairs with the repeat in the lagging template strand (step F). Continued synthesis also leads to a perfect inverted repeat in the leading nascent strand

Fig. 3 Intermolecular strand switching can occur within quasi-palindromic repeats forming perfect inverted repeats. A quasi-palindromic sequence, including d(CXG)n, d(CCTG)n • d(CAGG)n, and d(ATTCT)n • d(AGAAT)n repeats can form various degrees of mispaired hairpin structures in one or both strands. The self-complementary base-pairing potential can lead to an intramolecular or an intermolecular strand switch. For the intramolecular strand switch, during leading-strand synthesis the nascent strand can unpair (step B) and form a mispaired hairpin region (denoted by the shaded region of the helix) (step C). Continued synthesis down the hairpin can lead to the formation of a perfect inverted repeat (denoted by the thicker line) (step D). For the intermolecular strand switch, following unwinding (step E), the 3' end of the nascent strand pairs with the repeat in the lagging template strand (step F). Continued synthesis also leads to a perfect inverted repeat in the leading nascent strand

Fig. 4 Complex expansion mutation associated with an intermolecular strand switch during replication of d(ATTCT)n • d(AGAAT)n repeats. The A+T-rich spinocerebellar ataxia type 10 (SCA10) repeat undergoes both intermolecular and intramolecular strand switch events in Escherichia coli, creating an inverted repeat region associated with complex expansion mutations. In addition, the plasmid containing the expansion is a dimer. A model is presented for the expansion, inversion, and plasmid dimerization. An intermolecular strand switch from the leading to the lagging template occurs (step B). The dark line represents the d(ATTCT)n strand, while the light line represents the d(AGAAT)n strand. Arrow heads at the ends of lines represent the 3' end of a nascent DNA strand. Arrow tails at the ends of lines represent the 5' end of a DNA strand. Replication following the strand switch results in the formation of an inverted repeat region (contiguous dark and light line) (step C). Dissociation of the nascent 3' end from the lagging template strand and reassociation with the leading template strand results in an unpaired 3' end inherent with the expansion (step D). Primer-template pairing within flanking direct repeats, used for cloning the repeats, occurs concomitant with the formation of a hairpin or a loop (step E). A strand exchange occurs (step E) with the nascent lagging strand (synthesized in step D). Following introduction of a nick (at the large arrow) a Holliday junction is formed (steps E-G). Branch migration occurs (step G) and a nick is introduced into the lagging template strand (step H). The lagging template then becomes joined to the leading nascent strand (step I). Synthesis from the 3' end of the lagging template strand restores the crossover replication fork and continued replication leads to plasmid dimerization (step I). This complex molecular event provides a good example of the degree to which the properties of a simple DNA repeat sequence can direct complex genetic alterations a hairpin on continued DNA synthesis. Replication that follows the strand switch within a quasi-palindrome results in the formation of a perfect inverted repeat (Ripley 1982; Sinden et al. 1999; van Noort et al. 2003).

Results for one quasi-palindrome correction mutation in E. coli indicated that an intermolecular strand switch specific for the leading strand occurred (Rosche et al. 1997), while an intramolecular strand switch was implicated to explain another mutation (Viswanathan et al. 2000). Therefore, quasipalindrome corrections occurring in either the leading or the lagging strands have been identified in different mutational systems (Rosche et al. 1997, 1998; Viswanathan et al. 2000; Yoshiyama et al. 2001; Yoshiyama and Maki 2003). During replication of plasmid in E. coli containing the SCA10 repeat tract, which contains weak quasi-palindromic repeat symmetry, similar complex expansion mutations with the general sequence, d(TATTC)5-11 ■ d(GAATA)9-35, were observed regardless of the initial orientation of the repeat tract (either d(AGAAT)23 ■ d(ATTCT)23 or d(ATTCT)24 ■ d(AGAAT)24 ). This mutation was also coupled with plasmid dimerization (Hashem VI, Edwards SF, Klysik EA, Pytlos MJ, Sinden RR, unpublished). Insight into an explanation for this result stems from the fact that only a strand switch of the nascent Py-rich strand can produce the inverted repeat found in the complex expansion mutations. For the two different orientations of the repeat tract, the nascent Py-rich strand comprises the leading nascent strand for the d(AGAAT)23 ■ d(ATTCT)23 orientation, while it comprises the lagging nascent strand for the d(ATTCT)24 ■ d(AGAAT)24 orientation. Thus, to form the inverted repeat in the two different repeat orientations, the strand switch must occur in the leading strand for one orientation and in the lagging strand for the other orientation (Hashem VI, Edwards SF, Klysik EA, Pytlos MJ, Sinden RR, unpublished). In the d(ATTCT)24 ■ d(AGAAT)24 orientation, a simple slippage to lengthen the repeat tract must occur prior to the strand switch to generate the observed product (Fig. 4). This is the first example of a DNA sequence that can support both an intermolecular and an intramolecular strand switch during leading or lagging strand synthesis. The instability associated with d(ATTCT) ■ d(AGAAT) repeats, and even disease-associated triplet or tetranu-cleotide repeats, in human cells may be linked to aberrant replication. In the following a model is presented in which aberrant replication initiation leading to amplification may result in repeat expansion (as well as repeat deletion).

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