In Disease Associated DNA Repeats

As first proposed by Streisinger, primer-template misalignment can occur within a run of direct repeats (Streisinger et al. 1966). Mutations associated with primer-template misalignment have been established in many model systems (Kunkel and Soni 1988; Ripley 1990; Kunkel 1990; Papanicolaou and Ripley 1991; Rosche et al. 1998; Sinden et al. 1999; Bebenek and Kunkel 2000; van Noort et al. 2003). Misalignment can occur within runs of repeats (Streisinger et al. 1966; Wierdl et al. 1997; Kroutil and Kunkel 1999; Hashem et al. 2002) or between distant direct repeats (Drake et al. 1983; Ripley et al. 1986) (Fig. 2). In the case of triplet repeats, a simple slippage may result in a 3-nt loopout (Fig. 2, panel A). In cells, a 3-nt slippage, and/or repair of the loopout, can be very different for opposite orientations of the repeat with respect to the origin of replication (Hashem et al. 2002). Large slippage events

Fig. 2 Replication slippage can result in deletions and duplications (expansions). A A 3-bp misalignment can occur by unwinding of the primer end of the nascent strand from the template (step 2), followed by reannealing 3 nt to the 5' side on the template, resulting in a 3-nt loopout in the nascent strand (step 3). Continued synthesis would result in a 3-bp expansion in the nascent strand, if not repaired by mismatch or excision repair-type activities. B Primer-template misalignment between short direct repeats can also occur over large distances. In the case of disease-associated repeats, misalignment can occur anywhere within the repeat tract. When DNA repeats can form stable hairpins, they can promote slippage in the nascent strand leading to expansion (steps 2, 3), or in the template strand leading to deletion (steps 4, 5)

Fig. 2 Replication slippage can result in deletions and duplications (expansions). A A 3-bp misalignment can occur by unwinding of the primer end of the nascent strand from the template (step 2), followed by reannealing 3 nt to the 5' side on the template, resulting in a 3-nt loopout in the nascent strand (step 3). Continued synthesis would result in a 3-bp expansion in the nascent strand, if not repaired by mismatch or excision repair-type activities. B Primer-template misalignment between short direct repeats can also occur over large distances. In the case of disease-associated repeats, misalignment can occur anywhere within the repeat tract. When DNA repeats can form stable hairpins, they can promote slippage in the nascent strand leading to expansion (steps 2, 3), or in the template strand leading to deletion (steps 4, 5)

may occur within a long repeat tract, resulting in a backwards slippage and the formation of a hairpin in the leading nascent strand (Fig. 2, panel B, pathway to expansion), and continued replication would lead to expansion by the length of the slippage. A forward slippage, perhaps directed by hairpin formation in the lagging template strand, could lead to deletion in the lagging nascent strand (Fig. 2, panel B, pathway to deletion). This type of mutation can be influenced dramatically by DNA symmetry elements, especially inverted repeats. Inverted repeats can fold into hairpins that can promote deletion between flanking direct repeats in the lagging template strand (Trinh and Sinden 1991, 1993; Rosche et al. 1995), or direct duplications when hairpins form in the leading nascent strand (Hashem and Sinden 2005).

For primer-template misalignment to occur, DNA polymerization must stop and the polymerase must presumably dissociate from the DNA. It is not known what feature of DNA, either sequence or structure, might be involved in mediating this pausing or stopping. Polymerase may pause at random, or exhibit preferred pause sites, as occurs in vitro, where pause sites are associated with misalignment mutations (Papanicolaou and Ripley 1989, 1991). In the case of disease-associated DNA repeats, pausing might be enhanced or promoted by the formation of an alternative DNA structure, of the types discussed before. Moreover, as leading and lagging strand replications are believed to be coordinated, structure formation in the lagging strand may stop leading strand synthesis, and vice versa. Once the nascent 3' terminus dissociates from the template, it is free to anneal at any location containing complementary base pairs. Essentially nothing is known about the events and mechanics associated with polymerase dissociation. Similar frequencies of duplication and deletion between direct repeats spaced 17 bp apart lead to the suggestion that at least 20 bp become unpaired in the initial dissociation event (Trinh and Sinden 1993).

Because the size of potential expansion or deletion is limited to the length of the repeat tract minus the length of the segment used as a template, expansion by this model is necessarily less than the length of the repeat; thus, only expansion by less than a factor of 2 is possible. In previous reviews, we have discussed the possibility of reiterative DNA synthesis (Kornberg et al. 1964), perhaps caused by an alternative DNA structure block to DNA replication in the leading or lagging strand (Sinden and Wells 1992; Wells and Sinden 1993; Sinden 1999). Repeated slippage during replication has been observed in vitro with several enzymes, including human polymerase P (Petruska et al. 1998; Hartenstine et al. 2000; Kobayashi et al. 2002; Heidenfelder et al. 2003; Heidenfelder and Topal 2003; Ruggiero and Topal 2004).

Instabilities occurring throughout life in certain tissues in humans and in mice are consistent with the possibility that simple slipped misalignment occurs during replication. In humans, the expanded d(CTG) repeat is unstable and shows a bias toward continued expansion in germline and somatic tissues during life (Wong et al. 1995; Martorell et al. 1998). Mice also show repeat instabilities throughout life (Mangiarini et al. 1997; Monckton et al. 1997; Sato et al. 1999; Seznec et al. 2000; Fortune et al. 2000). Repeat heterogeneity in E. coli, especially in mismatch repair deficient strains, is consistent with slipped misalignment during replication of repeats in bacteria (Schumacher et al. 1998; Schmidt et al. 2000; Parniewski et al. 2000). Thus, slipped misalignment may be the simplest mechanism for repeat instability and it could be operable for all repeats.

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