Abstract
The occurrence of triplet-repeat expansion (TRE) during transmission of genetic information is involved in many neurological and neuromuscular diseases including Fragile X syndrome and myotonic dystrophy. DNA slippage during replicative synthesis appears to cause TRE. The causes of DNA slippage, however, remain mostly unknown. We investigated the effects of abasic sites on the occurrence of TRE during DNA replication in vitro using Escherichia coli Klenow polymerase I as the model polymerase. Here we show that a single abasic site analog, synthesized in the triplet-repeat tract at the 5' end of the template strand, induced dramatic TRE during DNA synthesis. The amount of TRE induced decreased when the abasic site was moved to the middle of the repeat tract, consistent with effectively decreasing the length of the repeat tract. Placing the abasic site in the primer did not induce TRE. TRE was sequence-dependent. The damage-induced increase in growing strand TRE depended on the sequence of the growing strand repeat as AAT approximately ATT > CAG > CTG. The expansions required replication from a primer complementary to the repeat tract. The expanded tracts were sequenced and contained multiple additions of the original repeat. The results imply that DNA damage can play a significant role in generating TRE in vivo.
Highlights
The occurrence of triplet-repeat expansion (TRE)1 during transmission of genetic information is involved in many diseases including Fragile X syndrome [1,2,3], the most common form of mental retardation; myotonic dystrophy, a neuromuscular disorder; and several neurodegenerative disorders [1, 2]
DNA synthesis and TRE were detectable only when annealed primer-template was used for DNA replication
Low Mg2ϩ concentration was used to generate dramatic TRE [29] similar to the type II TREs observed in Fragile X syndrome and myotonic dystrophy [1,2,3]
Summary
TRE, triplet-repeat expansion; THF, tetrahydrofuran; pol I, polymerase I. intermediates [12, 13]. If pausing induces slippage, the occurrence within the repeat tract of DNA damage that blocks DNA replication could profoundly effect TRE. Replication of triplet-repeat tracts in vitro shows TRE (26 –29). The abasic site analog tetrahydrofuran (THF) was used as the model DNA damage lesion. Abasic sites and THF block DNA replication THF was synthesized in the triplet-repeat tracts separately in the template and in the primer strands. Replication using E. coli Klenow pol I as the model polymerase and primed from within the repeat tract demonstrated that THF in the template strand greatly enhanced TRE during DNA synthesis
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