Abstract
Trinucleotide repeat (TNR) instability can cause a variety of human genetic diseases including myotonic dystrophy and Huntington's disease. Recent genetic data show that instability of the CAG/CTG repeat DNA is dependent on its length and replication origin. In yeast, the RAD27 (human FEN-1 homologue) null mutant has a high expansion frequency at the TNR loci. We demonstrate here that FEN-1 processes the 5'-flap DNA of CTG/CAG repeats, which is dependent on the length in vitro. FEN-1 protein can cleave the 5'-flap DNA containing triplet repeating sequence up to 21 repeats, but the activity decreases with increasing size of flap above 11 repeats. In addition, FEN-1 processing of 5'-flap DNA depends on sequence, which play a role in the replication origin-dependent TNR instability. Interestingly, FEN-1 can cleave the 5'-flap DNA of CTG repeats better than CAG repeats possibly through the flap-structure. Our biochemical data of FEN-1's activity with triplet repeat DNA clearly shows length dependence, and aids our understanding on the mechanism of TNR instability.
Highlights
The genetic instability of trinucleotide repeats (TNR) is important for a number of human inherited neuromuscular and neurological diseases, such as Huntington's disease (HD), muscular myotonic dystrophy (MMD), Fragile X syndrome and Spinocerebellar ataxia (Warren and Nelson, 1993)
The expansion of TNR in the particular loci of human genome can cause the genetic diseases while normal individuals can maintain a short range of TNR. e.g. 10-35 CTGn: 5' (CTG) repeats in the case of Huntington's disease (Oostra and Willems, 1995)
The mechanism of TNR expansion is not clear so far, but it has been suggested that the realignment or slippage mediated by the characteristics of the repeat sequence on the Okazaki fragment can lead to the instability of TNR. (Gordenin et al, 1997)
Summary
The genetic instability of trinucleotide repeats (TNR) is important for a number of human inherited neuromuscular and neurological diseases, such as Huntington's disease (HD), muscular myotonic dystrophy (MMD), Fragile X syndrome and Spinocerebellar ataxia (Warren and Nelson, 1993). The expansion of TNR in the particular loci of human genome can cause the genetic diseases while normal individuals can maintain a short range of TNR. The mechanism of TNR expansion is not clear so far, but it has been suggested that the realignment or slippage mediated by the characteristics of the repeat sequence on the Okazaki fragment can lead to the instability of TNR. During lagging strand synthesis, which has a greater chance to form ss regions compared to leading strand synthesis, the unusual structural formation (hairpintype) from ss DNA can give rise to replication errors at the TNR loci. There is evidence to suggest that triplet repeat instability is dependent on the character of repeat DNA itself, such as its length and sequence, in E.coli and yeast (Kang et al, 1996; Freudenreich et al, 1997)
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