Pausing of DNA Synthesis in Vitro at Specific Loci in CTG and CGG Triplet Repeats from Human Hereditary Disease Genes

Seongman Kang,Keiichi Ohshima,Miho Shimizu,Sorour Amirhaeri,Robert D Wells

doi:10.1074/jbc.270.45.27014

Abstract

Several human hereditary neuromuscular disease genes are associated with the expansion of CTG or CGG triplet repeats. The DNA syntheses of CTG triplets ranging from 17 to 180 and CGG repeats from 9 to 160 repeats in length were studied in vitro. Primer extensions using the Klenow fragment of DNA polymerase I, the modified T7 DNA polymerase (Sequenase), or the human DNA polymerase beta paused strongly at specific loci in the CTG repeats. The pausings were abolished by heating at 70 degrees C. As the length of the triplet repeats in duplex DNA, but not in single-stranded DNA, was increased, the magnitude of pausing increased. The location of the pause sites was determined by the distance between the site of primer hybridization and the beginning of the triplet repeats. CGG triplet repeats also showed similar, but not identical, patterns of pausings. These results indicate that appropriate lengths of the triplets adopt a non-B conformation(s) that blocks DNA polymerase progression; the resultant idling polymerase may catalyze slippages to give expanded sequences and hence provide the molecular basis for this non-Mendelian genetic process. These mechanisms, if present in human cells, may be related to the etiology of certain neuromuscular diseases such as myotonic dystrophy and Fragile X syndrome.

Highlights

CTG and CGG trinucleotide repeat expansions are associated with a number of human hereditary genetic disease genes including human myotonic dystrophy [1,2,3], Kennedy’s disease [4], spinocerebellar ataxia type I [5], Huntington’s disease [6], dentatorubral-pallidoluysian atrophy [7, 8], Haw River syndrome [9], Machado-Joseph disease [10], and Fragile X and XE syndromes [11,12,13]
We show that CTG and CGG triplet repeat sequences, which originated from hereditary genetic disease patients, have unorthodox properties; DNA polymerases pause at specific locations in these sequences
Our in vitro experiments show aberrant DNA synthesis in massive CTG and CGG repeat sequences; pausings of DNA polymerases occur at specific loci

Summary

Introduction

CTG and CGG trinucleotide repeat expansions are associated with a number of human hereditary genetic disease genes including human myotonic dystrophy [1,2,3], Kennedy’s disease [4], spinocerebellar ataxia type I [5], Huntington’s disease [6], dentatorubral-pallidoluysian atrophy [7, 8], Haw River syndrome [9], Machado-Joseph disease [10], and Fragile X and XE syndromes [11,12,13]. Fragile X syndrome, the most frequent inherited mental retardation, contains multiple CGG repeats within the mRNA noncoding region of a Fragile X associated gene (FMR-1) while, for the other hereditary diseases, the CTG repeat is located within the genes and encodes a tract of glutamines (4 –13). These highly polymorphic triplet repeats have been shown to range from 5 to 37 copies on normal chromosomes, whereas carriers and affected individuals have more than 39 copies [15], and the largest expansion observed is 2000 or more copies of the repeats in some Fragile X syndrome and myotonic dystrophy patients [14]. Our results suggest that non-B conformations of the triplet repeats may be responsible for the pausing and that these properties are related to the etiology of some hereditary genetic diseases

Methods

Results

Conclusion