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.
Subject(s)
Humans , Base Sequence , DNA, Single-Stranded/metabolism , Endodeoxyribonucleases/genetics , Flap Endonucleases , Gene Expression Regulation , Genetic Diseases, Inborn/genetics , Nucleic Acid Conformation , Trinucleotide Repeat Expansion , Trinucleotide RepeatsABSTRACT
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.
Subject(s)
Humans , Base Sequence , DNA, Single-Stranded/metabolism , Endodeoxyribonucleases/genetics , Flap Endonucleases , Gene Expression Regulation , Genetic Diseases, Inborn/genetics , Nucleic Acid Conformation , Trinucleotide Repeat Expansion , Trinucleotide RepeatsABSTRACT
The mechanism of Agrobacterium mediated genetic transformation of plants is dependent upon certain genetic function of the chromosome of the bacterium as well as on Ti-plasmid borne vir loci and the border sequences of T-DNA. The organisationally variable forms of the naturally occurring border sequences amongst Ti-plasmid types are differentially responsive to gene products of vir loci concerned with T-strand production. Additionally, the production of stable transformants is dependent upon vir gene products effective after T strands are produced. The interaction of border sequences from different strains of Agrobacterium with vir proteins encoded by various helper plasmids revealed that functional differences do exist amongst vir gene products contained in the type of helper plasmids used.