|Ph.D Student||Cohen )Ganelin( Esther|
|Subject||Mechanisms of Tetraplex-DNA Binding and Destablilization by|
the hnRNP-Related Protein CBF-A
|Department||Department of Medicine||Supervisors||PROF. Michael Fry|
|PROF. Pnina Shomer|
Fragile X syndrome, the most common hereditary form of mental retardation is initiated by the folding of a d(CGG)n trinucleotide repeat sequence within the Fragile X Mental Retardation 1 (FMR1) gene into hairpin or tetraplex structures. Formation of such structures prompt polymerase slippage and expansion of the repeat sequence. As a consequence of d(CGG)n expansion, expression of the FMR1 alleles is silenced and their replication is delayed. Stable FMR1 d(CGG)n runs in normal individuals consist of 6-52 d(CGG) repeats that are separated by a single d(AGG) triplet with an average periodicity of 10 repeats. By contrast, a full mutation involves loss of the interspersed d(AGG) triplets and expansion of the monotonous d(CGG) repeats to >200-2,000 copies.
In the first part of this thesis we show that interspersion of d(AGG) within a d(CGG)18 run reduces the extent of formation of G’2 tetraplex structures of the repeat sequence and significantly diminishes their thermal stability.
Cellular proteins that affect the stability of d(CGG)n secondary structures might modulate expansion of the repeat sequence. Rat liver hnRNP-related protein, designated qTBP42 (Quadruplex Telomeric DNA Binding Protein 42) was isolated and characterized in our laboratory. This protein strongly binds and stabilizes tetraplex forms of the telomeric DNA sequence d(TTAGGG)n while it paradoxically destabilizes G'2 tetraplex structures of the fragile X syndrome expanded sequence d(CGG)n. Partial amino acid sequence of qTBP42 is fully homologous to segments of CBF-A, a CArG box binding transcriptional repressor. In this thesis we demonstrate that recombinant CBF-A is physically and immunologically indistinguishable from qTBP42 and that it also conversely destabilizes tetraplex d(CGG)n while stabilizing tetraplex telomeric DNA.
Systematic deletion of five evolutionary-conserved motifs in CBF-A; two pairs of RNP1 and RNP2 boxes and an ATP/GTP binding domain, revealed that either RNP11 or the ATP/GTP binding box are necessary and sufficient for the unwinding of tetraplex d(CGG)n. By contrast, the RNP21 box represses the destabilizing activity. Inhibition by RNP21 is annulled only when both RNP11 and ATP/GTP binding box are present. Neither the RNP boxes nor the ATP/GTP motif are exclusively required for binding tetraplex telomeric DNA.
Selective disruption of secondary structures of the expanded d(CGG)n tract in FMR1 by CBF-A or a derivative thereof might be utilized to reinitiate FMR1 expression in fragile X syndrome cells.