טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentOren-Jazan Hely
SubjectTetraplex DNA Destabilizing Proteins from Human cells
DepartmentDepartment of Medicine
Supervisor Professor Michael Fry


Abstract

Expansion of a d(CGG)n trinucleotide repeat sequence in the 5'-UTR of the FMR1 gene silences FMR1 causing Fragile X syndrome. The d(CGG)n run forms hairpin and tetrahelical structures that engender slippage of DNA polymerase, d(CGG) expansion and blocking of the transcription and replication of the FMR1 gene. Resolution by proteins of these secondary structures might, therefore, promote FMR1 replication and transcription.

Ku protein (Ku) binds and stabilizes a bimolecular tetraplex of d(CGG)n whereas WRN protein, that possesses both helicase and exonuclease activities, destabilizes bimolecular tetraplex forms of d(CGG)n and physically interacts with Ku.

In the first part of this thesis we studied the effect of the binding of Ku to alternate DNA substrates on their unwinding or degradation by WRN helicase and exonuclease, respectively. We showed that Ku diminished the activities of both WRN helicase and exonuclease. Ku inhibited unwinding by WRN helicase of partial DNA duplex and G'2 3'-tail d(CGG)7 and slowed the degradation of G'2 3'-tail d(CGG)7 by the exonuclease through its direct association with the WRN protein. By contrast, the degradation by WRN exonuclease of partial DNA duplex and X-junction DNA was inhibited through the binding of Ku to these DNA substrates.

In the second part of the work we describe the purification and characterization of a new protein from human cells, designated human Tetraplex Destabilizing Protein (hTDP), that destabilized G'2 3'-tail d(CGG)7 and stabilized a bimolecular tetraplex of the telomeric DNA sequence d(TTAGGG)n.