טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBen-Yosef Ron
SubjectCharacterization of the Function of the Protein Translin
in the Yeast Schizosaccaromyces pombe
DepartmentDepartment of Biology
Supervisor Professor Haim Manor
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

Translin is a human protein that specifically binds G-rich single stranded DNA and RNA sequences. TRAX is a Translin paralogue which binds Translin, but does not bind nucleic acids. Both proteins are highly conserved in eukaryotic evolution including the fission yeast S. pombe. We reasoned that S. pombe would be suitable for genetic and biochemical analysis of the functions of the Translin and TRAX which have not yet been defined. Therefore we initiated such studies. We have already found that: (1) Like the human Translin, the nucleic acid binding form of the S. pombe Translin (spTranslin) is a homooctamer. (2) The spTranslin binds RNA with much higher affinity than single stranded DNA. (3) spTranslin specifically binds the S. pombe TRAX (spTRAX).

In my research, I deleted the spTranslin and spTRAX genes from the yeast cells and found that the genes are not essential for cells proliferation. I also found that the deletion did not increase the sensitivity of the cells to Methyl methanesulfonate and Hydroxyurea. These data indicated that spTranslin and spTRAX are not directly involved in DNA repair or replication. Southern blot analyses of telomeric repeats showed that spTranslin and/or spTRAX deletion did not affect telomere length, as previously suggested based on the ability of Translin to bind telomeric repeats in vitro. On the other hand, genetic assays did provide evidence for the involvement of spTranslin in the maintenance of d(GT)n·d(AC)n repeats, as suggested by its relatively high affinity to such repeats.

Another part of my research was aimed at identifying proteins that form specific complexes with spTranslin and/or spTRAX; for I expected that such identification may provide clues as to the functions of Translin and TRAX. I generated S. pombe strains carrying TAP tagged spTranslin and purified native complexes containing the tagged spTranslin from these cells by tandem affinity chromatography. Mass spectrometry revealed the presence of 27 ribosomal proteins from both ribosomal subunits in these complexes. Therefore, it appeared likely that spTranslin is specifically associated with ribosomes. Subsequently, I resolved ribosomes and polysomes from soluble proteins by sedimentation in sucrose gradients. I found that spTranslin is primarily associated with ribosomal subunits.

In summary, my data indicate that Translin may be involved in maintaining the integrity of d(GT)n·d(AC)n repeats in the S. pombe genome. The association with ribosomes suggests that Translin and TRAX may also play a role in regulation of protein translation in S. pombe.