Research Interests

Anticodon nuclease – Inhibition of protein synthesis underlies various regulatory, cytotoxic and antiviral phenomena. Such an effect can be achieved by removal of just one tRNA species, as does the tRNALys-specific anticodon nuclease (ACNase). ACNase is normally kept inactive in the uninfected bacterial cell, associated with the DNA restriction-modification enzyme EcoprrI (Fig. 1). The phage T4-coded peptide Stp, whose intended function is to inhibit the DNA restriction activity of EcoprrI, activates the latent ACNase. The consequent disruption of tRNALys can block protein synthesis and thus prevent the spread of the phage. However, anti-apoptotic RNA repair enzymes encoded by the phage, polynucleotide kinase (Pnk) and RNA ligase (Rli), normally offset the damage and the virus prevails. Currently we investigate structural and functional attributes of the isolated ACNase protein trying to understand how ACNase is shielded and activated and how it recognizes and cleaves a specific tRNA species. We also explore the possible application of ACNase as a means to disrupt tRNALys molecules utilized by HIV to prime the synthesis of the proviral DNA.

Fig. 1. Cleavage and ligation of tRNALys by activated ACNase in T4-infected E. coli. PrrC, the ACNase core protein is complexed with the DNA restriction modification enzyme EcoprrI that stabilizes PrrC and masks its activity. Stp, the T4 encoded peptide inhibitor of EcoprrI activates ACNase but the phage-encoded RNA repair enzymes (Pnk & Rli) offset the damage.

Eukaryal DNA replication - Existing cellular genomes replicate according to similar rules: parental duplex DNA unwinds from defined chromosomal sites and complementary DNA is synthesized on the exposed template strands in a semi-discontinuous fashion. Yet, eukaryal and bacterial organisms mediate these reactions using evolutionarily unrelated sets of proteins. Despite the marked differences in their structures and mode of action it has been long believed that DNA unwinding and complementary DNA synthesis proceed essentially in the same way in all domains of life.
The identification and characterization of an early intermediate in eukaryal DNA synthesis termed RNA-DNA primer led us to propose that the synthesis of the eukaryal discontinuous DNA strand differs from the bacterial paradigm in a fundamental way. Specifically, eukaryal Okazaki fragments are formed by repair like processing of a contiguous array containing several RDP units (Nested Discontinuity Model, on the right in Fig. 2).

In the current version of the Nested Discontinuity Model we also propose that eukaryal parental DNA is unwound in small RDP-size steps and that the short DNA template portion exposed is rendered double stranded before unwinding resumes. One consequence of this mechanism is greater symmetry of the eukaryal replication fork compared to the bacterial in that the two daughter arms are more or less equal in double-stranded DNA content. Interestingly, phylogenetic data suggest that the specific attributes of the eukaryal machinery could date back to an archaeal ancestor containing a rudimentary form of chromatin structure. Since nucleosomes form only on double stranded DNA, greater symmetry of the ancestral eukaryal/archaeal replication fork could facilitate the emergence of chromatin structure. Current studies in our laboratory address specific predictions of this model by attempts to identify specific interactions between defined DNA chain intermediates and proteins engaged in their synthesis and maturation.

Fig 2. Two models of eukaryal lagging DNA strand synthesis. In the model shown on the left, an RNA-DNA primer synthesized by DNA polymerase alpha-primase is continuously extended by the elongating DNA polymerase delta up to the full length of the Okazaki fragment. In the Nested Discontinuity Model on the right, a contiguous array of RNA-DNA primers is converted into an Okazaki fragment in a repair like manner that entails the removal of intervening RNA moieties, gap filling and ligation of the processed DNA segments.

Selected Publications

1. Amitsur, M., Benjamin, S., Rosner, R., Chapman-Shimshoni, D., Meidler, R., Blanga, S. and Kaufmann, G. (2003). Bacteriophage T4-encoded Stp can be replaced as activator of anticodon nuclease by a normal host cell metabolite. Mol. Microbiol. 50, 129-143. e-format
2. Jiang,Y., Blanga,S., Amitsur,M., Meidler,R., Krivosheyev,E., Sundaram,M., Bajji,A.C., Davis,D.R., and Kaufmann,G. (2002). Structural features of tRNALys favored by anticodon nuclease as inferred from reactivities of anticodon stem and loop substrate analogs. J. Biol. Chem. 277, 3836-3841. e-format
3. Jiang,Y., Meidler,R., Amitsur,M., and Kaufmann,G. (2001). Specific Interaction between Anticodon Nuclease and the tRNA(Lys) Wobble Base. J. Mol. Biol. 305, 377-388. e-format
4. Kaufmann,G. (2000). Anticodon nucleases. Trends Biochem. Sci. 25, 70-74. e-format
5. Meidler,R., Morad,I., Amitsur,M., Inokuchi,H., and Kaufmann,G. (1999). Detection of Anticodon Nuclease Residues Involved in tRNALys Cleavage Specificity. J. Mol. Biol. 287, 499-510. e-format
6. Penner,M., Morad,I., Snyder,L., and Kaufmann,G. (1995). Phage T4-coded Stp: double-edged effector of coupled DNA and tRNA-restriction systems. J. Mol. Biol. 249, 857-868. e-format

DNA REPLICATION

1. Kaufmann,G. and Nethanel, N. Did an early version of the eukaryal replisome enable the emergence of chromatin? Progress in Nucleic Acids Res. Mol.Biol. 77, in press.
2. Mass,G., Nethanel,T., and Kaufmann,G. (1998). The middle subunit of replication protein A contacts RNA-DNA primers within replicating SV40 chromosomes. Mol. Cell. Biol. 18, 6399-6410. e-format
3. Mass,G., Nethanel,T., Lavrik,O.I., Wold,M.S., and Kaufmann,G. (2001). Replication protein A modulates its interface with the primed DNA template during RNA-DNA primer elongation in replicating SV40 chromosomes. Nucleic Acids Res. 29, 3892-3899. e-format
4. Zlotkin,T., Kaufmann,G., Jiang,Y., Lee,M.Y.W.T., Uitto,L., Syvaoja,J., Dornreiter,I., Fanning,E., and Nethanel,T. (1996). DNA polymerase epsilon may be dispensable for SV40- but not cellular-DNA replication. EMBO Journal 15, 2298-2305. e-format
   

Courses

Graduate Courses organized and taught by Gabriel Kaufmann
The Graduate Program in Molecular Biology consists of courses taught by me in collaboration with guest speakers and the participating students who discuss current research problems presented at the beginning of the course. The specific subjects may change from year to year. Two subjects taught in recent years are listed below.

1. DNA transactions. – The maintenance and propagation of DNA genomes requires precise coordination of many processes including DNA replication, DNA repair, DNA recombination as well as DNA damage responsive signaling pathways. The course focuses on recent advances in the understanding of these processes and the implication of the knowledge gained to the study of cancer and other human disorders. Speakers in recent years covered DNA replication (Gabriel Kaufmann), DNA repair and damage tolerance (Zvi Livneh, Weizmann Institute), DNA damage responsive checkpoints and a related human disorder (Yossi Shiloh, Tel Aviv Medical School), Eukaryotic replication origins (Rolf Knippers, University of Konstanz), Werner Syndrome helicase and the Fragile X chromosome syndrome Michael Fry, Technion Medical School, Ectopic recombination and its avoidance (Martin Kupiec, Tel Aviv University), Telomerase (Anat Krauskopf, Tel Aviv University; Haim Manor, Technion), Homologous recombination (Amikam Cohen, Hebrew University Medical School).
2. Catalytic RNA – The discovery of catalytic RNA molecules rekindled interest in structural-functional and evolutionary aspects of RNA. In the last two decades we witnessed the elucidation of the structure and catalytic mechanisms of several natural ribozymes culminating with the spectacular elucidation of the ribosome crystal structure and demonstration that the peptide bond forming center in it is composed entirely of RNA. Other, artificial ribozymes, selected in test-tube evolution experiments increased the repertoire of known RNA catalyzed reactions and lend further credence to the hypothesis according that early forms of life were based on RNA genomes and catalytic RNA phenotype. The course is taught by the organizer (Gabriel Kaufmann, Tel Aviv University), Prof. Gadi Schuster (Haifa Technion), Dr. Gil Ast (Tel Aviv University Medical School), Prof. Shula Michaeli (Bar-Ilan University), Dr. Nayef Jarrous (Hebrew University Medical School) and Prof. Ada Yonath (Weizmann Institute-Max Planck Institute, Hamburg).