Michael Leffak, Ph.D.
Brage Golding Distinguished Professor of Research
Lab: 165 Diggs Lab
Office: 150 Diggs Lab
Ph.D.: 1976 City University of New York (Hsueh-jei Li)
Postdoctoral: Princeton University (Harold Weintraub)
The c-myc replicator
Our laboratory is involved in the study of proteins and DNA sequences that control DNA replication in eucaryotic cells, and the relationship of replication to human disease. The primary model system we use is the human c-myc origin of replication. Replication begins within 3-5 kb upstream of the c-myc gene (4, 6, 7, 11, 14-16), and a 2.4 kb segment of this region acts as an autonomously replicating origin element in plasmids in vitro (1) and in vivo (12, 13, 15). The same 2.4 kb segment of the c-myc origin acts to induce replication in flanking chromosomal DNA when transposed to an ectopic chromosomal location (4, 9, 10).
We have carried out real-time PCR quantitation of the replication activity of a series of c-myc replicator constructs integrated by FLP recombinase at a unique HeLa chromosomal location (9, 10). These experiments have identified regions of the c-myc replicator essential for chromosomal replication origin activity (9), including the 3′ transcription factor binding domain, and the DNA unwinding element (DUE) (Figure 1).
An inducible transcription factor binding site could restore ectopic origin activity to the inactive replicator deleted for the 3′ transcription factor binding domain, (4), and we have recently shown that a heterologous DUE can restore origin activity to the c-myc inactive replicator deleted for the endogenous DUE (8). Using chromatin immunoprecipitation we have analyzed the cell cycle-dependent binding of the origin recognition complex (ORC), Cdc6 and the MCM helicase complex to the c-myc origin, and found that ORC and Cdc6 bind predominantly at sites flanking the DUE while the MCM helicase is more closely associated with the DNA unwinding element (Figure 1) (3). Alteration of the c-myc origin chromatin structure by deletion of a positioned nucleosome or by histone hyperacetylation respectively, eliminated origin activity (3) or led to a dispersed pattern of MCM binding and replication initiation (6). Our current work continues to use the ectopic c-myc replicator to analyze the effect of disease-related non-canonical DNA sequences (triplexes, unwound DNA, trinucleotide repeats) on replication and chromosome instability.
Figure 2: DUE-B (His tagged) co-localizes with the hSM splicing factor in nuclear speckles. With appreciation to Nadia Katrangi and Dr. Paula Bubulya for immunohistochemistry. [Click on the image to view a larger version.]
Stemming from the demonstration that a DUE is an essential structure for c-myc replicator activity, and that AT-richness is not sufficient for DUE activity, we used the c-myc DUE in a yeast one hybrid screen to isolate the c-myc DNA unwinding element binding protein, DUE-B (2), with a predicted mass of 23.4 kDa. Based on homology to yeast proteins DUE-B was previously classified as a histidyl tRNA synthetase, however the human protein is approximately 60 amino acids longer than its orthologs in yeast or worms, and is primarily nuclear. Within the nucleus, two populations of DUE-B are evident. A fraction of DUE-B is chromatin-bound in G1 phase cells, and localizes to replication origin loci, including c-myc and lamin B2. DUE-B is also bound to a copy of the c-myc replication origin integrated at an ectopic chromosomal site. Strikingly, deletion of the c-myc DUE from the ectopic c-myc origin eliminated DUE-B binding in vivo, while replacement of the DUE with a functionally equivalent but heterologus DUE sequence restored DUE-B binding.
In asynchronously growing cells, a second population of DUE-B molecules localizes to nuclear speckles, which are depot sites of RNA processing enzymes (Figure 2).
DUE-B levels are constant over the cell cycle although the protein is preferentially phosphorylated in cells arrested early in S phase. Inhibition of DUE-B protein expression slowed HeLa cell progression from G1 to S phase, and induced cell death. In Xenopus egg extracts baculovirus expressed DUE-B inhibits the initiation of chromatin replication at the RPA loading step, even in the presence of endogenous DUE-B, suggesting that differential covalent modification of these proteins can alter their effect on replication. Immunodepletion of DUE-B from Xenopus egg extracts eliminated DNA replication while recombinant DUE-B expressed in HeLa cells restored replication activity, suggesting that DUE-B plays an important role in replication in vivo.
Figure 3: (A) DUE-B monomer, (B) DUE-B dimer. [Click on the image to view a larger version.]
We have also determined the 2.0 Å crystal structure of DUE-B (Figure 3) (5), and have carried out complementary biochemical characterization of its biological activity in vitro. The structure corresponds to a dimer of the N-terminal domain of the full-length protein and contains many of the structural elements of the nucleotide binding Rossman fold. A single magnesium ion resides in the putative active site cavity, which could serve to facilitate the ATP hydrolytic activity of this protein. The structure also demonstrates a notable similarity to those of tRNA editing enzymes, which may relate to the localization of a fraction of DUE-B molecules in nuclear speckles. Consistent with its structural homology to tRNA editing/proofreading enzymes, the N-terminal core of DUE-B displays both D-aminoacyl tRNA deacylase (proofreading) activity and ATPase activity. A single amino acid mutant of DUE-B, directed to the putative active site by the crystal structure, eliminated both tRNA proofreading and ATPase activities. We have further demonstrated that the C-terminal portion of the enzyme is disordered, and not essential for dimerization. However, this region is essential for DNA binding in vitro and becomes ordered in the presence of DNA. Work is continuing to examine the role of DUE-B in DNA replication.
- Berberich, S., A. Trivedi, D. C. Daniel, E. M. Johnson, and M. Leffak. 1995. In vitro replication of plasmids containing human c-myc DNA. J Mol Biol 245: 92-109. [Abstract]
- Casper, J. M., M. G. Kemp, M. Ghosh, G. M. Randall, A. Vaillant, and M. Leffak. 2005. The c-myc DNA-unwinding element-binding protein modulates the assembly of DNA replication complexes in vitro. J Biol Chem 280:13071-83. [Abstract]
- Ghosh, M., M. Kemp, G. Liu, M. Ritzi, A. Schepers, and M. Leffak. 2006. Differential Binding of Replication Proteins across the Human c-myc Replicator. Mol Cell Biol 26:5270-83. [Abstract]
- Ghosh, M., G. Liu, G. Randall, J. Bevington, and M. Leffak. 2004. Transcription factor binding and induced transcription alter chromosomal c-myc replicator activity. Mol Cell Biol 24:10193-207. [Abstract]
- Kemp, M., B. Bae, J. P. Yu, M. Ghosh, M. Leffak, and S. Nair. Structure and function of the c-myc DNA unwinding element binding protein DUE-B. [Abstract]
- Kemp, M., Bae, B., Yu, J.P., Ghosh, M., Leffak, M. and Nair, S. 2007. Structure and Function of the c-myc DNA-unwinding Element-binding Protein DUE-B. J. Biol. Chem. 282: 10441-10448. [Abstract]
- Leffak, M., and C. D. James. 1989. Opposite replication polarity of the germ line c-myc gene in HeLa cells compared with that of two Burkitt lymphoma cell lines. Mol Cell Biol 9:586-93. [Abstract]
- Liu, G., J. J. Bissler, R. R. Sinden, and M. Leffak. Liu, G., Bissler, J., Sinden, R. and Leffak, M. (2007) Unstable Spinocerebellar Ataxia Type 10 (ATTCT)•(AGAAT) Repeats Are Associated with Aberrant Replication at the ATX10 Locus and Replication Origin-Dependent Expansion at an Ectopic Site in Human Cells. Mol. Cell. Biol. 27, 7828-7838. [Abstract]
- Liu, G., M. Malott, and M. Leffak. 2003. Multiple functional elements comprise a mammalian chromosomal replicator. Mol Cell Biol 23:1832-42. [Abstract]
- Malott, M., and M. Leffak. 1999. Activity of the c-myc replicator at an ectopic chromosomal location. Mol Cell Biol 19:5685-95. [Abstract]
- McWhinney, C., and M. Leffak. 1990. Autonomous replication of a DNA fragment containing the chromosomal replication origin of the human c-myc gene. Nucleic Acids Res 18:1233-1242. [Abstract]
- McWhinney, C., and M. Leffak. 1988. Episomal persistence of a plasmid containing human c-myc DNA, p. 467-471. In B. Stillman and T. Kelly (ed.), Cancer Cells, vol. 6. CSH Laboratory Press, New York.
- McWhinney, C., S. E. Waltz, and M. Leffak. 1995. Cis-acting effects of sequences within 2.4-kb upstream of the human c-myc gene on autonomous plasmid replication in HeLa cells. DNA Cell Biol 14:565-79. [Abstract]
- Tao, L., Z. Dong, M. Leffak, M. Zannis-Hadjopoulos, and G. Price. 2000. Major DNA replication initiation sites in the c-myc locus in human cells. J Cell Biochem 78:442-57. [Abstract]
- Trivedi, A., S. E. Waltz, S. Kamath, and M. Leffak. 1998. Multiple initiations in the c-myc replication origin independent of chromosomal location. DNA Cell Biol 17:885-96. [Abstract]
- Waltz, S. E., A. A. Trivedi, and M. Leffak. 1996. DNA replication initiates non-randomly at multiple sites near the c-myc gene in HeLa cells. Nucleic Acids Res 24:1887-94. [Abstract]