Michael Leffak, PhD

Department:
Biochemistry/Molecular Biology-COSM
Title:
Professor, Biochemistry & Molecular Biology: Vice Chair of Research, Biochemistry & Molecular Biology
Address:
Diggs Laboratory 165, 3640 Colonel Glenn Hwy, Dayton, OH 45435-0001
Curriculum Vitae: 
CV.pdf 172.3 KB
Education History: 

Ph.D.: 1976 City University of New York (Hsueh-jei Li)
Postdoctoral: Princeton University (Harold Weintraub)

Academics

Teaching: 

My primary teaching responsibilites are in the graduate student introductory course in biochemistry and molecular biology (Molecular Biochemistry I, BMB/BMS7500), the advanced courses Molecular Basis of Inherited Disease (BMB/BMS7670) and Molecular Biology of the Nucleus (BMB/BMS7600), and the graduate level class in Research Ethics (BMB/BMS7030).

Research statement: 

   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, and a 2.4 kb segment of this region acts as an autonomously replicating origin element in plasmids in vitro and in vivo. 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.

   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. These experiments have identified regions of the c-myc replicator essential for chromosomal replication origin activity, including the 3? transcription factor binding domain, and the DNA unwinding element (DUE).

   An inducible transcription factor binding site could restore ectopic origin activity to the inactive replicator deleted for the 3? transcription factor binding domain, and we have shown that a heterologous microsatellite DUE (ATTCT)n can restore origin activity to the c-myc inactive replicator deleted for the endogenous DUE. 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. Alteration of the c-myc origin chromatin structure by deletion of a positioned nucleosome or by histone hyperacetylation, respectively, eliminated origin activity or led to a dispersed pattern of MCM binding and replication initiation.

   Our current work continues to use the ectopic c-myc replicator to analyze the effect of disease-related non-Watson-Crick DNA sequences (triplexes, unwound DNA, trinucleotide repeats) on replication and chromosome instability. The goals of this work are to identify the causes and consequences of break induced replication at microsatellite DNAs.

   Microsatellite DNAs are a diverse set of short (1-9 bp) sequences tandemly repeated up to ~40-50 times. These abundant repeats (~3% of the genome) are hotspots for DNA double strand breaks (DSBs). Microsatellite instability is responsible for nonrecurrent hypermutation and complex genome rearrangements (CGRs) in more than forty neurodegenerative diseases and multiple cancers. There is strong evidence of replication-dependent microsatellite DSB recombination at CGR junctions in model organisms and tumors.

   Microsatellites co-localize with hotspots of DSBs, indels, and genome rearrangements. Indeed, the majority of cytogenetic lesions resulting from impediments in replication fork progression occur at recurrent non-random sites. The formation non-B DNA secondary structures (including hairpins, triplexes (H-DNA), guanine quadruplexes (G4), AT-rich “abnormal” DNA structures) by microsatellites causes replication-dependent genome instability.

   Replication induced DSBs have been attributed to (CNG)n, (GAA)n, and AT-rich repeats at chromosomal fragile sites (CFS). However, we still do not know the mechanisms by which microsatellite structures cause chromosome breaks.

   To measure replication-dependent DSBs at microsatellite repeats, we devised the dual fluorescence (DF) set of constructs in which the c-myc replication origin is adjacent to a microsatellite repeat and flanked by fluorescent reporter genes (dTomato, eGFP). All DF constructs were integrated individually at the same genomic target site (Flp recombinase target site, FRT). DNA double strand breaks in DF cells can be detected by flow cytometry due to the loss of one or both fluorescent reporter genes.

   Using clonal cell lines derived from chromosomal integration of the DF constructs into the human genome, we have shown that non-Watson-Crick structures represent blocks to replication fork progress and lead to DNA double strand breaks and break-induced replication.

   Invasion of the sister chromatid by the broken DNA strands leads to highly mutagenic copying of the homologous sister template. Alternatively, invasion of nonhomologous chromosomal targets results in chromosomal translocations. Both the clustered mutations and the chromosomal translocations caused by break-induced replication are characteristic of the genome instability seen in human tumors.

   By the manipulation of various microsatellite DNA sequences and the proteins involved in replication fork stabilization and repair, we can identify the causes and consequences of genome instability that arise from the replication of non-Watson-Crick DNA structures.

Professional

Publications: 

Publications (peer-reviewed; excluding abstracts):

  1. Leffak, I.M., Hwan, J.C., Li, H-j., and Shih, T.Y. (1974) Circular Dichroism and Thermal Denaturation Studies of Nucleohistone IIb2. Biochemistry 13, 1116-1120.
  2. Hwan, J.C., Leffak, I.M., Li, H-j., Huang, P.C., and Mura, C. (1975) Studies on the Interaction between Histone V (f2C) and Deoxyribonucleic Acids. Biochemistry 14, 1390-1395.
  3. Leffak, I.M., and Li, H-j. (1977) Thermal Denaturation and Circular Dichroism Studies of Histone-DNA Complexes. Biochemistry 16, 5869-5878.
  4. Hwan, J.C., Leffak, I.M., Li, H-j., Huang, P.C., and Mura, C. (1975) Studies on the Interaction between Histone V (f2C) and Deoxyribonucleic Acids. Biochemistry 14, 1390-1395.
  5. Leffak, I.M., and Li, H-j. (1977) Thermal Denaturation and Circular Dichroism Studies of Histone-DNA Complexes. Biochemistry 16, 5869-5878.
  6. Leffak, I.M., Grainger, R.M., and Weintraub, H. (1977) Conservative Assembly of Nucleosomal Histones. Cell 12, 837-845.
  7. Weintraub, H., Flint, S.J., Leffak, I.M., Groudine, M., and Grainger, R.M. (1978) The Generation and Propagation of Variegated Chromosome Structures. Cold Spring Harbor Symp. Quant. Biol. 42, 401-408.
  8. Leffak, I.M., and Li, H-j. (1981) Sequence Sensitivity of Histone Binding. Biochim. Biophys. Acta 656, 86-92.
  9. Trempe, J.P., and Leffak, I.M. (1982) Assembly of Semihistone A24. Nucleic Acids Res. 10, 5467-5481.
  10. Leffak, I.M. (1983) Stability of the Conservative Mode of Nucleosome Assembly. Nucleic Acids Res.11, 2717-2732.
  11. Leffak, I.M. (1983) Chromatin Assembled in the Presence of Cytosine Arabinoside has a Short Nucleosome Repeat Length. Nucleic Acids Res. 11, 5451-5466.
  12. Leffak, I.M. (1983) Decreased Protein Staining After Chemical Crosslinking. Anal. Biochem. 135, 95-101.
  13. Leffak, I.M. (1984) Conservative Segregation of Nucleosome Core Histones. Nature 307, 82-85.
  14. James, C.D., and Leffak, I.M. (1984) Replacement Synthesis Labeling of Recombinant DNA Molecules Using the E. Coli Exonuclease III/DNA Polymerase Enzyme Pair. Anal. Biochem. 141, 33-37.
  15. Trempe, J.P., and Leffak, I.M. (1985) Histone H1 and HMG 14/17 are Deposited Nonrandomly in the Nucleus. Nucleic Acids Res. 13, 4853-4869.
  16. James, C.D., and Leffak, I.M. (1986) Replication Polarity through the Avian Alpha-Globin Locus. Mol. Cell. Biol. 6, 976-984.
  17. Kumar, S., and Leffak, I.M. (1986) Assembly of Active Chromatin. Biochemistry 25,2055-2060.
  18. Fink, P.C., Leffak, I.M., and Prochaska, L.J. (1987) Homology between the Gene Encoding Subunit III of Bovine Cytochrome C Oxidase and Bacterial Genomes. FEBS Lett. 214, 75-80.
  19. Leffak, M. (1988) Nonrandom Assembly of Chromatin During Hydroxyurea Inhibition of DNA Synthesis. Biochemistry 27, 686-691.
  20. Trempe, J.P., Lindstrom, Y.I., and Leffak, M. (1988) Opposite Replication Polarities of Transcribed and Nontranscribed Histone H5 Genes. Mol. Cell. Biol. 8, 1657-1663.
  21. McWhinney, C.D., and Leffak, M. (1988) Episomal Persistence of a Plasmid Containing Human c-myc DNA. Cancer Cells 6, 467-472.
  22. Leffak, M., and James, C.D. (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, 586-593.
  23. Kumar, S., and Leffak, M. (1989) DNA Topology of the Ordered Chromatin Domain 5' to the Human c-myc Gene. Nucleic Acids Res. 17, 2819-2833.
  24. McWhinney, C., and Leffak, M. (1990) Autonomous Replication of a DNA Fragment Containing the Chromosomal Replication Origin of the Human c-myc Gene. Nucleic Acids Res. 18, 1233-1242.
  25. Kumar, S., and Leffak, M. (1991) Conserved Chromatin Structure in c-myc 5' Flanking DNA After Viral Transduction. J. Mol. Biol. 222, 45-57.
  26. Fink, P., Zhao, Y., Leffak, M. and Prochaska, L. (1991) Nucleotide Sequence of a 23S and a 5S-like rRNA Gene from the Thermophilic Bacillus Species Strain PS3. Nucleic Acids Res. 19, 6334.
  27. Fink, P., Zhao, Y., Prochaska, L. and Leffak, M. (1991) Sequence of a tRNA Gene Cluster from the Thermophilic bacillus species strain PS3. Nucleic Acids Res. 19, 5437.
  28. Berberich, S., and Leffak, M. (1993) DNase-Sensitive Chromatin Structure Near a Chromosomal Origin of Bidirectional Replication of the Avian Alpha Globin Locus. DNA and Cell Biol. 12, 703-714.
  29. Itoh-Lindstrom, Y., and Leffak, M. Alteration of in vitro DNA synthesis in the alpha globin locus of chick embryo fibroblasts due to in vivo activity of Rous sarcoma virus pp60src. (1994) Nucleic Acids Res. 22, 498-505.
  30. Messing, S.L., Darrow, R., Leffak, M., Fleischman, D., and Organisciak, D. (1994) Visible light induced damage to retinal DNA in vivo. Invest. Opthalmol. Vis. Sci. 35, 2138.
  31. Berberich, S., Trivedi, A, Daniel, D., Johnson, E. and Leffak, M. (1995) In vitro replication of plasmids containing human c-myc DNA. J. Mol. Biol. 245, 92-109.
  32. McWhinney, C., Waltz, S.E. and Leffak, M. (1995) Cis-Acting Sequence Effects on Autonomous Replication of Plasmids Containing 5' Flanking DNA of the Human c-myc Gene. DNA and Cell Biol. 14, 565-579.
  33. Organisciak, D., Kutty, R., Leffak, M., Wong, P., Messing, S., Wiggert, B., Darrow, R., and Chader, G. (1995) Oxidative Damage and Responses in Retinal Nuclei Arising from Intense Light Exposure. In: Degenerative Diseases of the Retina. R. Anderson, ed. New York: Plenum Press; 9-17.
  34. Waltz, S.E., Trivedi, A. and Leffak, M. (1996) DNA Replication Initiates Nonrandomly at Multiple Sites Near the c-myc Gene in HeLa Cells. Nucleic Acids Res. 24, 1887-1894.
  35. Khaira, P., James, C.D., and Leffak, M. (1998) Amplification of the Human c-myc Gene in Three Burkitt Lymphoma Cell Lines. Gene 211, 101-108.
  36. Trivedi, A., Waltz, S., Kamath, S. and Leffak, M. (1998) Multiple Initiations in the c-myc Replication Origin Independent of Chromosomal Location. DNA and Cell Biology 17, 885-896.
  37. Jones, D., and Leffak, M. (1999) A Bifunctional Regulatory Element of the Human ApoA-I Gene Responsive to a Distal Enhancer. DNA and Cell Biology 18, 107-119.
  38. Specht, S., Leffak, M., Darrow, R.M. and Organisciak, D.T. (1999) Visible Light Induced Damage to Rat Retinal DNA. Photochem. and Photobiol. 69, 91-98.
  39. Malott, M. and Leffak, M. (1999) Activity of the c-myc Replicator at an Ectopic Chromosomal Location. Mol.Cell. Biol. 19, 5685-5695.
  40. Rein, T., Kobayashi, T., Malott, M., Leffak, M., and DePamphilis, M.L. (1999) DNA Methylation at Mammalian Replication Origins. J. Biol. Chem. 274, 25792-25800.
  41. Tao, L., Dong, Z., Leffak, M., Zannis-Hadjopoulos, M., Price, G. (2000) Major DNA replication initiation sites in the c-myc locus in human cells. J. Cell. Biochem. 78, 442-457.
  42. Specht, S., Darrow, R., Organisciak, D., and Leffak, M. (2000) DNA Damage to Photoreceptor Cells Following Intense Light Exposure. Photochem. & Photobiol. 71, 559-66.
  43. Kamath, S., and Leffak, M. (2001) Multiple Sites of Replication Initiation in the Human ß-Globin Gene Locus. Nucleic Acids Res. 29, 809-817.
  44. Dhar, S., Yoshida, K., Machida, Y., Khaira, P., Chaudhuri, B., Wohlschlegel, J., Leffak, M., Yates, J., and Dutta, A. (2001) Replication from oriP of Epstein-Barr Virus Requires Human ORC and Is Inhibited by Geminin. Cell 106, 287-296.
  45. Liu, G., Malott, M., and Leffak, M. (2003) Multiple Functional Elements Comprise a Mammalian Chromosomal Replicator. Mol. Cell. Biol. 23, 1832-1842.
  46. Potaman, V.N., Bissler, J.,J., Hashem, V.I., Oussatcheva, E.A., Lu, L., Shlyakhtenko, L.S., Lyubchenko, Y.L., Matsuura, T., Ashizawa, T., Leffak, M., Benham, C.J., and Sinden, R.R. (2003) Unwound structures in SCA10 (ATTCT)n•(AGAAT)n repeats. J. Mol. Biol. 326, 1095-1111.
  47. Ghosh, M., Liu, G., Randall, G., Bevington, J. and Leffak, M. (2004) Transcription Factor Binding and Induced Transcription Alter Chromosomal c‑myc Replicator Activity (2004) Mol. Cell. Biol. 24, 10193-10207.
  48. Kemp, M.G., Ghosh, M., Liu, G., and Leffak, M. The Histone Deacetylase Inhibitor Trichostatin A Alters the Pattern of DNA Replication Origin Activity in Human Cells (2005) Nucleic Acids Res. 33; 325-336.
  49. Casper, J., Kemp, M., Ghosh, M., Randall, G., Vaillant, A., and Leffak, M. (2005) The c-myc DNA Unwinding Element Binding Protein Modulates the Assembly of DNA Replication Complexes In Vitro. J. Biol. Chem. 280; 13071-13083.
  50. Moaddel, R., Price, G.B., Juteau, J., Leffak, M., and Wainer, I.W. (2005) The synthesis and initial characterization of an immobilized DNA unwinding element binding protein chromatographic stationary phase. J. Chromatogr. 820:197-203.
  51. Potaman, V.N.  Pytlos, M.J., Hashem, V.I., Bissler, J.J., Leffak, M., and Sinden, R.R.  (2006) DNA Structures and Genetic Instabilities Associated with SCA10 (ATTCT)n•(AGAAT)n Repeats Suggests a DNA Amplification Model for Repeat Expansion, ppg. 460-477. In Genetic Instabilities and Neurological Diseases  (Eds. R.D. Wells and T. Ashizawa), Academic Press, Boston, MA.
  52. Ghosh, M., Kemp, M., Liu, G., Ritzi, M., Schepers, A., Leffak, M. (2006) Differential binding of replication proteins across the human c‑myc replicator. Mol. Cell. Biol. 26, 5270-5283.
  53. 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. (2007) J. Biol. Chem. 282: 10441-10448.
  54. 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.
  55. Chowdhury, A., Liu, G., Kemp, M., Chen, X., Katrangi, N., Myers, S., Yao, J., Bubulya, P., and Leffak, M. (2010) The DNA Unwinding Element Binding Protein DUE-B Interacts with Cdc45 in Preinitiation Complex Formation. Mol. Cell. Biol. 30, 1495–1507.
  56. Liu, G., Chen, X., Bissler, J., Sinden, R., and Leffak, M. (2010) Replication dependent DNA hairpin formation at CTG/CAG repeats in human cells. Nature Chem. Biol. 6, 652-659.
  57. Xu, Y., and Leffak, M. (2010) ATRIP from TopBP1 to ATR - in vitro activation of a DNA damage checkpoint. Proc Natl Acad Sci U S A 107, 13561-2.
  58. Liu, G., Leffak, M. (2012) Instability of (CTG)n*(CAG)n trinucleotide repeats and DNA synthesis. Cell Biosci 2:7-19.
  59. Liu, G., Chen, X., Gao, Y., Lewis, T., Barthelemy, J., and Leffak, M. (2012) Altered replication promotes instability of DMPK (CTG)n·(CAG)n repeats. Mol Cell Biol 32, 1618-32.
  60. Wang, Z., Kim, E. Leffak, M. Xu, Y.J. (2012) Treslin, DUE-B, and GEMC1 cannot complement Sld3 mutants in fission yeast. FEMS Yeast Research 12, 486-90.
  61. Liu, G., Myers, S., Chen, X., Bissler, J., Sinden, R., Leffak, M. (2012) Replication Fork Stalling and Checkpoint Activation by a PKD1 Locus Mirror Repeat Polypurine-Polypyrimidine (Pu-Py) Tract. J Biol Chem 287, 33412-33423.
  62. Liu, G., Chen, X., and Leffak, M. (2013) Oligodeoxynucleotide Binding to (CTG)×(CAG) Microsatellite Repeats Inhibits Replication Fork Stalling, Hairpin Formation, and Genome Instability. Mol Cell Biol 33, 571-581.
  63. Chen, X., Liu, G., and Leffak, M. (2013). Activation of a human chromosomal replication origin by protein tethering. Nucleic Acids Res 41, 6460-6474.
  64. Leffak, M. (2013) Hypothesis: Local dNTP depletion as the cause of microsatellite repeat instability during replication. Bioessays 35, 305.
  65. Gao, Y., Yao, J., Poudel, S., Romer, E., Abu-Niaaj, L., and Leffak, M. (2014) Protein phosphatase 2A and Cdc7 kinase regulate the DNA unwinding element-binding protein in replication initiation. J Biol Chem 289, 35987-36000.
  66. Virts, E., Jankowska, A., Mackay, C., Glaas, M., Wiek, C., Kelich, S., Lottmann, N., Kennedy, F., Marchal, C., Lehnert, E., Scharf, R., Dufour, C., Lanciotti, M., Farruggia, P., Santoro, A., Savasan, S., Scheckenbach, K., Schipper, J., Wagenmann, M., Lewis, T., Leffak, M., Hanenberg, H., et al. (2015). AluY-mediated germline deletion, duplication and somatic stem cell reversion in UBE2T defines a new subtype of Fanconi anemia. Hum Mol Genet 24:5093-108.
  67. Barthelemy, J., Hanenberg, H. and Leffak, M. (2016) FANCJ is essential to maintain microsatellite structure genome-wide during replication stress. Nucleic Acids Res 44:6803-6816.
  68. Guo J, Gu L, Leffak M, Li GM. (2016) MutS beta promotes trinucleotide repeat expansion by recruiting DNA polymerase beta to nascent (CAG)n or (CTG)n hairpins for error-prone DNA synthesis. Cell Res 26:775-786.
  69. Gadgil R, Barthelemy J, Lewis T, Leffak M. (2016). Replication stalling and DNA microsatellite instability. Biophys Chem 10.1016/j.bpc.2016.11.007.
  70. Leffak, M. (2017) Break-induced replication links microsatellite expansion to complex genome rearrangements. BioEssays 10.1002/bies.201700025.
  71. Poudel, S., Yao, J., Kemp, M.G. and Leffak, M. (2018) Interaction between DUE-B and Treslin is required to load Cdc45 on chromatin in human cells. J Biol Chem, 293, 14497-14506.
  72. Lewis, T., Barthelemy, J., Virts, E., Kennedy, F. M., Wiek, C., Linka, R. M., Gadgil, R., Hanenberg, H., and Leffak, M. (2018) DNA double strand breaks trigger Alu-mediated reversion of a FANCT duplication in a model of homologous recombination. Nucl. Acids Res. 47, 3503-3520.
  73. Gadgil, R. Y., Rider, S. D., Jr., Lewis, T., Barthelemy, J., and Leffak, M. (2020) Analysis of Trinucleotide Repeat Stability by Integration at a Chromosomal Ectopic Site. Methods Mol Biol 2056, 121-136.
  74. Gadgil, R. Y., Romer, E. J., Goodman, C. C., Rider, S. D., Jr., Damewood, F. J. t., Barthelemy, J. R., Shin-Ya, K., Hanenberg, H., and Leffak, M. (2020) Replication stress at microsatellites causes DNA double strand breaks and break induced replication. J Biol Chem. (2020) doi: 10.1074/jbc.RA120.013495.
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