Faculty and Research Interests
Subhasis Biswas, PhD
Science Center 306A
Fax: 856 566-6291
University of Washington, Seattle, WA
PhD , 1981
Mechanism of eukaryotic DNA replication: Much progress has been made in the identification of eukaryotic proteins that are likely involved in the various stages of chromosomal DNA replication. Chromosomal replication origins or ARSs of Saccharomyces cerevisiae are the best characterized in eukaryotes. ORC binding to replication origins has been shown to be required for the activation of the replication origins in Xenopus oocytes and Drosophila embryos. In addition, a number of other G1 phase proteins including Cdc6, Mcm 2-7 proteins, pol a are required for the initiation of DNA replication. Mcm 2-7 protein complex undergoes rearrangement to Mcm467 complex with DNA helicase activity, although its in vivo function as helicase remains unclear. Mcm and ORC proteins are also phosphorylated in the G1 to S transition. Recent studies also established that successful DNA synthesis and fork progression in the S-phase require participation of the DNA polymerases, a , d , and perhaps e . Therefore, in vitro analyses to determine putative roles of these proteins in DNA replication are essential.
Recent major findings from our laboratory are as follows: (i) Replication of supercoiled plasmid in S-phase nuclear extract of S. cerevisiae depends on pol a /primase complex and pol d , as well as Mcm467 helicase, but does not appear to require pol e . Perhaps long DNA templates are required to dissect the processive role of pol e , in the in vitro system, which is consistent with the findings of Fukui et al. (53). (ii) Yeast Topo I appeared to be essential for supporting in vitro plasmid replication. (iii) Recombinant ORC significantly stimulates DNA synthesis in the in vitro system and this stimulation required a supercoiled plasmid with a yeast ARS. (iv) Mcm467 complex is activated by the ARS specific sequences both in terms of DNA-dependent ATPase and DNA helicase activities.
Anthrax DNA helicase and primase inhibitors as novel antibiotics for Biodefense: This project is being carried out in collaboration with Microbiotix Inc., Worcester , MA . The long term goal of this project is to identify specific inhibitors against Bacillus anthracis (Anthrax) DNA helicase and DNA primase and develop them into novel antibiotics for biodefense. Development of new antibiotics will likely be effective against both natural and engineered-resistant forms bacterial pathogens and bioterrorist agents. Two key components of prokaryotic DNA replication pathways are DNA helicase (DnaB) and primase (DnaG), which act early and catalyze rate-limiting steps in DNA replication. The Anthrax DnaB gene has been cloned, expressed and partially purified. The DnaG gene has been cloned, and expressed. Active proteins are being purified and will be used for high throughput screening of of a library of >100,000 discreet small molecule compounds using FRET (fluorescence resonance energy transfer) based DNA helicase and DNA primase assays. The putative positive compounds will be analyzed for specificity of inhibition of DNA replication in bacteria.
1. Biswas-Fiss EE, Affet S, Ha M, Biswas SB. Retinoid Binding Properties of Nucleotide Binding Domain 1 of the Stargardt Disease Associated ABC Transporter, ABCA4.. J Biol Chem. 2012 Nov 9. [Epub ahead of print] PMID: 23144455
2. Shawna Murphy Rotoli, Esther Biswas-Fiss, and Subhasis Biswas (2012) Quantitative Analysis of the Mechanism of DNA Binding by Bacillus DnaA Protein. Biochimie. Vol. 94: In Press , Publication ID: 22974984 .
3. Biswas-Fiss E, Kukaritirat J, Biswas S.: Thermodynamic analysis of DNA binding by a Bacillus single stranded DNA binding protein. (2012) BMC Biochemistry. Vol. 13: pages 1-16; #10, Publication ID: 22698072 .
4. Biswas-Fiss, E. E., Kurpad, D. S., Joshi, K., and Biswas, S. B. Interaction of extracellular domain 2 of the human retina-specific ABC transporter (ABCA4) with all-trans retinal. J. Biol. Chem. published April 19, 2010 as doi:10.1074/jbc.M110.11289
5. Biswas, S.B., Shankar, D., Clark, J. & Biswas, E.E. (2009) Bacterial Replicative DNA Helicases, Chapter 12 in Bacterial DNA, DNA Polymerases and DNA Helicases , Edited by Walter D. Knudsen and Sam S. Bruns, Nova Publishers, Inc. Hauppauge, NY. ISBN 978-1-60741-094-2.
6. Biswas, S.B., Wudra, E., & Biswas, E.E. (2009) Mechanisms of DNA Binding and Regulation of Bacillus anthracis DNA primase, Biochemistry 48, 7373-7382. PMDI: 19583259
7. Aiello, D., Barnes, M. H., Biswas, E. E., Biswas, S. B., Gu, S., Williams, J. D., Bowlin, T. L., and Moir, D. T. (2009) Discovery, Characterization and Comparison of Inhibitors of Bacillus anthracis and Staphylococcus aureus Replicative DNA Helicases. Bioorganic& Medicinal Chemistry, Volume 17, Pages 4466-4476
8. Biswas E. E., Barnes, M., Moir D. T. and Biswas S. B. (2009) An essential DnaB helicase of Bacillus anthracis: identification, characterization, and mechanism of action. J. Bacteriol. 191, 249-260
9. Subhasis B. Biswas and Esther E. Biswas-Fiss (2006). Quantitative Analysis of Binding of Single-Stranded DNA by Escherichia coli DNAB Helicase and the DnaB-DnaC Complex. Biochemistry 45(38), 11505-11513; DOI:10,1021/bi06118d.
10. Biswas, SB, Khopde SM, and Biswas EE. (2005) Control of ATP-Dependent Binding of Saccharomyes cerevisiae Origin Recognition Complex to Autonomously Replicating DNA Sequences. Cell Cycle, 4, 113-120
11. Mitkova, A.V., Biswas-Fiss, E.E. & Biswas, S.B. (2005) Modulation of plasmid DNA replication in Saccharomyces cerevisiae in vitro by DNA polymerases and Mcm467 complex. JBC, 280, 6285-6292.
12. Esther E. Biswas, Sujata Khopde & Subhasis Biswas (2005) Mcm467 Complex of Saccharomyces cerevisiae is Preferentially Activated by the Autonomously Replicating DNA Sequences. Biochemistry, 44, 2916-2025.
13. Biswas, SB., Flowers, S, Biswas-Fiss, E.E. (2004) Quantitative analysis of nucleotide modulation of DNA binding by the DnaC protein of E.scherichia coli. Biochem. J., 379, 553-562.
14. Mitkova AV, Khopde SM, Biswas SB. (2003) Mechanism and stoichiometry of interaction of DnaG primase with DnaB helicase of Escherichia coli in RNA primer synthesis. J Biol Chem. 278, 52253-52261.
15. Biswas SB, Khopde SM, Zhu F. X, Biswas EE. (2003) Subunit interactions in the assembly of Saccharomyces cerevisiae DNA polymerase alpha. Nucleic Acids Res. 31, 2056-2065.
16. S. Flowers, E. E. Biswas, and S B. Biswas (2003) Conformational Dynamics of DnaB Helicase upon DNA and Nucleotide Binding: Analysis by Intrinsic Tryptophan Fluorescence Quenching. Biochemistry, 42, 1910-1921.
17. Khopde, S., Biswas, E.E. and Biswas, S.B. (2002) Affinity and sequence specificity of DNA binding and site selection for primer synthesis by Escherichia coli primase. Biochemistry, 41, 14820-14830.
18. Flowers, S., Biswas, E. E, and. Biswas*, S (2002) Mechanism of DNA Binding by E. coli DnaB helicase: Analysis of Conformational Transitions by Fluorescence Quenching. Biochemistry, In Press.
19. Suarez, T. C., Biswas S. B. , and Biswas, E. E. (2002) Biochemical Defects in Human ABCR Nucleotide Binding Domain 1 Mutants Associated with Macular Degeneration. Journal of Biological Chemistry, 277, 21759-21767.
20. Biswas, E. E., Chen, P-H., and Biswas*, S.B. (2002) Modulation of ATPase Activities of E. coli DnaB Helicase by Single Stranded DNA Binding Proteins. Nucleic Acids Research, 30, 2809-2816.
21. Mitkova, A., Biswas, E. E., and Biswas*, S.B. (2002) Cell Cycle Specific Plasmid DNA Replication in the Nuclear Extract of Saccharomyces cerevisiae: Modulation by Replication Protein A and Proliferating Cell Nuclear Antigen. Biochemistry, 41, 5255-5265.
22. Biswas, E. E. , Nagele, R.G. and Biswas*, S.B. (2001) A Novel Human Hexameric DNA Helicase: Expression, Purification and Characterization. Nucleic Acids Research, 29, 1733-1740.
23. Biswas E.E. and Biswas, S.B. (2000) The C-terminal Domain of the Human ABCR Protein is a Functional ATPase, Biochemistry, 39, 15879-15886.
24. Biswas, E. E., and Biswas, S. B. (2002) Molecular Basis and Functional Consequences of Genetic Mutations in Human ABCR Nucleotide Binding Domain 2. Journal of Biological Chemistry, Submitted for publication
25. Biswas, E. E. and Biswas*, S. B. (1999) Mechanism of DNA Binding by the DnaB helicase of Escherichia coli: Analysis of the Roles of Domain Gamma in DNA Binding. Biochemistry, 38, 10929-10939.
26. Biswas, E. E. and Biswas*, S. B. (1999) Mechanism of DnaB helicase of Escherichia coli: Structural Domains Involved in ATP Hydrolysis, DNA Binding, and Oligomerization. Biochemistry, 38, 10919-10928.