Faculty and Research Interests
Subhasis Biswas, PhD
Science Center 306A
Fax: 856 566-6291
University of Washington, Seattle, WA
PhD , 1981
Control of Replication of Human Papilloma Virus. Persistent infection is a known risk factor for progression to malignancy. Despite the high incidence of HPV infection and its associated malignancies, there is currently no effective antiviral agent available for treatment. Given their major role in HPV replication, the E1 and E2 proteins are important candidates for the development of new therapies. Thus, basic research on these HPV proteins will lead to the identification and validation of interactions that can be used for finding inhibitors that block critical stages of the viral life cycle. In a clinical setting, it may be useful to target various stages of the viral life cycle to achieve a more effective treatment of HPV infection and HPV-associated cancers.
HPV is classified into high-risk and low-risk types; low-risk types are correlated with benign lesions such as genital warts, whereas high-risk types are associated with the development of cancer. The goal of our research is to produce mechanistic insights into the functional roles of the low- and high-risk HPV proteins involved in DNA replication. The viral E2 protein, a DNA replication origin-binding protein, is the master regulator of HPV. By binding to it cognate sequences at the long control region of the viral
genome, E2 initiates viral replication, and activates or represses transcription of HPV-encoded oncogenes E6 and E7. Thus, E2 is the switch between viral replication and transcription. We observed that there is a hierarchy of E2 binding to its various recognition sequences in the origin. We are establishing a possible correlation between DNA replication, HPV genotype, and associated risk of carcinogenesis. The factors that regulate viral persistence are poorly understood, but are very important for understanding the consequences of infection in human.
Mechanism of Replication Initiation in Prokaryotes and Eukaryotes. Initiation of DNA replication in both prokaryotes and eukaryotes is the most crucial regulatory step for cell proliferation.
In prokaryotes, the DnaA protein, a single-subunit protein found in all prokaryotes, regulates the initiation of DNA replication. In conjunction with DnaB helicase, this pre-replication complex unwinds double stranded DNA (dsDNA) before beginning replication. We are currently focusing on these two enzymes in order to understand initiation of prokaryotic DNA replication. We have cloned, expressed and purified DnaA protein to study its interaction with the origin of replication. In addition, we are analyzing its modulation by regulatory proteins that control replication and sporulation. By utilizing mutants of DnaA protein as well as biophysical and molecular techniques, we are exploring mechanisms of various stages in the initiation of DNA replication.
In eukaryotes, prokaryotic DnaA and DnaB proteins are replaced by multimeric protein assemblies namely the origin recognition complex (ORC), the mini-chromosome maintenance proteins (MCM2-7), the cell division cycle 6 (Cdc6), and the chromatin licensing and DNA replication factor 1 (Cdt1). The individual proteins have been studied in recent years; however the co-expression of the protein complexes have been difficult. In order to fathom the functional roles of these proteins, they must be studied in their native forms. In vitro studies of such complexes are limited due to difficulties encountered in recombinant protein expression and purification. A recently developed Baculovirus Expression Vector System (BEVS) facilitates the production of a multi-protein complex. Using high capacity pACEBac and pIDX (pIDC, pIDK and pIDS) family of vectors, our approach has been to create constructs which harbor all genes required for the recombinant expression of MCM2-7, CDC6/CDT1 and ORC1-6 complexes. Subunit genes for each multimer were cloned into pACEBac and pIDX vectors. Baculovirus derived from the multi-gene single plasmid construct(s) are being used for simultaneous transfection of insect cell lines. Using this system, we have created in vivo assembly of multiple replication gene products and optimized production of these multi-protein complexes for systematic studies of these complexes in human DNA replication. We are using this system to study the regulation of initiation of eukaryotic DNA replication.
ABCA7 transporter and Alzheimer’s disease in African Americans. Alzheimer’s disease is the most common cause of dementia in the elderly and represents a major public health concern worldwide. At present, 5.3 million Americans suffer from Alzheimer’s disease and it costs $227 billion annually for treatment and care. African Americans are twice as likely to develop late-onset Alzheimer’s disease as Caucasians and the cause of this higher rate is not known. There is no cure for AD and the molecular basis of development of Alzheimer’s disease is poorly understood. Recent population genetic studies identified several genes that correlate to the development of Alzheimer’s disease. Among these genes, ABCA7 was linked to late-onset Alzheimer’s disease in African Americans. ABCA7 is a member of the ABCA membrane transporter family. Our hypothesis is that ABCA7 protein is involved in pumping out Aβ peptide from the neuronal cytoplasm preventing its accumulation in the healthy brain. Furthermore, we propose mutations in ABCA7 lead to defective transporters that fail to efficiently clear Aβ peptides from the neuronal cells, which causes memory loss and late-onset Alzheimer’s disease. In order to test these hypotheses, we plan to produce and purify ABCA7 protein as well as its four membrane external domains using recombinant DNA technology. Using the recombinant proteins, we shall study the properties of ABCA7 to dissect its role in the development of Alzheimer’s disease. A detailed understanding of ABCA7 and its genetic variants will aid in the determination of the role of this protein in the progression and pathology of Alzheimer’s disease, leading to more accurate prognoses and the development of more targeted therapies in the near future.
1. Yilmaz, G., Biswas E. E., & Biswas, S. B. (2016) Mechanism of Activation of the origin of DNA Replication in Human Papilloma Virus by E2 initiator protein, Biochemistry, To be submitted.
2. Patel, M, Bhatia, L. K., Patel, K., Yilmaz, G., Biswas E. E., & Biswas, S. B. (2016) Conformational Flexing of DnaA protein in Origin Activation. J. Biol. Chem., To be Submitted.
3. Rotoli, S. M., Biswas E. E., & Biswas, S. B. (2012) Quantitative Analysis of the Mechanism of DNA Binding by Bacillus DnaA Protein. Biochimie, 94, 2764-2775 PMID: 22974984
4. Biswas-Fiss E. E., Kukiratirat, J., & Biswas, S. B. (2012) Thermodynamic analysis of DNA Binding by a Bacillus Single Stranded DNA Binding Protein. BMC Biochemistry, 2012, 13:10. PMID: 22698072
5. 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
6. 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 .
7. 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 .
8. 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
9. 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.
10. 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
11. 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
12. 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
13. 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.
14. 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
15. 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.
16. 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.
17. 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.
18. 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.
19. 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.
20. 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.
21. 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.
22. 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.
23. 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.
24. 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.
25. 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.
26. 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.
27. 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.
28. 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
29. 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.
30. 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.