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Subhasis
Biswas, Ph.D.
E-mail: biswassb@umdnj.edu
Phone: 856 566-6270
Fax: 856 566-6291
Office: Science Center 306A
Address:
Department of Molecular Biology
UMDNJ-SOM
2 Medical Center Drive
Stratford, NJ 08084
Special Research Interests:
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.
Figure 1
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
Recent Publications:
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.
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
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.
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.
Biswas, S.B., 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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
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.
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. |
