|
The "reading" and "writing" of the genetic code underlies many
facets of life at the molecular level. The processes of evolution, cellular
adaptation/differentiation, and many aspects of pathogenesis are dependent upon
how enzymes recognize and process DNA and RNA. Our group is interested in
applying quantitative biochemical and biophysical measurements to the study of
enzymes and macromolecular complexes that engage nucleic acids. The major focus
of our research is in the study of proteins that are involved in DNA damage
tolerance and how these elements contribute to genomic instability.
The mechanism of action for Y-family DNA polymerases and interactions that are
important for facilitating their biological functions are of particular interest
to our group. These specialized polymerases appear to be important for
preventing replication fork stalling in the face of damaged template bases or
unusual secondary structures (e.g. G4 tetraplex structures, hairpins, and/or
Z-DNA). We are interested in understanding at the atomic scale how these enzymes
catalyze DNA synthesis when other polymerases are inhibited and how
protein-protein interactions between the Y-family polymerases and other
replisome-associated proteins, such as the Werner's syndrome protein, function
to alter the efficiency and outcome of specialized DNA synthesis events. Because
of their association with various cancers, as well as the unique structural and
functional properties used by these enzymes, we are attempting medium-throughput
screening of chemical libraries to identify small molecules and/or nucleoside
analogues that specifically modulate Y-family DNA polymerase activity.
In addition to understanding the kinetic and structural features defining
specialized DNA synthesis events, we are motivated to investigate how DNA damage
tolerance mechanisms contribute to the initiation and progression of highly
malignant primary brain tumors. Recent evidence has shown that both low- (i.e.
grade II) and high-grade (i.e. grades III and IV) primary gliomas exhibit
constitutively active DNA damage signaling. The relevance of Y-family DNA
polymerases to glioma development was highlighted by a study reporting that two
Y-family DNA polymerases (pol iota and kappa) were over-expressed in glioma
specimens from a cohort of Chinese patients. One of these enzymes (pol kappa)
was shown to be a statistically significant prognostic indicator for survival
(i.e. more enzyme yielded a shorter survival time). We are currently performing
proteomic analysis of glioma specimens obtained at UAMS, with a specific focus
on enriching for DNA damage response elements and identifying changes in the
PTMs associated with replication fork stress. These results will provide
valuable insight into how damage signaling is altered in primary brain tumors as
the lesion progresses to the highly malignant and invasive glioblastoma
multiforme (grade IV) and may help us better understand and perhaps attenuate
cellular mechanisms that contribute to chemo- and radio-resistance.
The core techniques employed by our group use biochemical/molecular biological
approaches with particular emphasis on structural and functional analysis of
enzymes and macromolecular complexes involved in the pathways described above.
We study the kinetic and biophysical characteristics of protein-protein
and protein-nucleic acid complexes using rapid chemical quench and stopped-flow
methods, as well as x-ray crystallography and mass spectrometric-based methods,
such as hydrogen-deuterium exchange mass spectrometry. Cell-culture experimental
systems are also employed to address critical questions concerning the
involvement of specific polymerases in mutagenic events, macromolecular complex
assembly/function (i.e. replication fork dynamics and RNA processing machinery)
and in testing the efficacy of compounds that modulate the activity of specific
enzymes. For a more detailed discussion of research objectives feel free to
contact me by phone, email or visit us at www.eofflab.org
Selected Publications:
Eoff, R.L., McGrath, C.E., Maddukuri, L., Salamanca-Pinzon, G.S.,
Marquez, V.E., Marnett, L.J., Guengerich, F.P., and Egli, M. (2010) "Selective
modulation of DNA polymerase activity by fixed conformation nucleoside
analogues" Angew. Chem. Int. Ed. Engl. 49, 7481-7485
Eoff, R.L., Choi, J-Y., and Guengerich, F.P. (2010) "Mechanistic
studies with DNA polymerases reveal complex outcomes following bypass of DNA
damage" J. Nucleic Acids 2010
Maddukuri, L., Eoff, R.L., Choi, J-Y., Rizzo, C.J., Guengerich, F.P.,
and Marnett, L.J. (2010) "In vitro bypass of the major malondialdehyde- and
basepropenal-derived DNA adduct by human DNA Y-family polymerases kappa, iota,
and Rev1" Biochemistry 49, 8415-8424.
Eoff, R.L., and Raney, K.D. (2010) "Kinetic mechanism for DNA
unwinding by multiple molecules of Dda helicase aligned on DNA" Biochemistry
49, 4543-4553.
Eoff, R.L., Ponce-Sanchez, R., and Guengerich, F.P. (2009)
"Conformational changes during nucleotide selection by Sulfolobus solfataricus
DNA polymerase Dpo4" J. Biol. Chem. 284, 21090-21099.
Irimia, A.*, Eoff, R.L.*, Guengerich, F.P., and Egli, M. (2009)
"Structural and functional elucidation of the mechanism promoting error-prone
synthesis by human DNA polymerase - opposite the
7,8-Dihydro-8-oxo-2'-deoxyguanosine adduct" J. Biol. Chem. 284,
22467-22480
|
E-mail:
|
rleoff@uams.edu |
|
Office:
|
(501) 686-8343
Biomedical
Research Center 1 Room 421E |
|
Labs: |
(501)
603-1004
Biomedical Research
Center 1 Room B416 |
|
FAX: |
(501) 686-8169 |
|
