PNNL-13501 - Pacific Northwest National Laboratory

expression systems for several years; and 3) the cost of a
mammalian expression protein factory is too costly at the
present time.
Results and Accomplishments
Several proteins were successfully produced in active
form. One of these proteins, XPA, has been used
successfully in several projects.
1. Single-molecule XPA-DNA binding measurements.
This is the first demonstration of a DNA binding
protein to be bound to a bona fide damaging lesion.
Manuscripts for this single-molecule XPA work and
our conventional spectrofluorimetry studies are in
preparation. The underlying work demonstrates the
proteins and DNA substrates are functionally active.
2. We have provided DNA substrate, XPA, and RPA to
Biomolecular Interaction. Active DNA repair
proteins provide an attractive test system for this
project, especially because results can be compared
to the single-molecule and conventional spectroscopy
studies.
3. Partial proteolysis and domain mapping were
successful. We demonstrated that partial proteolysis
fragments of XPA can be quickly and accurately
characterized at both N- and C- termini by mass
spectrometry, eliminating the need for N-terminal
sequencing. The underlying scientific assumption is
that regions readily cleaved by partial proteolysis are
accessible and/or potentially disordered. Our results
agree with fragmentary nuclear magnetic resonance
structural information on XPA. Potentially, all future
proteins prepared in the Protein Factory could be
proteolytically mapped by the methods we developed
for XPA and tested by the program PONDR (under
refinement at Washington State University).
4. A shorter version of the XPA protein was also
successfully expressed and purified. This fragment is
larger than the ~1/3 of XPA whose NMR structure
was determined independently by Japanese
investigators and Mike Kennedy. This new XPA
protein has not yet been characterized for DNA
binding, spectrofluorimetry, or single-molecule work.
These studies may provide new insights into the
damage recognition problem by demonstrating
functions or lack of functions of the missing regions.
5. Careful spectrofluoremetric equilibrium and kinetic
measurements of XPA binding fluorescently labeled
oligonucleotides were made.
6. Remaining stocks of poly (AOP-ribose) polymerase
are being studied in Sam Wilson’s laboratory at the
National Institute of Environmental Health Sciences
for a role in base excision repair. A manuscript is in
preparation.
Summary and Conclusions
We demonstrated the necessary capabilities to clone,
express, purify, and characterize recombinant proteins.
The combination of partial proteolysis, mass
spectrometry, and a program that predicts protein
flexibility or disorder offers an important characterization
tool to map unstructured regions of proteins.
Development of fluorescent spectroscopy capabilities
with a DNA repair protein offers an alternative
characterization technology widely applicable to other
proteins and complexes.
Publications
Brooks PJ, DS Wise, DA Berry, JV Kosmoski,
MJ Smerdon, RL Somers, H Mackie, AY Spoonde,
EJ Ackerman, K Coleman, RE Tarone, and JH Robbins.
2000. “The oxidative DNA lesion 8,5’-cyclo-2’deoxyadenosine
is repaired by the nucleotide excision
repair pathway and blocks gene expression in mammalian
cells.” J. Biol. Chem. 275:22355-62.
Iakoucheva IM, AM Kimzey, CD Masselon, JE Bruce,
EC Garner, CJ Brown, AK Dunker, RD Smith, and
EJ Ackerman. 2000. “Identification of intrinsic order
and disorder in the DNA damage-recognition protein
XPA by time-resolved proteolysis and Fourier transform
ion cyclotron resonance mass spectrometry.” Protein Sci.
(submitted).
Biosciences and Biotechnology 65