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The Role of Proliferating Cell Nuclear Antigen
(PCNA) Protein in Radiation-Mediated Cleavage of
Oligonucleotides by Titanium Dioxide DNA
Nanocomposites In Vitro Angela Babbo, Tatjana
Paunesku, Aiguo Wu, Eric Brown, Jennifer Link,
Cecille Cruz, Yao Wong, Gayle E. Woloschak Robert
H. Lurie Comprehensive Cancer Center,
Northwestern University, 303 E. Chicago Ave.,
Chicago, IL 60611, USA Experimental Facilities
Division, Argonne National Laboratory, 9700 South
Cass Avenue, Argonne, IL 60439, USA.
Objectives
Methods
Results

• In the course of ongoing studies using Titanium
  Dioxide (TiO2)DNA oligonucleotide
  nanocomposites, we looked at the possibility of
  using radiolabeled proliferating cell nuclear
  antigen (PCNA) protein as the source for
  nanocomposite mediated DNA scission. TiO2
  oligonucleotide nanocomposites are made of 45 Å
  nanoparticles of TiO2 conjugated to
  oligonucleotide DNA(s).
• Exposure to electromagnetic radiation of energy
  greater than 3.2 eV results in charge separation
  in semiconductor TiO2 leading to accumulation of
  electropositive holes in DNA resulting in DNA
  cleavage. This reaction can be mediated by 32P
  radionuclide attached to DNA helix created with
  the nanocomposite.
• Now we are determining whether 32P phosphorylated
  PCNA can achieve the same result. PCNA is a
  ring-like protein that slides along the DNA
  molecule and serves as a scaffold for DNA
  polymerases involved in DNA replication and
  repair.
• The ability of PCNA to form stable trimer ring
  structures on oligonucleotide substrates forming
  Y forked duplexes is well documented.
  Similarly, we found that the PCNA ring is stable
  on a DNA conjugated to a nanoparticle, where the
  nanoparticle serves the role of the fork.

• The amount of DNA cleaved from the nanocomposites
  was determined by PAGE. The frequency of DNA
  cleavage can be visualized through the quantity
  of double-stranded DNA in the gel.
• Our results using 32P-PCNA show a lack of double
  stranded hybrid, and thus, a lack of DNA cleavage
  when the 32P is on the PCNA.

• We engineered the phosphorylation amino acid
  sequence at the N-terminus of PCNA and
  radiolabeled the purified PCNA protein. We
  allowed this 32P labeled PCNA to assemble on the
  nanocomposite, looking for the excitation of
  TiO2, and DNA cleavage.
• A TiO2 nanocomposite with an attached 50-mer was
  used in combination with complementary
  oligonucleotides, with or without addition of the
  radiolabeled PCNA.
• A complementary radiolabeled oligonucleotide was
  used to monitor the scission reaction, and as a
  positive control.
• The nanocomposites were annealed with the
  non-labeled complementary oligonucleotides, the
  32P-PCNA was added to half of the tubes, and
  incubated at room temperature overnight.
• Finally, radiolabeled scission monitoring
  oligonucleotides were added to the tubes,
  annealed, and run on an 8 polyacrylamide gel
  (PAG). The gels were visualized by Storage
  Phosphor imaging.

PCNA protein binds oligonucleotide attached to
nanoparticle
Excitation of the TiO2 can be achieved by
exposure to a radiolabeled oligonucleotide
Wells 1 to 5 contain mixes of nanocomposite and
radiolabeled complementary oligonucleotide
incubated for increasing duration of time (30 to
270 minutes). Arrows point to a cleavage product.
Top nanocomposite alone Bottom nanocomposite
with PCNA protein bound to it.
Conclusions
Excitation of the TiO2 can NOT be achieved by
exposure to a radiolabeled DNA binding protein

• Based on our results, it appears that 32P has to
  be located in a DNA hybrid with the nanocomposite
  in order to cause DNA scission from the TiO2
  nanoparticle.
• Presence of 32P on a DNA-binding protein can not
  serve this role. One possible explanation is that
  the radiolabeled phosphorus on the N-terminus of
  the PCNA protein is not in a position favorable
  for excitation of TiO2.
• We will look into this possibility by engineering
  the phosphorylation sequence in other locations
  on the PCNA protein, and then use atomic force
  microscopy to visualize the orientation of PCNA
  on the nanocomposite.
• Another possible explanation is that the TiO2
  excitation by 32P depends on charge transfer in
  the DNA hybrid and that this process can not be
  initiated by 32P located on the DNA sliding clamp
  PCNA.

Well 1 Band mobility controlit contains only
the radiolabeled oligonucleotide used in these
experiments for the final 30 minutes before gel
electrophoresis.0-mer Wells 2, 5, 8 contain
oligonucleotide T2 (not-attached to TiO2
nanoparticle) mixed with nothing (2),
complementary 20-mer (5) mixed with
complementary 30-mer (8) Wells 3, 6 and 9
Contain oligonucleotide T2 attached to TiO2
nanoparticle mixed with nothing (3),
complementary 20-mer (6) mixed with
complementary 30-mer (9) Wells 4, 7 and 10
Contain radiolabeled PCNA-ph recombinant protein
and oligonucleotide T2 attached to TiO2
nanoparticle mixed with nothing (4),
complementary 20-mer (7) mixed with
complementary 30-mer (10
A diagram of TiO2-DNA oligonucleotide induced DNA
scission Excitation by 32P attached to a
complimentary oligo.
References Paunesku, T., Rajh, T., Wiederrecht,
G., Maser, J., Vogt, S., Stojicevic, N., Protic,
M., Lai, B., Oryhon, J., Thurnauer, M., and
Woloschak, G. Biology of TiO2 -oligonucleotide
nanocomposites. Nat. Materials 2003
2(5)343-6. Acknowledgements TiO2 nanoparticles
were kindly provided by T. Rajh and M. Thurnauer,
Chemistry division, Argonne National Laboratory,
USA and by M. Aslam and V. Dravid, Materials
Science and Engineering, Northwestern University.
TiO2 nanocrystallites bound to DNA oligos exhibit
semiconducting properties through both
constituents (metal oxide and DNA oligo). Charge
pairs separation leads to accumulation of
electrons in the conduction band of TiO2
accumulation of electropositive holes on the DNA
leading to DNA cleavage.