Interaction among proteins of the Eukaryotic DNA Replication Machinary :
Genetic Analysis of PCNA in CHO cells resistant to aphidicolin.

Kendra Taylor and [Nawin C. Mishra]{.underline}

Dept. of Biological Sciences

The University of South Carolina

700 Sumter St. Columbia , SC 29208

[ABSTRACT]{.underline}

One of the major findings of the human genome project is the fact that
there are much less number of genes ( 35 thousand ) than previously
suggested ( 100 thousand ) in human; this necessarily means that most
genes and their products must interact during our physiology to
determine a particular phenotype . Thus interaction of proteins becomes
a rule rather than exceptions. Therefore analysis of global protein
interaction can provide an insight into the molecular basis of human
diseases and their possible alleviation with designer drugs. We have
chosen to study such interactions of proteins involved in DNA
replication since an accurate duplication of DNA is crucial for the
survival of an organism. A large number of proteins are involved in the
replication of the genomic blue print. Among them DNA polymerases are
the most important. A number of DNA polymerases have been shown to
participate in DNA synthesis during DNA replication, repair and
recombination.

**We are therefore interested in examining the global interaction of
these proteins in the replication machinery of mammalian cells. In order
to do so we have created mutants of CHO cells and human cells that are
altered in the major proteins of the DNA replication machinery. We have
selected these mutants by their ability to grow in the presence of DNA
polymerase inhibitors and anti cancer drugs such as aphidicolin and
Ara-C and Ara-A. We have shown that these mutants (called AphR for their
dominant mutation conferring resistance to aphidicolin and Ara-C) to
possess DNA polymerases of the alpha family that are altered in their
DNA polymerizing and proof reading activities and show resistance to
these drugs both *in vivo* and *in vitro* assays. We have also found
that some of these mutants have corresponding compensatory mutations in
PCNA. We have focused our attention to PCNA because it is a very small
protein (30 Kdal) and that it interacts with over a dozen proteins
involved in DNA replication (i.e. DNA polymerases delta and epsilon and
RF-C ) , cell cycle control ( p21 ) and DNA repair . We have cloned and
expressed DNA pol delta and PCNA (both wild type and mutant ) genes of
CHO cells . The expressed PCNA and DNA pol delta proteins have been
identified by western analysis in our laboratory.**

**This provides us the opportunity to examine the interaction of these
proteins particularly DNA pol delta and PCNA in the AphR mutants of the
CHO and human cells available exclusively in our laboratory.**

**In order to examine and establish the nature of protein interactions
we are attempting the determination of the nucleotide sequences and
inferred amino acid sequences of PCNA, DNA pol delta and RF-C from a
number of AphR mutants in order to establish the nature of changes in
these genes and proteins that are needed for their proper interaction
under physiological conditions. We have chosen to examine PCNA and DNA
pol delta since these proteins work very closely in the replication
machinery. We have shown that in a particular AphR mutant of CHO cells,
a change in DNA pol delta is accompanied with a change in PCNA. It has
been further shown that in the yeast PCNA mutants, their wild type
growth can be restored by a compensatory mutation in RF-C. In view of
these findings we plan to clone and determine the nuleotide sequences of
these genes from a number of AphR mutants available in our laboratory in
order to establish the compensatory nature of these mutations and to
examine the hypothesis that these proteins must change their 3D
structures for global interaction in the replication machinery. If this
is true, then in a mutant when DNA pol delta changes then PCNA must make
corresponding compensatory changes. We plan to clone these genes via
RT-PCR and then determine the nucleotide sequences using automated DNA
sequencing system available in our dept. We have successfully utilized
these methods and systems to clone DNA pol delta and PCNA. We plan to do
this for a large number of mutants to generate a profile of compensatory
changes in these proteins in order to identify domains involved in the
interaction of these proteins. We plan to present some of our findings
for the discussion.( Supported by a DoE grant, contractNo. DE-FG09-90ER
61040 to NCM )**