Project Title:
16,
Molecular and Cellular Radiobiology
The Chemistry and Function of DNA Polymerases
Technical Progress
in FY 1973:
(Cont'd.)
RX -03-02- (a)
Chromatography on DEAE-cellulose reveals at least two hybrid templatedependent polymerases in HeLa cell extracts separable from DNA dependent
polymerases and a terminal transferase,
Last year attempts to detect hybrid template-directed DNA polymerase
in mammary tumor tissue from dibenzanthracene-treated rats were unsuccessful.
Now in a limited number of cases some activity has been found in these tissues.
Preliminary results indicate that activity in the rat tumor tissue is confined
primarily to the cell nucleus, in contrast to chick embryo heart where about
90% of the activity appears in a "cytoplasm" fraction.
The significance of this
observation is not apparent at present.
The anticipated studies on hybrid-dependent polymerases in Lymphocytes,
described a year ago, have not been carried out because sufficient personnel
was not available and because significant progress in other laboratories made
thts seem like a less promising area for our efforts than those areas actually
pursued.
17.
Expected Results in FY 1974:
A real understanding of the function of hybrid-dependent polymerase in
chick embryo tissues can probably only be attained with a pure enzyme.
For
this reason the purification will continue to be pursued using such techniques
as: improved ion exchange chromatography using gradient elution, density gradier
centrifugation, chromatography on DNA-cellulose, and, possibly, preparative
disc electrophoresis.
In addition to using specific activity as a measure of
purification, the purification will be monitored by analytical disc electro-
phoresis, electrofocusing and analytical ultracentrifugation.
A major problem
in purification has been instability of the enzyme as purification proceeds.
A concentrated effort will be made to find conditions preventing loss of
enzyme activity.
Instability prevents an accurate determination of the molec™
lar weight of the purified enzyme.
The search for a natural heteropolymer which would serve as template-priz.
for the reaction will be pursued more vigorously.
or
are
pol
With the HeLa enzyme, standardization by DEAE cellulose chromatography
hocellulose chromatography will be undertaken to detect if there
etuations in the amount of the two (or more?) hybrid template-dependent
iases with the cell cycle.
Tissues from radiation- and dibenzanthracene-induced tumors will be
examined for their ability to incorporate tritiated thymidine ( from tritiated
Since the amount
thymidine triphosphate) into particles of approximately 70S.
of incorporation at best would be expected to be small, this study will be
{See Continuation Sheet)
1119281
RX-221