Removal of uv-induced pyrimidine dimers from the replicated and unreplicated DNA of human fibroblasts Page: 2 of 4


Cell Culture and Radioactive Labeling: Normal human
fibroblasts, HSBP were routinely grown in Dulbecco's modified
medium plus 10% fetal calf serum at 370 in an atmosphere
containing 5% CO2. Cells grown to a monolayer in 75 cm2
flasks were subcultured at a ratio of 1:3. 3H-thymidine
(s.a. 3 Ci mmol) at 0.03 pCi/ml or 32P ortho phosphate at
0.5 pCi/ml were added and the cells grown to a monolayer.
Cell Plating UV Irradiation and Density Labelling: Cells
were plated at 7.105 per 150 mm petri dish and forty hours
later UV irradiated. Some 3H labeled cells were sampled to
determine the dimers induced, others were incubated in medium
containing Fluorodeoxyuridine (0.25 ug/ml) and bromodeoxy-
uridine (3 ug/ml). 32P prelabeled cells for repair replica-
tion studies were similarly incubated except that 3H tlhjmidine
(s.a. 55 Ci/mmol) at 5 uCi/ml was also included in the medium.
Extraction of DNA, Separation of Replicated and Unrepli-
cated Portions: Cells were lysed with 0.5 M EDTA, 0.5%
sarkosyl plus 200 ug/ml Proteinase K (Merck), and left at 370
for 8 hrs prior to phenol extraction. Cesium chloride (1.25
g/ml) was dissolved in each sample and centrifuged in a 40
rotor of a Beckman 5-50 at 30,000 rpm for 40 hours. Gradients
were collected as 30 fractions of 0.13 ml, 10 1 of each
fraction acid precipitated to determine the heavy/light and
light/light peaks, and these portions pooled and dialysed
against .011 TrisHCl, .001M EDTA, .04M NaCl pH 8.0.
Estimation of Pyrimidine Dimers: This was carried out by
determining the number of UV endonuclease sensitive sites (7).
Determination of Repair Replication: Unreplicated and
replicated DNA from 32P prelabeled cells was obtained as
above. The light strand from heavy/light DNA, and both light
strands from light/light DNA were further purified by 2
centrifugations in alkaline cesium chloride (8). -H activity
found banding with light 32P labeled strands was used as a
measure of repair.
The Effect of UV on DNA Synthesis: Table 1 gives the
amounts of DNA synthesised in cells during 12 or 24 hours
after 5 or 10 J-m-2. Cells receiving 5 J-m-2 by 24 hours
have synthesized as much as unirradiated controls, but after
10 J.m-2 only half this amount is made during the same period.

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Waters, R. Removal of uv-induced pyrimidine dimers from the replicated and unreplicated DNA of human fibroblasts, article, January 1, 1978; Tennessee. ( accessed March 20, 2019), University of North Texas Libraries, Digital Library,; crediting UNT Libraries Government Documents Department.

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