TY - JOUR
T1 - An analysis of DNA replication in synchronized CHO cells treated with benzo[a]pyrene diol epoxide
AU - Yamanishi, Doug T.
AU - Bowden, G. Tim
AU - Cress, Anne E.
N1 - Funding Information: This work was supported by USPHS Grants CA-269072, CA-31010 and CA23074. The authors wish to thank Debbie Bradley-Dunlop and Betsy Hurd for their excellent technical assistance and Sally Anderson for her secretarial help.
PY - 1987/10/9
Y1 - 1987/10/9
N2 - Synchronized Chinese hamster ovary (CHO) cells treated with (±)7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-dihydrobenzo[a]pyrene (BP diol epoxide I) were used to test the 'block-gap' model of replicative bypass repair in mammalian cells. One feature of the model is that carcinogenic or mutagenic DNA adducts act as blocks to the DNA replication fork on the leading strand. Using synchronized CHO cells, the rate of S phase progression by BrdUrd labeling of newly replicated DNA was measured. The rate of S phase progression was reduced by 22% and 42%, when the cells were treated at the G1/S boundary with 0.33 and 0.66 μM BP diol epoxide I, respectively. Using the pH step alkaline elution assay, it was found that the reduced rate of S phase progression was due to a delay in the appearance of multiple replicon size nascent DNA. This observation was consistent with the frequency of BP-DNA adducts present in the leading strand. A second feature of the 'block-gap' model is that the adduct-induced blockage on the lagging strand will produce gaps. It was determined by the use of high-resolution agarose gel electrophoresis, that the ligation of Okazaki size replication intermediates was blocked in a dose-dependent manner in BP diol epoxide I treated, synchronized CHO cells. These data are consistent with a block to the leading strand of DNA replication at DNA-carcinogen adducts. An inhibition of the ligation of Okazaki size fragments by BP diol epoxide I implies a block to replication of the DNA lagging strand leading to gap formation. The data presented here are, therefore, supportive of the 'block-gap' model of replicative bypass repair in carcinogen damaged mammalian cells.
AB - Synchronized Chinese hamster ovary (CHO) cells treated with (±)7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-dihydrobenzo[a]pyrene (BP diol epoxide I) were used to test the 'block-gap' model of replicative bypass repair in mammalian cells. One feature of the model is that carcinogenic or mutagenic DNA adducts act as blocks to the DNA replication fork on the leading strand. Using synchronized CHO cells, the rate of S phase progression by BrdUrd labeling of newly replicated DNA was measured. The rate of S phase progression was reduced by 22% and 42%, when the cells were treated at the G1/S boundary with 0.33 and 0.66 μM BP diol epoxide I, respectively. Using the pH step alkaline elution assay, it was found that the reduced rate of S phase progression was due to a delay in the appearance of multiple replicon size nascent DNA. This observation was consistent with the frequency of BP-DNA adducts present in the leading strand. A second feature of the 'block-gap' model is that the adduct-induced blockage on the lagging strand will produce gaps. It was determined by the use of high-resolution agarose gel electrophoresis, that the ligation of Okazaki size replication intermediates was blocked in a dose-dependent manner in BP diol epoxide I treated, synchronized CHO cells. These data are consistent with a block to the leading strand of DNA replication at DNA-carcinogen adducts. An inhibition of the ligation of Okazaki size fragments by BP diol epoxide I implies a block to replication of the DNA lagging strand leading to gap formation. The data presented here are, therefore, supportive of the 'block-gap' model of replicative bypass repair in carcinogen damaged mammalian cells.
KW - (CHO cell)
KW - Benzo[a]pyrene diol epoxide
KW - Block-gap model
KW - DNA replication
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U2 - 10.1016/0167-4781(87)90092-3
DO - 10.1016/0167-4781(87)90092-3
M3 - Article
C2 - 3115292
SN - 0167-4781
VL - 910
SP - 34
EP - 42
JO - BBA - Gene Structure and Expression
JF - BBA - Gene Structure and Expression
IS - 1
ER -