Concentration dependent alterations of DNA replication initiation and elongation by benzo[a]pyrene diol epoxide

Abstract

Vero cells treated with various concentrations of (+/-)7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy, 7,8,9,10-dihydrobenzo[a]pyrene (BP-diol epoxide I) exhibited dose-dependent inhibition in both the rate of DNA synthesis and in the size of nascent DNA. The maximum inhibition was seen 2--3 h after addition of BP-diol epoxide I. A recovery in both the rate of synthesis and size of nascent DNA was observed 5--10 h after treatment. The pH step alkaline elution assay which separates different nascent DNA replication intermediates was used to investigate whether the inhibition and recovery noted above could be accounted for by alterations in DNA replication initiation (DNA synthesis within a replicon) or elongation (rejoining of replicons). At lowest dose studied (0.166 muM BP-diol epoxide I) a reversible inhibition in DNA initiation was observed. At the higher dose levels (0.66 muM and 1.66 muM BP-diol epoxide I) inhibition of both DNA initiation and elongation were observed and inhibition of elongation predominated. The inhibition in elongation was detected by an increase in the relative amount of low molecular weight nascent DNA associated with DNA synthesis within a replicon and a relative decrease in the higher molecular weight elongated DNA. A reversal in the inhibition of both initiation and elongation was noted.

Critical Role for Mouse Hus1 in an S-Phase DNA Damage Cell Cycle Checkpoint

“…In both yeasts and higher eukaryotes, checkpoint-mediated regulation of DNA synthesis following DNA damage is achieved primarily through control of DNA replication initiation, usually by inhibition of late firing origins of replication in damaged cells (34,49,50,52,55). Accordingly, inhibition of DNA replication in cells treated with low doses of BPDE is associated with reduced initiation of DNA synthesis (3,6,10,31). Whether failure of this specific regulatory mechanism is the basis for the S-phase checkpoint defect in Hus1-null cells will require further investigation.…”

Benzo[alpha]pyrene diol epoxide I binds to DNA at replication forks.

“…Sites of carcinogen modification of DNA were identified by polyclonal rabbit antibodies elicited against DNA modified with B[aJP diol epoxide-I in vitro. This antigenic DNA contained trans-(7R)-N2- [10-(713,8a,9a-trihydroxy-7,8,9,10-tetrahydrobenzo[a] In the past, information concerning the effects of B[a]P diol epoxide-I exposure on DNA replication was obtained primarily from studies of the size distribution of nascent DNA molecules by using alkaline sucrose gradient sedimentation or alkaline elution (13,(18)(19)(20)(21) Eagle containing 10% (vol/vol) heat-inactivated fetal bovine serum, NaHCO3 at 2.2 g/liter, and Hepes at 6 g/liter (pH 7.2). Cells were incubated in a humidified atmosphere of 5% C02/95% air at 37°C.…”

“…In subcellular systems in vitro DNA synthesis is blocked at each DNA adduct encountered by the polymerase (15)(16)(17). In eukaryotic systems DNA adducts block DNA chain elongation and cause gaps to be formed in nascent DNA (13,(18)(19)(20)(21). However, in most nonlethal cases, DNA synthesis resumes with time, and gaps, as well as B[a]P diol epoxide-I-DNA adducts, decrease or disappear.…”

“…In the past, information concerning the effects of B[a]P diol epoxide-I exposure on DNA replication was obtained primarily from studies of the size distribution of nascent DNA molecules by using alkaline sucrose gradient sedimentation or alkaline elution (13,(18)(19)(20)(21). Such studies depended on detection of changes in large populations of DNA molecules, and some correlated the number of B[a]P diol epoxide-I adducts present in DNA with the reduction in either the size of nascent DNA fragments (18) or the rate of DNA synthesis (21).…”

DNA–carcinogen interaction: covalent DNA-adducts of benzo(a)pyrene 7, 8-dihydrodiol 9, 10-epoxides studied by biochemical and biophysical techniques

“…Since the two enantiomers exhibit different nucleotide-binding specificities, this implies that DNA binding per se rather than binding to a particular base is the major factor in BPDE-induced toxicity. It is well documented from studies using both non-synchronized and synchronized cells that racemic anti-BPDE efficiently blocks DNA synthesis by interfering with both replicon initiation and synthesis in active replicons (Bowden et al 1982;Cordeiro-Stone et al 1986;Yamanishi et al 1987)-…”