Mechanism of phage phiX174 DNA inactivation by benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide.

Abstract

A previous report from this laboratory has shown that certain derivatives of polycyclic aromatic hydrocarbons bind to 4X174 DNA and render it noninfectious. The present work describes the relationship between the extent of 4X174 DNA binding by (±) -anti-benzo [alpyrene-7,8-dihydrodiol-9,10- (1-3), it reacts covalently with nucleic acids (4, 5), and DNA conjugated with anti-BPdiolepoxide has been isolated from cultured cells exposed to BP (2, 6). In addition, several studies have shown that BP-diolepoxide is unusually active as a mutagenic agent for mammalian (1, 7) and bacterial cells (8,9), and a more potent carcinogen than the parent hydrocarbon in newborn mice (10).We have described a bacteriophage assay system for evaluation of the biological activity of polycyclic aromatic hydrocarbons (PAH) and related compounds (11)(12)(13) (14), except that the infected cell lysates obtained by lysozyme treatment were subjected to two successive centrifugations (15 min at 17,000 X g and 2 hr at 100,00 X g) for sedimentation of phage. The resuspended phages were then purified as reported (14). DNA was isolated from purified viruses by several extractions with phenol containing 1% sodium dodecyl sulfate, followed by dialysis against 10 mM Tris (pH 7.5)/1 mM EDTA. The concentration of OX DNA was determined on the basis that, in a 1-cm light path, 1 A260 unit is equivalent to 36 jsg/ml or 1.3 X 1013 molecules per ml (14).Radioactive 4X174am3 DNA was prepared by the same procedure described above, except that infection was carried out in the presence of [3H]thymidine (6 Ci/mmol) at a concentration of 20 nmol/ml. Isolation of DNA from the radioactive virus by phenol extraction gave a specific activity of 3 X 104 cpm/,ug of DNA.Infectivity Assay of gbX174am3 DNA. The infectivity of OX174am3 DNA was assayed by absorption with spheroplasts prepared from E. coli K12W1485 and plating with its sensitive host, E. coli HF4714, for plaque development, as reported (11, 13). Preparation of Radioactive Replicative Intermediates RF1 and RF2. 3H-Labeled 4X174am.3 phage, prepared as described above, was used to infect E. coli C in the presence of chloramphenicol (15 ug/ml). After 10 min of infection, DNA was extracted from the infected cells by the procedure of Brutlag et al. (15). The cell lysate was adjusted to contain 1 M NaCl and was extracted once with phenol; the nucleic acid was precipitated from the aqueous phase with ethanol. After resuspension in Tris/EDTA and sedimentation in a neutral sucrose gradient, the pellet showed two radioactive peaks, 21 S and 16 S, corresponding to the qX174 DNA replicative intermediates RF1 and RF2, respectively.

Termination of in vitro DNA synthesis at AAF adducts in the DNA

“…In developing this in vitro system to measure termination of DNA synthesis at lesions in the DNA, we had two major objectives: to determine whether a particular lesion is a block to synthesis by DNA polymerases, and to be able to detect (5,7,13) have concluded that UV-induced pyrimidine dimers and adducts of BPDE in DNA are blocks to synthesis by pol I and pol III of E. coli. However, none of these systems have been able to eliminate the problem of illegitimate priming; that is, if a DNA template contains multiple 3'-OH termini that can act as primers for DNA polymerase, synthesis may reinitiate downstream from a site of termination.…”

“…The presence of pyrimidine dimers or benzo(a)pyrene diolepoxide (BPDE) residues in this template produces an absolute block to synthesis catalyzed by DNA polymerases I and III from E. coli (5,7). On OX174…”

Disruption of transcription in vitro and gene expression in vivo by DNA adducts derived from a benzo[a]pyrene diol epoxide located in heterologous sequences

“…While the long term effects have been widely studied, much less is known about acute effects of BPDE-DNA adducts in Previous studies indicated a high affinity of the transcripcells. In vitro studies have amply demonstrated that BPDEtion factor Sp1 for DNA adducts derived from benzo[a]-DNA adducts represent strong blocks to the progression of pyrene diol epoxide (BPDE) in sequences that are not both DNA polymerase and RNA polymerase (12)(13)(14)(15). Thus, normal binding sites for Sp1.…”

The interaction of metabolic systems with damaged DNA.

“…The ability to use a completely sequenced DNA (Sanger et al 1977) is a major dvantage and both Radmans group (Caillet-Fauquet et al 1977) and group led by S. Weiss at the University of Chicago (Hsu et al 1977) have used X174 DNA to show that a single pyrimidine dimer or a single benzpyrene residue can be an absolute block to DNA synthesis catalyzed by E. coli DNA polymerase.…”