Inhibition and Recovery of Dna Synthesis in Uv‐irradiated Chinese Hamster V‐79 Cells†

Dahle, Dan; Griffiths, Tony; Carpenter, J. G.

doi:10.1111/j.1751-1097.1980.tb04003.x

Cited by 44 publications

(22 citation statements)

References 33 publications

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“…DNA fiber autoradiography of mammalian cells is consistent with blockage of replication forks (4,8,10). Most data on SV40 replication are also consistent with the blocking of replication fork progression by pyrimidine dimers in the template strand (3,9,19,25,26) Their interpretation seems inconsistent with data showing that most label incorporated after UV irradiation is near the origin of replication, as if dimers block fork progression (25).…”

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confidence: 80%

Pyrimidine dimers block simian virus 40 replication forks.

Berger¹,

Edenberg²

1986

Mol. Cell. Biol.

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UV light produces lesions, predominantly pyrimidine dimers, which inhibit DNA replication in mammalian cells. The mechanism of inhibition is controversial: is synthesis of a daughter strand halted at a lesion while the replication fork moves on and reinitiates downstream, or is fork progression itself blocked for some time at the site of a lesion? We directly addressed this question by using electron microscopy to examine the distances of replication forks from the origin in unirradiated and UV-irradiated simian virus 40 chromosomes. If UV lesions block replication fork progression, the forks should be asymmetrically located in a large fraction of the irradiated molecules; if replication forks move rapidly past lesions, the forks should be symmetrically located. A large fraction of the simian virus 40 replication forks in irradiated molecules were asymmetrically located, demonstrating that UV lesions present at the frequency of pyrimidine dimers block replication forks. As a mechanism for this fork blockage, we propose that polymerization of the leading strand makes a significant contribution to the energetics of fork movement, so any lesion in the template for the leading strand which blocks polymerization should also block fork movement.A variety of chemical and physical agents damage DNA within cells, causing inhibition of DNA replication and transcription, alteration of gene expression, mutagenesis, carcinogenesis, or cell death. Inhibition of DNA replication, when it occurs, is crucial to the fate of the cell: although cells may tolerate some mutations, they must replicate their full complement of DNA before dividing. The structures that result from attempting to replicate a damaged DNA template are the substrates for subsequent repair and recovery processes. Understanding the initial interaction between lesions and the replication fork will therefore provide insight into the mechanisms of recovery.UV light is one of the best-studied of DNA-damaging agents. The primary lesion produced by UV irradiation is the cis-syn cyclobutane pyrimidine dimer (31). The mechanism by which UV inhibits eucaryotic DNA replication remains controversial, especially the question of whether dimers inhibit all, most, or no replication forks. The earliest model (16) proposed that a pyrimidine dimer in the template blocked elongation of a daughter strand without affecting the progression (unwinding and movement) of the replication fork, which reinitiated synthesis about 1,000 nucleotides downstream, leaving a long single-stranded gap that could later be filled de novo (Fig. 1B). This resembles a situation in Escherichia coli, except that in E. coli the gaps are filled by recombination (23,24). An alternative hypothesis (7) is that lesions block the movement of the replication fork itself, as in Fig. 1C. With increasing awareness of the semidiscontinuous nature of DNA replication in mammalian cells (Fig. 1A), the latter model was modified to suggest that lesions in the template for the continuously synthesized (leading) strand block...

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confidence: 80%

Pyrimidine dimers block simian virus 40 replication forks.

Berger¹,

Edenberg²

1986

Mol. Cell. Biol.

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“…There is a transient fluence-dependent reduction in the rate of ["]-thymidine incorporation when mammalian cells are exposed to 254 nm light (Dahle et al, 1980). At low fluences ( < 2 Jim'), this reduction appears to be due primarily to inhibition of replicon initiation (Kaufmann et al, 1980).…”

Section: Introductionmentioning

confidence: 99%

“…At low fluences ( < 2 Jim'), this reduction appears to be due primarily to inhibition of replicon initiation (Kaufmann et al, 1980). At higher fluences, the reduction appears to be mediated by blockage of DNA fork progression by UV-induced DNA lesions (Edenberg, 1976;Dahle et al, 1980). In most mammalian cells, rates of DNA replication return to normal levels several hours after exposure.…”

Section: Introductionmentioning

confidence: 99%

“…Excision repair is involved in this recovery, but even rodent cells, which exhibit low levels of repair and therefore retain many pyrimidine dimers, exhibit recovery of normal rates of replication (Meechan et al, 1986). Consequently, other types of repair and tolerance processes such as the activation of alternative sites of replicon initiation, (Griffiths and Ling, 1985), bypass of the lesion (Lehmann, 1972;Dahle et al, 1980;Griffiths and Ling, 1985), and translesion synthesis (Meyn et al, 1976) help to overcome the reduction in thymidine incorporation. Another mechanism for repairing pyrimidine dimers, enzymatic photoreactivation, is found in prokaryotes and diverse eukaryotes including insects (Muraoka et ul., 1980).…”

Section: Introductionmentioning

confidence: 99%

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Effects of Ultraviolet Light on Thymidine Incorporation and Dna Chain Elongation in Photoreactivable Insect Cells

1989

Photochem & Photobiology

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An insect cell line, IAL-PID2, was exposed to UV and analyzed for its ability to incorporate [3H]-thymidine and to elongate replicon-sized DNA fragments. After exposure to 5 or 10 J/m2 UV, the cells exhibited a rapid and prolonged depression in the rate of thymidine incorporation. Photoreactivation reduced this depression but did not entirely reverse it. For exposures of 5 J/m2 or above, full recovery did not occur until 18 h after exposure. The blockage of fork progression after UV exposure was fluence-dependent, with replication segments after exposure to 20 J/m2 being shorter than those observed after exposure to 10 J/m2. Immediately after exposure to either 10 or 20 J/m2, photoreactivation reversed blockage of fork progression, indicating that the (5-6) cyclobutyl pyrimidine dimer is responsible for blockage. This also indicates that blockage of fork progression may not be the only factor responsible for the prolonged depression seen in thymidine incorporation. Three hours after exposure to either 10 or 20 J/m2, replication segments were still significantly shorter than control segments. Photoreactivation completely reversed blockage after exposure to 10 J/m2, but did not completely reverse blockage after exposure to 20 J/m2, indicating that at such fluences, other lesions may play a role in UV-induced blockage of fork progression.

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“…The sum of the individual sensitivities for all regions of the SV40 genome is equal to the total sensitivity of viral DNA subjected to random damage. Our results allow us to estimate the propor-Introduction Damage in DNA disrupts DNA replication (Rasmussen and Painter, 1964;Dahle et al, 1980;Sarasin and Hanawalt, 1980), RNA synthesis (Saurbier and Hercules, 1978;Hackett et al, 1981;Mayne, 1984) and certain specific protein DNA interactions (Siebenlist et al, 1980;Ptashne et al, 1980;Cleaver, 1983). All lesions in the genome play some role in the inhibition of DNA replication.…”

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confidence: 98%

Ultraviolet radiation inactivates SV40 by disrupting at least four genetic functions.

Brown¹,

Cerutti²

1986

The EMBO Journal

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We have examined the relative sensitivities of different genetic functions to damage induced by u.v. light. For this purpose we have introduced defined amounts of damage into specific regions of the genome of SV40 and have determined the effect of the damage on the survival of transfected viral DNA. We found that the lethal effect of the damage depends on its location within the viral genome. The region most sensitive to damage contains the transcriptional promotors and enhancers for the early and late viral genes plus part of the origin of DNA replication. Lesions within this regulatory region are 3.2‐fold more effective in inactivating viral DNA than is the same amount of damage randomly distributed throughout the viral genome. The region least sensitive to damage lies within the coding portion of the viral coat protein genes, which are expressed only late in infection and would therefore be transcribed from undamaged progeny viral genomes, provided DNA replication occurs. Damage within this region is only 45% as effective in inactivating viral DNA as are randomly distributed lesions. Thus there is a 7‐fold difference in the lethal effect of DNA damage within the most sensitive and least sensitive regions of the viral genome. Intermediate sensitivities are observed within the transcribed portion of the viral A gene, coding for the T antigen whose expression is required early in infection, and in a region at the terminus of DNA replication. The sum of the individual sensitivities for all regions of the SV40 genome is equal to the total sensitivity of viral DNA subjected to random damage.(ABSTRACT TRUNCATED AT 250 WORDS)

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Inhibition and Recovery of Dna Synthesis in Uv‐irradiated Chinese Hamster V‐79 Cells†

Cited by 44 publications

References 33 publications

Pyrimidine dimers block simian virus 40 replication forks.

Pyrimidine dimers block simian virus 40 replication forks.

Effects of Ultraviolet Light on Thymidine Incorporation and Dna Chain Elongation in Photoreactivable Insect Cells

Ultraviolet radiation inactivates SV40 by disrupting at least four genetic functions.

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