Radiation effects on DNA synthesis in a defined chromosomal replicon.

Abstract

It has recently been shown that the tumor suppressor p53 mediates a signal transduction pathway that responds to DNA damage by arresting cells in the late G1 period of the cell cycle. However, the operation of this pathway alone cannot explain the 50%o reduction in the rate of DNA synthesis that occurs within 30 min of irradiation of an asynchronous cell population. We are using the amplified dihydrofolate reductase (DHFR) domain in the methotrexate-resistant CHO cell line, CHOC 400, as a model replicon in which to study this acute radiation effect. We first show that the CHOC 400 cell line retains the classical acute-phase response but does not display the late G1 arrest that characterizes the p53-mediated checkpoint. Using a two-dimensional gel replicon-mapping method, we then show that when asynchronous cultures are irradiated with 900 cGy, initiation in the DHFR locus is completely inhibited within 30 min and does not resume for 3 to 4 h. Since initiation in this locus occurs throughout the first 2 h of the S period, this result implies the existence of a p53-independent S-phase damage-sensing pathway that functions at the level of individual origins. Results obtained with the replication inhibitor mimosine define a position near the GJ1S boundary beyond which cells are unable to prevent initiation at early-firing origins in response to irradiation. This is the first direct demonstration at a defined chromosomal origin that radiation quantitatively down-regulates initiation.The effects of ionizing radiation on DNA have been studied intensively for decades. An understanding of these effects and the cellular response to them is required to comprehend the genetic basis of radiation-induced mutation and to develop novel approaches to enhance the effectiveness of radiotherapy for the treatment of cancer and other diseases.Mammalian cells have elaborated complex mechanisms to deal with assaults on the integrity of genomic DNA. It is well known, for example, that cells down-regulate DNA replication ([3H]thymidine incorporation) by 50 to 55% within 30 min of a challenge with high-dose ionizing radiation (25,31,38); replication rates return to normal within a few hours (38). Presumably, this transient arrest of DNA synthesis allows the removal of adducts or the repair of strand breaks, or both, before these lesions can be permanently fixed by DNA replication into catastrophic chromosome breaks or mutations. The radiation dose-response curve has a steep initial component at lower doses, which is thought to result from inhibition of new initiations at origins of replication; a much smaller, refractile component is observed at high doses, which is thought to reflect direct effects on chain elongation (25,31,40). Because of the consequent high residual rate of [3H]thymidine incorporation by unaffected replication forks, it has been difficult to assess the extent to which initiation is specifically inhibited at any given dose of radiation.Various models have been proposed to explain how damage to the template itself, or c...

The role of ATM in DNA damage responses and cancer

“…This acute response to IR is distinguishable from the G1 cell cycle checkpoint by its independence of p53 status (Larner et al, 1994). The continued progression of AT cells through G1 and S ultimately leads to a sustained accumulation of cells in the G2 (Beamish and Lavin, 1994).…”

ATM: A mediator of multiple responses to genotoxic stress

“…This inhibition re¯ects regulation at the level of replicon initiation (Larner et al, 1994), and most likely allows repair of the damage before the lesions are permanently ®xed by DNA replication into irreparable chromosomal breaks.…”

A p53-independent damage-sensing mechanism that functions as a checkpoint at the G 1 /S transition in Chinese hamster ovary cells

“…13; unpublished observations). Thus, it is possible that mimosine-or aphidicolinblocked cells have passed an important cell cycle checkpoint after which they are relatively refractile to DNA damage for some time period.…”

“…However, in response to a moderate dose of ionizing radiation, the overall rate of [ 3 H]thymidine incorporation in an asynchronously growing mammalian cell culture is reduced to Ϸ50% of pre-irradiation values within 1.5-2 hr (11)(12)(13)(14)(15). It has been suggested that this rapid response functions by inhibiting subsequent initiation at origins of replication, while allowing forks already in progress to continue (12).…”

“…It has been suggested that this rapid response functions by inhibiting subsequent initiation at origins of replication, while allowing forks already in progress to continue (12). Recently, this was shown directly by demonstrating the loss of replication bubbles from the amplified dihydrofolate reductase (DHFR) locus in the methotrexate-resistant Chinese hamster ovary (CHO) cell line, CHOC 400, using a two-dimensional (2D) gel replicon mapping technique (13). Since CHOC 400 cells have a mutated p53 gene (16) and lack the radiation-sensitive G 1 checkpoint (13,16), we have proposed the existence of a p53-independent S-phase damage-sensing pathway (13).…”