Eric Ackerman
eackerman@pnl.gov
Pacific Northwest National Laboratory
Website: http://www.sysbio.org/resources/staff/ackerman.stm

Why this Project?

Even low doses (0.1 Gy) exert measurable effects on DNA repair. The first-known oxidative lesion repaired only by nucleotide excision repair found in normal cells is cyclo-dA. This lesion is found in normal cells and thought to be a byproduct of oxidative metabolism. When this lesion occurs, it stimulates repair. If repair is stimulated by low dose radiation, there are some implications for human health. For example, do some individuals exhibit a greater, lower, or no stimulation to certain DNA lesions? If there are population polymorphism that influence DNA repair, then it would be possible to use our assay for screening individuals for repair sensitivity.

Project Goals

To determine whether low dose differentially affects different lesions in the nucleotide excision repair pathway.
To determine if more than one pathway is responsible for these differential effects of low dose radiation on repair.
To gain mechanistic insights into the repair of DNA lesions.
To correlate the repair stimulation effects seen at higher doses (10-25 Gy) with cell death.
To determine if damage is attributed solely to the nucleotide excision repair process or two different non end-joining repair lesions.

Experimental Approach

Human fibroblasts are used to develop a functional assay to measure the effects of low dose radiation on the repair of many different lesions representative of those found in cells as consequences of normal oxidative metabolism, as well as those caused by radiation.
The sensitivity of our assay is adequate to determine the lower dose boundary for stimulation of repair by low dose radiation. However, because of equipment limitations, doses below 0.05-0.1 Gy have not been tested, so it is not known if even lower doses might also stimulate repair.
Our assay is host-cell reactivation, so the damage to the DNA is placed in a reporter gene. If damage is removed, luminescence is detected and quantitated, but if the damaged is not removed, then there is no luminescence.

Outcomes

Interestingly, the amount of DNA repair increases at low doses between 10-50 rads, plateaus, and then increases even further at higher doses in the dose region below where radiation-induced lethality would be expected. Varying parameters such as transfection protocols or DNA quantities do not appreciably changed the overall significant result that repair is stimulated at 1-5 Gy, followed by a plateau, and then followed by another stimulation near 10 Gy.