Björn Rydberg, Sophia Chernikova and Priscilla K. Cooper
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley,
CA 94720.
Joel Bedford
Department of Radiological Health Sciences, Colorado State University,
Fort Collins, CO.
Double-strand break misrejoining. DNA double-strand breaks (DSBs) that mis-rejoin with wrong DNA ends are the cause of important cytogenetic alterations, such as chromosome aberrations and large deletions. Using a hybridization assay to determine the integrity of large restriction fragments, we have found that the misrejoining frequency of DSBs in human fibroblasts is highly dose dependent for low LET radiation but nearly independent of dose for high LET radiation. Our studies have been done with a number of different high LET particles including 1 GeV Fe ions from the Alternating Gradient Synchrotron at Brookhaven National Laboratory.
Normal human fibroblasts at the G0 stage of the cell cycle were irradiated with 10-160 Gy, incubated for various times at 37 °C, trypsinized, and embedded in agarose plugs. After lysis, the DNA was restricted with NotI and separated by pulsed field gel electrophoresis. Southern blots were probed for a unique 3.2 Mbp NotI fragment. Comparison of the intact full size band with a smear of broken or mis-rejoined restriction fragments was determined on each lane to estimate DSB induction and joining of correct ends. A conventional FAR assay was used to estimate total rejoining, and the difference between total rejoining and correct rejoining was calculated as misrejoining.
Our data show that DSBs induced by high LET radiation, such as Fe HZE ions, are more likely than DSBs induced by X-rays to undergo misrejoining with wrong DNA ends at doses below 80 Gy. This result is consistent with the idea that two DSBs need to be close to each other within the cell nucleus in order to have a possibility to mis-rejoin. At low doses of X-rays, randomly induced DSBs are not likely to be in close proximity. In contrast, a proportion of DSBs induced by the Fe ions and other high LET particles will be distributed along the track of the particle independent of dose, presumably in sufficient proximity for a misrejoining mechanism. This data is used in computational studies linking DSB misrejoining to formation of chromosome aberrations at low doses (NSCORT, project 1).
Recently we have complemented this study with measurements of chromosome breaks and interchanges using the Fe ion beam at the AGS. Premature chromosome condensation (PCC) was used to measure the initial damage and to follow repair. This data will be compared with the misrejoining data and also with previously obtained data on non-rejoined DSBs.
Relevance of base damage at complex DSBs. High LET radiation produces complex damage clusters that can involve multiple lesions, including DNA breaks and altered bases. In particular, DSBs produced by high LET radiation are likely to have associated base damage that may need special processing. We tested this idea by studying DSB rejoining and misrejoining after 1 GeV Fe ion irradiation of an XP-G mutant fibroblast cell line that is deficient both in transcription-coupled repair and global genome repair of oxidative base damage by base excision repair. We found that DSB repair was normal, both in extent and fidelity, in comparison with a control fibroblast. Processing of DNA ends therefore is likely to primarily involve processes other than base excision repair, at least after high doses. However, these results do not rule out a contribution of XPG-mediated processes to DSB rejoining of importance after low doses of IR or in transcriptionally active regions of the genome.
Chromatid-type aberrations induced by base damage. DSBs induced in the G1 phase of the cell cycle give rise to chromosome type aberrations in the next mitosis. In contrast, for mechanistic reasons unrepaired base damages or single-strand breaks are expected to give rise to chromatid type aberrations. To determine whether deficient repair of IR-induced base damage introduced in G1 does in fact result in excess chromatid-type aberrations at the first mitosis, we irradiated a G1 population of an XP-G fibroblast cell line deficient in transcription-coupled repair of oxidative base damage with X-rays or with 1 GeV Fe ions at the AGS. Results of the scoring of aberrations in the XP-G cells in comparison to a normal cell line will be presented.
Work supported by the U. S. National Aeronautics and Space Administration (NASA) to Project 2 of the NSCORT in Radiation Health at the Lawrence Berkeley National Laboratory.