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DOE Lowdose Radiation Program Workshop IV

Abstract

Title: Mechanisms of Low-Dose Inducible DNA Repair and the Adaptive Response

Authors: Janice Pluth, Hengameh Zahed Kargaran, Stacey Gauny, and Priscilla K. Cooper

Institutions: Life Sciences Division, Lawrence Berkeley National Laboratory

Neither the underlying mechanism nor the generality and extent of a protective
effect to subsequent radiation-induced or endogenous damage (the radio-adaptive
response) that can be induced by exposure to low level ionizing radiation (LLIR) is well understood at present. Such information is essential for development of meaningful models for assessing risk associated with exposure to low doses of ionizing radiation. Although DNA damage signaling and DNA repair processes appear likely to be involved, the detailed nature and regulation of repair processes responsive to LLIR is still largely unknown. Furthermore, important interconnections between the relevant repair pathways and with other essential DNA transactions -- including replication, transcription, chromatin dynamics, and cell cycle progression -- largely remain to be elucidated. While double-strand breaks (DSBs) are the most deleterious DNA damage produced by IR, a very much larger number of diverse base damages are directly produced, and these can be processed into DSBs either by replication or, when closely opposed, by lesion removal processes. An LLIR-inducible base excision repair (BER) process in human cells identified by M. Weinfeld (Le et al., Science 280:1066-1069, 1998) and shown to remove
thymine glycols (Tg) more rapidly after a low priming dose of IR is thus a likely
candidate for involvement in the adaptive response.

Preliminary studies by the Weinfeld group in collaboration with us have shown
that human cells lacking transcription-coupled repair (TCR) because of mutations in the multi-functional repair protein XPG or in the Cockayne syndrome protein CSB do not exhibit LLIR-inducible BER. Our studies are aimed at understanding whether these and other proteins required for inducible BER are also critical for the adaptive response. We hypothesize that the salient damage induced both by LLIR and endogenous sources is oxidative base damage and strand breaks that, when encountered by a replication fork, result indirectly in double-strand breaks (DSBs). Further, we propose that recombinational repair coordinated with recruitment of the transcription-coupled base excision repair (TC-BER) machinery is required for removal of the lesion and restoration of fork progression. To test this hypothesis for the mechanism underlying the adaptive response, we have proposed to (1) characterize the roles of TCR-related proteins in responses to LLIR; (2) develop a highly sensitive assay to quantify the adaptive response; and (3) examine the possibility that TCR proteins are recruited to replication forks stalled by oxidative lesions. To date we have optimized a rapid and accurate FACS-based assay to quantitate histone ?H2AX as a measure of DSBs and have shown a linear (R2 =0.99)
dose response relationship down to 10 cGy. We are currently working out conditions to accurately detect doses under 10 cGy as well. An adaptive response regimen of
exposures is being tested on various normal and TCR-defective lines. Once conditions for an optimal adaptive response have been identified, the FACS-based assay will be used to detect differences in response between primed and naive cells after exposure to a challenge dose. We have also used immunofluorescence techniques to identify proteins that localize to stalled replication forks. We have observed that XPG co-localizes with Mre11 and NBS1, components of the MRN complex, which forms foci during S phase and in response to DNA damage. XPG foci formation is enhanced after exposure to hydroxyurea (HU), an agent known to cause stalled replication forks. These results imply that XPG may be important in resolving stalled replication forks. Our recent demonstration that XPG-defective cells are more sensitive to H2O2 than their complemented counterparts suggests that this function involves responses to oxidative damage, in agreement with our hypothesis for the mechanism underlying the adaptive response.