A specific feature of ionising radiation is the formation of clustered DNA damage, where two or more lesions form within one to two helical turns of the DNA induced by a single radiation track. The complexity of ionising radiation-induced DNA damage increases with increasing ionisation density and it has been shown that complex DNA damage has reduced efficiency of repairability. In mammalian cells, base excision repair (BER) is the predominant pathway for the repair of non-DSB clustered DNA lesions and is split into two sub-pathways known as short patch (SP) BER and long patch (LP) BER. SP-BER is the predominant pathway, especially in the repair of isolated DNA lesions. However, LP-BER is thought to play a greater role in the repair of radiation-induced clustered lesions. In this study, cell lines were generated stably expressing the fluorescently tagged BER proteins, XRCC1-YFP (marker for SP-BER) or FEN1-GFP (marker for LP-BER). The recruitment and loss of XRCC1-YFP and FEN1-GFP to sites of DNA damage induced by both ultrasoft X-ray (USX), a form of low linear energy transfer (LET) radiation, and near infrared (NIR) laser microbeam irradiation (‘mimic’ high LET radiation) was visualised in real-time and the decay kinetics of the fluorescently-tagged proteins determined. The half-life of fluorescence decay of FEN1-GFP following USX irradiation was longer than that of XRCC1-YFP, indicating that LP-BER is a slower process than SP-BER. Additionally, the fluorescence decay of XRCC1-YFP after NIR laser microbeam irradiation was fitted by bi-exponential decays with a fast component and a slow component, reflecting the involvement of XRCC1 in the repair of different types of DNA damage. In contrast to USX irradiation, where the XRCC1-YFP fluorescence decay reached background levels by 20 min, XRCC1-YFP still persisted at some of the NIR laser induced DNA damage sites even after 4 hours. This is consistent with the fact that the laser induces more complex damage that presents a major challenge to the repair proteins, persisting for much longer than the simple damage caused by low LET USX irradiation. Persistent, unrepaired DNA damage can potentially lead to mutations and replication-induced DSBs if it persists into S-phase. PARP1 inhibition reduced the recruitment of XRCC1 to DNA damage sites. However, a considerable amount of XRCC1 was still detected at the DNA damage sites, leading to the conclusion that there is a subset of DNA damage that requires XRCC1 but not PARP1 for repair. Understanding how clustered damage is repaired by the BER pathway can aid the design of future therapies which can be used in combination with radiotherapy to enhance the radiosensitisation effect. Knockdown of FEN1 was investigated and found to radiosensitise A549 (adenocarcinoma) cells, possibly as a result of an excess of unrepaired radiation-induced lesions requiring LP-BER for repair, although FEN1 knockdown alone induced cell death in non-cancerous BEAS-2B cells.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:588443 |
| Date | January 2013 |
| Creators | Cooper, Sarah Louise Pamela |
| Contributors | O'Neill, Peter; Lomax, Martine E. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:2c4b3d1e-67c3-4da5-8967-3080b1ebf27a |
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