Excision repair deficiencies in man and mice; lessons from CSA and Fen1 mutants
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AbstractThousands of DNA lesions, in form of chemical modifications, base loss and single-strand breaks are estimated to occur in every cell per day. Excision repair pathways and cell cycle checkpoints have evolved as part of the cellular response to DNA damage. Base excision repair (BER) can remove subtle DNA lesions, while nucleotide excision repair (NER) can remove more bulky helix distorting DNA damage. In paper I, we characterized two Cockayne syndrome (CS) patients deficient in a sub- pathway of NER, transcription coupled (TC) NER. We hypothesized that the underlying mutation most probably would be found in one of the known CS proteins, CSA or CSB. Molecular analysis confirmed our hypothesis, and a new splice site mutation was identified in the CSA gene. Moreover, we reviewed on the known human mutations in the CSA protein, at the time of publication, and their possible correlation to clinical findings. In the discussion of this thesis, an update on CS proteins, their known human mutations and clinical characteristics is further reviewed. Still, a molecular explanation of the CS pathology is lacking, and the role of the CS proteins in TC-NER and possibly oxidative damage repair needs further investigation. Flap endonuclease 1 (FEN1) is essential in mammalian long-patch (LP) BER and in removal of RNA primers in lagging strand DNA replication. Thus, it could be hypothesized that FEN1 deficiency would have detrimental consequences for cell survival and health of mutated mice. In paper II we show that Fen1 mutations in mice result in severe phenotypes in form of embryonic lethality and early cancer development. An update on FEN1’s role and regulation in the cell, and possible mechanisms causing cancer, is given in the discussion of this thesis. Moreover, in paper III we characterized Fen1 knock-in mice with a yellow fluorescent protein (YFP) tag fused to FEN1, in order to study FEN1-YFP kinetics in BER and DNA replication in vivo. For the first time, the kinetics of the FEN1-YFP protein in LP-BER, expressed from the Fen1-YFP gene at an endogenous level, could be investigated in living cells, following highly localized laser irradiation. This micro-irradiation method produces a high concentration (local damage) and wide spectrum of DNA lesions, including LP-BER substrates for FEN1. We found that FEN1-YFP is rapidly recruited to DNA damaged areas and were able to follow ongoing repair through the progressive disappearance of FEN1’s flap substrate. Inhibition of PARP disrupted FEN1 accumulation at DNA lesions, indicating that PARP is needed for FEN1 recruitment to DNA repair intermediates in LP-BER. Fluorescence recovery after photobleaching (FRAP) measurements following local damage allowed us to study the kinetics of FEN1 binding and unbinding its flap substrate. FRAP after global damage allowed us to measure the proportion of FEN1 binding at the moment of bleaching, and to estimate how long the FEN1 molecules stay bound to the substrate. We found that FEN1 binding after local damage is very short lived. In line with FEN1’s role in DNA replication and its interaction with PCNA, we compared the (co)localization of FEN1 and PCNA in S-phase DNA replication foci.
List of papers
|Paper I. Kleppa L, Kanavin ØJ, Klungland A, Strømme P. A novel splice site mutation in the Cockayne syndrome group A gene in two siblings with Cockayne syndrome. Neuroscience, (2007), 145:1397-1406. The paper is removed from the thesis in DUO due to publisher restrictions. The published version is available at: https://doi.org/10.1016/j.neuroscience.2006.09.025|
|Paper II. Larsen E, Kleppa L, Meza TJ, Meza Zepeda LA, Rada C, Castellanos CG, Lien GF, Nesse GJ, Neuberger MS, Laerdahl JK, William Doughty R, Klungland A. Early-Onset Lymphoma and Extensive Embryonic Apoptosis in Two Domain-Specific Fen1 Mice Mutants. Cancer Res. (2008), 68:4571-4579. The paper is removed from the thesis in DUO due to publisher restrictions. The published version is available at: https://doi.org/10.1158/0008-5472.CAN-08-0168|
|Paper III. Kleppa L, Mari PO, Larsen E, Flor Lien G, Godon C, Theil AF, Nesse GJ, Wiksen H, Vermeulen W, Giglia Mari G, Klungland A. In vivo kinetics and PARP1 dependence of FEN1 in base excision repair. Published as: Kinetics of endogenous mouse FEN1 in base excision repair. Nucl. Acids Res. (2012) First published online: July 18, 2012. The paper is removed from the thesis in DUO due to publisher restrictions. The published version is available at: https://doi.org/10.1093/nar/gks673|