Analysis of the function of LSH in DNA damage repair
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DNA damage from both normal metabolic activities and environmental factors such as UV and radiation can cause as many as 1 million individual lesions to the DNA per cell per day (Lodish et al 2004). Cells respond to this continuous damage by employing many, highly efficient DNA repair mechanisms and undergo apoptosis when normal DNA repair fails. Of the many types of DNA damage that can occur, double strand breaks (DSBs) are the most toxic (Featherstone & Jackson 1999). A single unrepaired DSB is enough to induce cellular apoptosis and several mechanisms have developed to repair DSBs. The recognition, signalling and repair of DSBs involve large multi-‐subunit complexes that bind to both the DNA and modified histone tails, which require modification of the chromatin in order to access their bind sites and function effectively (Allard et al 2004). Consequently several chromatin-‐remodelling proteins have been implicated in DSB repair (van Attikum et al 2004, Chai et al 2005). LSH (Lymphoid specific helicase) is a putative chromatin-‐remodelling enzyme that interacts with DNA methyltransferases and has been connected to DNA methylation (Myant & Stancheva, 2008). Knockouts of LSH or its homologues in A. thaliana and M. musculus show a reduction in DNA methylation of 60-‐70% (Jeddeloh et al 1999, Dennis et al 2001). However in addition to this phenotype, knockout A. thaliana also have an increased sensitivity to DNA damage (Shaked et al 2006). A homologue of LSH has also been identified in S. cerevisiae, which interacts with known repair proteins (Collins et al 2007) and may be involved in DSB repair. Although the majority of Lsh-‐/-‐ mice die shortly after birth, 40% of the line produced by Sun et al survive and show unexplained premature aging (Sun et al 2004). As premature aging is a hallmark of increased acquisition of DNA damage there is the possibility of a conserved role for LSH in mammalian DNA damage repair. Here I show that LSH depleted mammalian cells have an increased sensitivity specifically to DSB inducing agents and show increased levels of apoptosis. Further analysis shows that cells lacking LSH repair DSBs slower, indicating a novel role for LSH in mammalian repair of DSB. I performed an in depth analysis of the DSB defects in LSH depleted cells in an attempt to elucidate the function of LSH in DSB repair. I found that LSH depleted cells can correctly recognise DSBs but recruit downstream signalling and repair factors, such as γH2AX, less efficiently. I show that reduced recruitment of downstream DSB repair factors is not accompanied by extended cell cycle checkpoint signalling. This suggests that LSH depleted cells continue through the mitosis with unrepaired DSBs, which most likely leads to apoptosis and the increased sensitivity to DSB inducing agents. These experiments also showed that recruitment of DSB signalling and repair factors is not impaired equally at all breaks, and I present a model system created to quantitatively compare individually breaks between WT and LSH depleted cells to identify DSB that require LSH for efficient repair. I also preformed an analysis of Lsh-/- MEFs containing WT or catalytic null mutant LSH rescue constructs and I show that WT but not catalytic null LSH can restore efficient DSB repair. These studies identify a novel role for LSH in mammalian DSB repair and demonstrate the importance of its catalytic activity.