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The Influence of Histone Chaperones and Histone Modifying Proteins In 1 The Influence of Histone Chaperones and Histone Modifying Proteins in Regulating the DNA Damage Checkpoint Senior Thesis Presented to The Faculty of the School of Arts and Sciences Brandeis University Undergraduate Program in Biology James Haber, Advisor In partial fulfillment of the requirements for the degree of Bachelor of Science By Lizabeth J. Katsnelson April 2015 Copyright by Lizabeth J. Katsnelson Committee members: Name: Dr. James E. Haber Signature: _____________________________ Name: Dr. Susan Lovett Signature: _____________________________ Name: Dr. James Morris Signature: _____________________________ 2 Acknowledgments People say, “It takes a village to raise a child” and just like raising a child, it takes a village to finish a senior thesis. There are so many people I would like to thank for helping and supporting me throughout this process. First, I would like to thank my advisor, Dr. James Haber, for allowing me to work on this project in his laboratory and for believing in my skills as a young scientist. I would also like to thank the rest of the members of my thesis committee, Dr. Susan Lovett and Dr. James Morris, for reading and assessing my thesis. This project and thesis would have never come together without the extraordinary support from my mentor, Michael Tsabar. Michael’s mentorship was an invaluable aspect to my growth as a researcher and I am truly grateful to have had him as my guide during this journey. I would also like to thank all the other members of the Haber lab, both past and current, who were always willing to provide guidance to me and all the other undergraduates in the lab. Lastly, I would like to thank my friends and family for all their support. I could not have accomplished any of this with out the love and support of both my parents, my grandmother, and my sister, who all encouraged me to go to Brandeis and always believed in my abilities. 3 Abstract DNA double stranded breaks (DSBs) occur constantly, and are highly deleterious, which is why eukaryotic cells have evolved mechanisms of repair to ensure genomic stability. A crucial aspect of the DNA damage response is recognition of the lesion and activation of the DNA damage checkpoint, which prevents cells from dividing prior to repairing the lesion. Following a DSB in yeast, the two PI3-like protein kinases Mec1 and Tel1 phosphorylate a cascade of downstream effectors that lead to cell cycle arrest. A key target for Mec1 regulated phosphorylation is Rad53, which, following Mec1 mediated phosphorylation undergoes autophosphorylation. Following repair, cells must properly deactivate the checkpoint in order to continue the cell cycle, a process termed recovery. Recovery defective cells fail to deactivate the checkpoint and therefore cannot resume cell cycle progression, despite being able to repair. This study focuses on the role of histone chaperones and histone modifying proteins in the deactivation of the checkpoint, specifically Asf1, CAF-1, Rtt109, and Rtt101. Asf1 and CAF-1 are histone H3-H4 chaperones. Interestingly, Asf1 binds Rad53 prior to suffering damage. Once Rad53 is phosphorylated during the damage response, its affinity to Asf1 is greatly reduced. Rtt109 is a histone acetyl transferase and acetylates H3K56 after Asf1 binds H3, which then weakens Asf1-H3 interactions. Rtt101, a ubiquitin ligase, ubiquitylates H3K56ac to aide in the dissociation of Asf1 from H3. Therefore, the hypothesis proposed in this thesis is that dissociating H3-H4 from Asf1 is necessary to allow Asf1 to interact and sequester Rad53 following repair, thus deactivating the checkpoint and facilitating recovery. 4 Introduction The nucleus of eukaryotic cells holds genomic information in the form of chromatin, which is formed by repetitions of its functional unit, the nucleosome (Polo, 2015). Each nucleosome protects approximately 146 base pairs of DNA that wrap around a histone octamer. The histone octamer contains a (H3-H4)2 tetramer and two H2A-H2B dimers (Burgess and Zhang, 2013). Depositing H3-H4 molecules is the rate- limiting step in the formation of nucleosomes (Han, et al., 2013). Nucleosomes pose a barrier for DNA-related processes such as replication, repair, or transcription. In order to modify nucleosome affinity to DNA, histones can be post-translationally modified by acetylation, methylation, phosphorylation, and ubiquitylation to stimulate or disable certain cellular processes (Burgess and Zhang, 2013). DNA damage can result from many exogenous and endogenous sources such as radiation, reactive oxygen species, or replication across nicked DNA (Harrison and Haber, 2006). Double-strand breaks (DSBs) are the most detrimental form of DNA damage and failure to repair DSBs can result in genomic instability conducive to the development of many diseases, including cancer (Finn, et al., 2012). Eukaryotic cells have elaborate and highly conserved pathways of DNA damage sensing, signaling, and repair termed the DNA damage response (DDR) (Bradbury and Jackson, 2003). Not only does the DDR require recognition of the altered DNA structure, but also the recruitment of repair factors in order to initiate repair. Once the cell has repaired damaged DNA, the chromatin structure of the repaired DNA must be reassembled for the cell to return to its normal cell cycle (Hu et al., 2001). Failure to repair DSBs can result in cell death (apoptosis) or in the propagation of mutations that can lead to cancer. Even a single DSB 5 can lead to apoptosis, directly inactivate key genes, chromosomal translocations, or generate unstable chromosomal abnormalities, making the repair response very important (Bradbury and Jackson, 2003). These pathways are highly conserved from budding yeast (Saccharomyces cerevisiae) to humans, which is why significant findings in yeast may be extrapolated to vertebrates (Finn, et al., 2012). The DNA damage checkpoint is central to the DDR. When activated by damage, the cell cycle arrests to allow for proper repair (Finn, et al., 2012). In yeast, cell cycle arrest occurs at the G2/M checkpoint prior to anaphase and damaged DNA must be repaired before the cell can enter mitosis (Kim and Haber, 2009, Harrison and Haber, 2006). Although the DDR is highly conserved from yeast to humans, mammalian cells mainly arrest the cell cycle at the G1/S checkpoint, which is before DNA is replicated (Price and D’Andrea, 2013). The proteins involved in turning on, maintaining, and turning off the checkpoint are named checkpoint proteins. Checkpoint proteins were originally identified from experiments in which their loss of function caused defects in cell cycle progression or cell cycle arrest in response to exposure to DNA damaging agents (Branzei and Foiani, 2005). The checkpoint response acts as a signal transduction cascade in which sensor proteins first detect specific structures or lesions in the DNA. Then, protein kinases activate the transducers, transmit, and perhaps also amplify the checkpoint signal to the receivers. The receivers, which are essentially effectors, are downstream targets of the initial checkpoint. This includes the DNA repair apparatus and the cell cycle machinery responsible for modulating cell cycle progression in response to DNA damage (Branzei and Foiani, 2005). These processes are accompanied by alterations in chromatin structure by various kinases, histones, histone chaperones, 6 nucleosomes, and others (Tsabar and Haber, 2013). Eukaryotes respond to DNA damage by repairing through two major pathways called homologous recombination (HR) or non-homologous end-joining (NHEJ) (Bradbury and Jackson, 2003). In HR, after suffering a DSB, the DNA ends are processed in a 5’ to 3’ direction in a process termed resection, producing RPA bound 3’ single- stranded tails. In gene conversion (GC), one type of HR, the resected DNA ends recognize and pair with homologous sequences from a sister chromatid, an allelic locus, or at an ectopic location in the genome termed “donor” (Mehta and Haber, 2014). A filament of the Rad51 recombinase that forms on the resected ends facilitates the search for homology and pairing with homologous sequences. Binding of Rad51 is facilitated by Rad54 and Rad52, which are recruited to the DNA ends. Rad51 then mediates the search for homologous sequences and then once it is found, Rad51 catalyzes the reaction in which the damaged strand invades the donor. DNA polymerase extends the DNA from the 3’ terminus of the damaged molecule and the ends are ligated by DNA ligase I (Jackson, 2001). Another form of HR is single-strand annealing (SSA), which results in the formation of a deletion between homologous sequences flanking a DSB. Rad51 is not required for this process because the homologous sequence is on the same chromosome, which is why this is a deleterious form of HR and is error prone (Scully and Xie, 2013). Unlike HR, NHEJ requires little to no homology when ligating broken ends and normally results in small deletions or insertions. NHEJ can occur throughout the cell cycle, specifically in G1 when HR 5’ to 3’ resection of DSB ends is blocked (Mehta and Haber, 2014). During NHEJ, there is usually limited nuclease digestion followed by ligation of the broken ends. This may result in loss of some genetic material, but it is the 7 predominant pathway for DSB repair in mammals because it is efficient in removing potentially lethal DSBs (Bradbury and Jackson, 2003). In this paper, we focus on HR because the yeast models used in this project repair DSBs via an ectopic GC event or a SSA event. Response to DNA damage and activating the checkpoint in eukaryotic cells depends greatly on members of the phosphatidylinositol 3’ kinase-like kinase (PI3KK) family (Harrison and Haber, 2006). Mammalian cells contain two important DNA damage checkpoint PI3KK’s, Ataxia telangiectasia mutated protein (ATM) and ataxia telangiectasia and Rad3-related protein (ATR). The Saccharomyces cerevisiae orthologs to ATM and ATR are Tel1 and Mec1, respectively. In humans, ATM is the protein that is mutated in ataxia telangiectasia, which is a radiosensitivity and genome instability disorder categorized by progressive cerebellar degeneration and increased cancer incidence (Bradbury and Jackson, 2003).
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