Sirtuins Hst3p và Hst4p điều hòa ổn định bộ gen trong Saccharomyces cerevisiae

Regulation of genomic stability by S. cerevisiae sirtuins Hst3p and Hst4p by Ivana Celic A dissertation submitted to The Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland June, 2006 UMI Number: 3243300 UMI Microform 3243300 Copyright 2007 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.

ProQuest Information and Learning Company 300 North Zeeb Road P. Box 1346 Ann Arbor, MI 48106-1346 Abstract The Sir2 proteins, also known as sirtuins, represent a large and highly conserved family of NAD+-dependent protein deacetylases that control various fundamental biological processes. The baker’s yeast, Saccharomyces cerevisiae, has five members of this family, Sir2p and Hst1-4p, important for regulation of transcriptional silencing and genomic stability. Hst3p and Hst4p are two redundant sirtuins with the major role in maintenance of genomic stability.

They control genomic stability by regulating the level of acetylation of histone H3 lysine 56. This residue, present in the core of the nucleosome surface, is acetylated during the S phase of the cell cycle and contributes to the repair processes active during DNA replication. At the end of the S phase, K56 of histone H3 is deacetylated in a Hst3p- and Hst4p-dependent manner. Failure to deacetylate K56 leads to a growth defect, sensitivity to DNA-damaging agents and chromosome loss in hst3 hst4 cells.

These phenotypes can be suppressed by mutation of K56 into the nonacetylable residue arginine. Failure to deacetylate K56 also leads to activation of the DNA-damage response and renders hst3 hst4 cells sensitive to perturbations in DNA replication, repair and checkpoint function as is evident from numerous synthetic lethality interactions that hst3 hst4 cells display with mutations in the genes that regulate these processes. The growth defect of hst3 hst4 cells can be suppressed by overexpression of RFC1, the large subunit of RFC, a “clamp loader” that loads PCNA, the “sliding clamp” onto DNA during DNA replication. Interestingly, the growth defect of hst3 hst4 cells can also be suppressed by deletion of CTF18 and, somewhat less efficiently, RAD24 and ELG1.

CTF18, RAD24 and ELG1 all encode large subunits of alternative RFCs, each of ii which shares four smaller subunits (Rfc2p-Rfc5p) with Rfc1p. We propose that ongoing cycles of K56 acetylation and deacetylation contribute to the regulation of the functional equilibrium between different RFC complexes in the cell. (Thesis Advisor and Reader) Professor Department of Molecular Biology and Genetics Johns Hopkins University School of Medicine Brendan Cormack, Ph. (Thesis Reader) Associate Professor Department of Molecular Biology and Genetics Johns Hopkins University School of Medicine iii Acknowledgements This has been a very, very long ride and many people have helped me during this time in different ways.

I would like to thank my advisor, Jef Boeke, for giving me opportunity to work in his lab, for his patience and support over all these years. I am grateful to my committee members, Carol Greider, Cynthia Wolberger and Brendan Cormack for words of encouragement and letting me graduate after all. I am very grateful to Alain Verreault and his postdoctoral fellow Hiroshi Masumoto, who originally discovered that Hst3p and Hst4p regulated histone K56 acetylation, for their generosity in sharing results and willingness to collaborate. Also, many thanks go to Wendell Griffith, who has done some great MS analysis of K56 acetylation.

I thank the Boeke lab members for making the Boeke lab nice place to work. I had a great opportunity to overlap with some exceptional graduate students, Eric Bolton, Jeffrey Han and Siew-Loon Ooi, who thought me lot of little tricks of the trade. I thank to my friends, some close, some far away, who made these years more bearable. Special thanks goes to Jeff Han, for his support and many, many moments of happiness he brought in my life.

My deepest gratitude goes to my family, my parents, my sister and my grandmothers for being always there for me no matter what. My parents have sacrificed a lot to put me through school and ultimately to get me here and I will be always indebted for that. My sister and my grandmothers helped as much as they could. I am, also, very grateful to my sister for taking good care of my parents.

It has given me great peace of mind over these years. iv Table of Contents Page Title Page i Abstract ii Acknowledgments iv Table of Contents v List of Figures and Tables viii Chapter 1: Introduction 1 Chapter 2: The sirtuins Hst3p and Hst4p control histone H3 lysine 56 acetylation 23 Introduction 24 Results 26 Hst3 and Hst4 control histone H3 K56 acetylation during the cell cycle 26 The nicotinamide binding pocket of Hst3 is necessary for regulation of histone H3 K56 acetylation 27 The phenotypes of hst3 hst4 mutant cells are caused by high levels of H3 K56 acetylation 30 The histone chaperone Asf1p is needed for H3 K56 acetylation 32 Hst3p can trigger K56Ac deacetylation in mature chromatin 33 Discussion 35 Experimental Procedures 51 v Chapter 3: Cells lacking Hst3p and Hst4p activate the DNA damage response due to the presence of chronic DNA damage and display genetic interaction with genes involved in DNA metabolism 63 Introduction 64 Results 66 hst3 hst4 cells slow down cell-cycle progression in response to replication inhibition and DNA damage 66 hst3 hst4 cells activate DNA damage checkpoint in the absence of exogenous DNA damage 67 hst3 hst4 mutant depends on replication checkpoint for viability 69 Increased H2A serine 129 phosphorylation in hst3 hst4 cells 71 hst3 hst4 require subset of DNA repair proteins for viability 72 Overexpression of Rfc1p, large subunit of DNA clamp loader, suppresses phenotypes of hst3 hst4 cells 73 Discussion 76 Experimental procedures 96 References 107 Appendices 130 Appendix A: A phylogenetically conserved NAD+-dependent protein deacetylase activity in the Sir2 protein family 131 Appendix B: Chemistry of gene silencing: the mechanism of NAD+-dependent deacetylation reactions 138 Appendix C: Telomeric and rDNA silencing in Saccharomyces cerevisiae vi are dependent on a nuclear NAD+ salvage pathway 147 Appendix D: SIRT3, a human SIR2 homologue, is an NAD+-dependent deacetylase localized to mitochondria 161 Appendix E: Structure of a Sir2 enzyme bound to an acetylated p53 peptide 168 Appendix F: Sir2-dependent activation of acetyl-CoA synthetase by deacetylation of active lysine 182 Curriculum Vitae 186 vii List of Figures Page Figure 1. Phylogenetic tree of sirtuins. Proposed catalytic mechanism of Sir2 proteins.

Schematic alignment of S. Alignment of Hst3p, Hst4p and selected sirtuins. Hst3p and Hst4p control histone H3 K56 acetylation during the cell cycle. Effect of nicotinamide on the acetylation of histone H3 K56.

Mutation in invariant residues of Hst3p affects K56 acetylation level and function of Hst3p in vivo. The phenotypes of hst3 hst4 double mutants are substantially suppressed by mutation of histone H3 K56 into arginine. The hht1-K56R mutation suppresses the mitotic chromosome loss phenotype of hst3 hst4 mutant cells. The Asf1p histone chaperone is needed for H3 K56Ac.

Effects of rapid inactivation of Hst3p on H3 K56 deacetylation. Induction of Hst3p synthesis leads to H3 K56 deacetylation in mature chromatin. Abnormal distribution of histone H3 K56 acetylation with respect to replication forks is likely responsible for the DNA damage sensitivity of hst3 hst4 mutant cells. Drugs sensitivities of hst3 hst4 mutants.

Like wild-type cells, hst3 hst4 cells slow down DNA replication when exposed to MMS. Like wild-type cells, hst3 hst4 cells do not elongate their mitotic spindles when exposed to HU. Induction of RNR3 and HUG1 in hst3 hst4 cells. Hyperphosphorylation of Rad53p in hst3 hst4 cells.

hst3 hst4 cells required functional DNA replication checkpoint for viability. hst3 hst4 cells display increased level of histone H2A Ser128 phosphorylation. Synthetic lethality analysis with hst3 hst4 and suppression with H3 K56R. Overexpression of RFC1 suppresses growth defect, Ts phenotype and HU sensitivity of hst3 hst4 cells.

Overexpression of Rfc1p does not affect histone H3 K56 acetylation level in hst3 hst4 cells. Suppression of hst3 hst4 growth defect and Ts phenotype by inactivation of RAD24, but not RAD9. Suppression of hst3 hst4 growth defect and Ts phenotype by inactivation of alternative RFC complexes. List of the genes whose expression was changed in hst3 hst4 cells.

Synthetic lethal analysis with hst3 hst4. 95 ix Chapter 1 Introduction 1 Protein acetylation and deacetylation have emerged over the past decade or two as important posttranslational mechanisms regulating various aspects of cellular biology. This regulation is carried out through the action of protein acetylases, which modify the ε-NH2 of lysine residues and their counterparts, the protein deacetylases, which remove acetyl groups (Kouzarides, 2000; Kurdistani and Grunstein, 2003). The Sir2 family of proteins is an integral part of this regulatory network.

Sir2 proteins deacetylate lysine residues in histones and other proteins in a very unique enzymatic reaction that requires nicotinamide adenine dinucleotide (NAD+) (Imai et al., 2000; Landry et al., 2000b; Smith et al. The deacetylation reaction catalyzed by Sir2 proteins is absolutely dependent on NAD+. This is what differentiates Sir2 proteins as class III deacetylases, distinct from class I and class II deacetylases that don’t require NAD+ and deacetylate proteins through a more simple hydrolysis mechanism (Finnin et al. Presently, it is not known why sirtuins deacetylate proteins in such an energetically costly mechanism, but it has been suggested that sirtuins may represent a cellular NAD+ sensor linking metabolism to other aspects of cellular physiology, like genome stability, aging, apoptosis and differentiation.

Sir2 proteins or sirtuins form a large and highly conserved protein family. All three kingdoms of life, archea, bacteria and eukaryotes, are represented within the sirtuin family (Figure 1. Some organisms have only one sirtuin, while others have multiple paralogs, for example yeast with five (Brachmann et al., 1995) and mammals with 7 paralogs (Frye, 1999; Frye, 2000). All sirtuins share a conserved catalytic core domain necessary for the NAD+-dependent deacetylation reaction.

Sir2 proteins differ significantly in their N- and C-terminal extensions, which range from non- 2 existent, for example in archeal sirtuins, to very long ones, like in S. These extensions mediate member-specific functions like protein localization, as in the case of human SirT3 which is targeted to mitochondria by its N-terminal extension (Onyango et al., 2002; Schwer et al., 2002), formation of multiprotein complexes with specialized function, as in the case of S.cerevisiae Sir2p, which exists in the form of at least two functionally different protein complexes within a cell (Ghidelli et al., 2001) or they have an autoregulatory function, as in the case of S. cerevisiae Hst2p in which N and C-terminal domain have inhibitory activity by binding to the catalytic core intermolecularly and intramolecularly, respectively (Zhao et al. Phylogenetic tree of sirtuins (published with permission by William Hawse and Cynthia Wolberger).

4 Sirtuins catalyze NAD+-dependent deacetylation of lysine residues in proteins (Imai et al., 2000; Landry et al., 2000b; Smith et al. During this reaction, the glycosidic bond between the C1’ atom of ribose and nicotinamide moiety in NAD+ is cleaved to generate, in addition to the deacetylated lysine residue, free nicotinamide and acetyl-ADP ribose (APDR) (Sauve et al., 2001; Tanner et al., 2000; Tanny and Moazed, 2001). NMR and mass-spectrophotometric analysis of the Sir2 deacetylation product revealed that actual product is 2’- and 3’-acetyl-ADP ribose (Sauve et al. 2’-acetyl-ADP ribose is the first product of the reaction followed by non-enzymatic conversion to 3’- acetyl-ADP ribose to a final equilibrium ratio of 47:67 for the 2’ and 3’ stereoisomers.

Labeling experiments with H218O, MS and NMR analysis led Sauve et al. (Sauve et al., 2001) to propose the mechanism (Figure 1.2), which involves the nucleophilic attack by the carbonyl oxygen of the acetyl-lysine on the C1’ carbon of the nicotinamide ribose (N- ribose) in NAD+. This step involves formation of the highly reactive riboxacarbenium ion that captures the acyl oxygen of acetyl-lysine to generate a 1’-O-alkyl-amidate, followed by the attack of the N-ribose 2’hydroxyl (2’OH) to form a 1’, 2’-acyloxonium structure, which is the precursor for 2’-acetyl-ADP ribose formation.