The Cac1 subunit of histone chaperone CAF-1 organizes CAF-1-H3/H4 architecture and tetramerizes histones.

The histone chaperone Chromatin Assembly Factor 1 (CAF-1) deposits tetrameric (H3/H4)2 histones onto newly-synthesized DNA during DNA replication. To understand the mechanism of the tri-subunit CAF-1 complex in this process, we investigated the protein-protein interactions within the CAF-1-H3/H4 architecture using biophysical and biochemical approaches. Hydrogen/deuterium exchange and chemical cross-linking coupled to mass spectrometry reveal interactions that are essential for CAF-1 function in budding yeast, and importantly indicate that the Cac1 subunit functions as a scaffold within the CAF-1-H3/H4 complex. Cac1 alone not only binds H3/H4 with high affinity, but also promotes histone tetramerization independent of the other subunits. Moreover, we identify a minimal region in the C-terminus of Cac1, including the structured winged helix domain and glutamate/aspartate-rich domain, which is sufficient to induce (H3/H4)2 tetramerization. These findings reveal a key role of Cac1 in histone tetramerization, providing a new model for CAF-1-H3/H4 architecture and function during eukaryotic replication

Rapid Cdc13 turnover and telomere length homeostasis are controlled by Cdk1-mediated phosphorylation of Cdc13

Budding yeast telomerase is mainly activated by Tel1/Mec1 (yeast ATM/ATR) on Cdc13 from late S to G2 phase of the cell cycle. Here, we demonstrated that the telomerase-recruitment domain of Cdc13 is also phosphorylated by Cdk1 at the same cell cycle stage as the Tel1/Mec1-dependent regulation. Phosphor-specific gel analysis demonstrated that Cdk1 phosphorylates residues 308 and 336 of Cdc13. The residue T308 of Cdc13 is critical for efficient Mec1-mediated S306 phosphorylation in vitro. Phenotypic analysis in vivo revealed that the mutations in the Cdc13 S/TP motifs phosphorylated by Cdk1 caused cell cycle delay and telomere shortening and these phenotypes could be partially restored by the replacement with a negative charge residue. In the absence of Ku or Tel1, Cdk1-mediated phosphorylation of Cdc13 showed no effect on telomere length maintenance. Moreover, this Cdk1-mediated phosphorylation was required to promote the regular turnover of Cdc13. Together these results demonstrate that Cdk1 phosphorylates the telomerase recruitment domain of Cdc13, thereby preserves optimal function and expression level of Cdc13 for precise telomere replication and cell cycle progression

Rsp5 and Pph3 switch off the signals of phosphorylation of Cdc13

在酵母菌 Saccharomyces cerevisiae中，端粒體蛋白質Cdc13 的功能在於藉由延攬端粒酶延長端立體以及保護染色體末端結構。 在整個細胞分裂的過程中，蛋白質Cdc13 的表現量以及被磷酸化的程度從G1時期往G2/M 時期達到最高峰，而在繼續往G1時期會逐漸減少。在先前的研究指出，蛋白質Rsp5參與裂解單股DNA結合蛋白質Rpa1。而且，Cdc13和Rpa1的結構相類似。因此推測Cdc13的裂解可能經由蛋白質Rsp5所調控。此外，蛋白質Cdc13和Rad53都被DNA損傷反應蛋白激酶Mec1所磷酸化。 而且，Rad53的去磷酸化經由蛋白質Ptc2/Ptc3和Pph3所完成。在本篇研究中，我發現蛋白質Pph3在生體內(in vivo)調控Cdc13的去磷酸化。總結，這兩項發現可推測端粒酶延長端粒體長度的功能可能經由Rsp5 和Pph3對於Cdc13的表現量降低及去磷酸化程度來終止。In Saccharomyces cerevisiae, the telomere binding protein Cdc13 activates telomere replication by recruiting telomerase, and also performs an essential function in chromosome end protection. Through cell cycle progression, the protein expression level and phosphorylated status of Cdc13 is highest in G2/M phase and decreases in G1 phase. In previous studies, Rsp5 is involved in the degradation of the ssDNA binding protein, Rpa1. Since Cdc13 and Rpa1 share a similar domain organization, I speculate that Cdc13p level is regulated through proteasome-dependent degradation. I found that Rsp5 is a specific ubiquitin E3 ligase of Cdc13. Moreover, Cdc13 and Rad53 are both phosphorylated by the DNA damage-responsive protein kinase, Mec1. Rad53 dephosphorylation has been shown to be dependent on the presence of the PP2C-like phosphatase Ptc2/Ptc3 and PPh3. I found that Pph3 mediates Cdc13 dephosphorylation from G2/M to G1 phase. Take together; these findings suggest that both proteins degradation and dephosphorylation promote telomerase inactivation at the M phase. When cells need to stop telomere replication and enter the next stage of cell cycle.口試委員審定書. ii 文摘要. iii bstract. iv ontents. v igure Contents. vi hapter 1: Introduction of telomere . 1 hapter 2: Materials and Methods . 5 hapter 3: Results . 7 .1 Cdc13p degradation is proteasome-dependent 7 .2 Cdc13 protein level at steady state is increased in rsp5-1 strain. 7 .3 Rsp5p interact with Cdc13p in vivo and in vitro 8 .4 The stability of Cdc13-T308A is increased 9 .5 Pph3 mediates the dephosphorylation of Cdc13. 10 .6 Pph3 does not dephosphorylate the phosphorylation by Cdk1. 11 hapter 4: Discussion. 12 igures. 14 eferences. 2

PP2A and Aurora differentially modify Cdc13 to promote telomerase release from telomeres at G2/M phase

In yeast, the initiation of telomere replication at the late S phase involves in combined actions of kinases on Cdc13, the telomere binding protein. Cdc13 recruits telomerase to telomeres through its interaction with Est1, a component of telomerase. However, how cells terminate the function of telomerase at G2/M is still elusive. Here we show that the protein phosphatase 2A (PP2A) subunit Pph22 and the yeast Aurora kinase homologue Ipl1 coordinately inhibit telomerase at G2/M by dephosphorylating and phosphorylating the telomerase recruitment domain of Cdc13, respectively. While Pph22 removes Tel1/Mec1-mediated Cdc13 phosphorylation to reduce Cdc13-Est1 interaction, Ipl1-dependent Cdc13 phosphorylation elicits dissociation of Est1-TLC1, the template RNA component of telomerase. Failure of these regulations prevents telomerase from departing telomeres, causing perturbed telomere lengthening and prolonged M phase. Together our results demonstrate that differential and additive actions of PP2A and Aurora on Cdc13 limit telomerase action by removing active telomerase from telomeres at G2/M phase

A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9

Until recently, the favored method for making directed modifications to the budding yeast genome involved the introduction of a DNA template carrying the desired genetic changes along with a selectable marker, flanked by homology arms. This approach both limited the ability to make changes within genes due to disruption by the introduced selectable marker and prevented the use of that selectable marker for subsequent genomic manipulations. Following the discovery of CRISPR-Cas9-mediated genome editing, protocols were developed for modifying any DNA region of interest in a similar single transformation step without the need for a permanent selectable marker. This approach involves the generation of a DNA double-strand break (DSB) at the desired genomic location by the Cas9 nuclease, expressed on a plasmid which also expresses the guide RNA (gRNA) sequence directing the location of the DSB. The DSB is subsequently repaired via homologous recombination using a PCR-derived DNA repair template. Here, we describe in detail an improved method for incorporation of the gRNA-encoding DNA sequences into the Cas9 expression plasmid. Using Golden Gate cloning, annealed oligonucleotides bearing unique single-strand DNA overhangs are ligated into directional restriction enzyme sites. We describe the use of this CRISPR-Cas9 genome editing protocol to introduce multiple types of directed genetic changes into the yeast genome

phosphorylation of Cdc13

Cdk1-mediate

The histone chaperone ASF1 regulates the activation of ATM and DNA-PKcs in response to DNA double-strand breaks

<p>The Ataxia-telangiectasia mutated (ATM) kinase and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are activated by DNA double-strand breaks (DSBs). These DSBs occur in the context of chromatin but how chromatin influences the activation of these kinases is not known. Here we show that loss of the replication-dependent chromatin assembly factors ASF1A/B or CAF-1 compromises ATM activation, while augmenting DNA-PKcs activation, in response to DNA DSBs. Cells deficient in ASF1A/B or CAF-1 exhibit reduced histone H4 lysine 16 acetylation (H4K16ac), a histone mark known to promote ATM activation. ASF1A interacts with the histone acetyl transferase, hMOF that mediates H4K16ac. ASF1A depletion leads to increased recruitment of DNA-PKcs to DSBs. We propose normal chromatin assembly and H4K16ac during DNA replication is required to regulate ATM and DNA-PKcs activity in response to the subsequent induction of DNA DSBs.</p

Yeast Cip1 is activated by environmental stress to inhibit Cdk1–G1 cyclins via Mcm1 and Msn2/4

Upon environmental changes, proliferating cells delay cell cycle to prevent further damage accumulation. Yeast Cip1 is a Cdk1 and Cln2-associated protein. However, the function and regulation of Cip1 are still poorly understood. Here we report that Cip1 expression is co-regulated by the cell-cycle-mediated factor Mcm1 and the stress-mediated factors Msn2/4. Overexpression of Cip1 arrests cell cycle through inhibition of Cdk1-G1 cyclin complexes at G1 stage and the stress-activated protein kinase-dependent Cip1 T65, T69, and T73 phosphorylation may strengthen the Cip1and Cdk1-G1 cyclin interaction. Cip1 accumulation mainly targets Cdk1-Cln3 complex to prevent Whi5 phosphorylation and inhibit early G1 progression. Under osmotic stress, Cip1 expression triggers transient G1 delay which plays a functionally redundant role with another hyperosmolar activated CKI, Sic1. These findings indicate that Cip1 functions similarly to mammalian p21 as a stress-induced CDK inhibitor to decelerate cell cycle through G1 cyclins to cope with environmental stresses.A G1 cell cycle regulatory kinase Cip1 has been identified in budding yeast but how this is regulated is unclear. Here the authors identify cell cycle (Mcm1) and stress-mediated (Msn 2/4) transcription factors as regulating Cip1, causing stress induced CDK inhibition and delay in cell cycle progression