Transcriptional activation of budding yeast DDI2/3 through chemical modifications of Fzf1.

DDI2 and DDI3 (DDI2/3) are two identical genes in Saccharomyces cerevisiae encoding cyanamide (CY) hydratase. They are not only highly induced by CY, but also by a DNA-damaging agent methyl methanesulfonate (MMS), and the regulatory mechanism is unknown. In this study, we performed a modified genome-wide genetic synthetic array screen and identified Fzf1 as a zinc-finger transcriptional activator required for CY/MMS-induced DDI2/3 expression. Fzf1 binds to a DDI2/3 promoter consensus sequence CS2 in vivo and in vitro, and this interaction was enhanced in response to the CY treatment. Indeed, experimental over production of Fzf1 alone was sufficient to induce DDI2/3 expression; however, CY and MMS treatments did not cause the accumulation or apparent alteration in migration of cellular Fzf1. To test a hypothesis that Fzf1 is activated by covalent modification of CY and MMS, we performed mass spectrometry of CY/MMS-treated Fzf1 and detected a few modified lysine residues. Amino acid substitutions of these residues revealed that Fzf1-K70A completely abolished MMS-induced and reduced CY-induced DDI2/3 expression, indicating that the Fzf1-K70 methylation activates Fzf1. This study collectively reveals a novel regulatory mechanism by which Fzf1 is activated by chemical modifications and in turn induces the expression of its target genes for detoxification.

Structure of Ddi2, a highly inducible detoxifying metalloenzyme from Saccharomyces cerevisiae.

Cyanamide (H2N-CN) is used to break bud dormancy in woody plants and to deter alcohol use in humans. The biological effects of cyanamide in both these cases require the enzyme catalase. We previously demonstrated that Saccharomyces cerevisiae exposed to cyanamide resulted in strong induction of DDI2 gene expression. Ddi2 enzymatically hydrates cyanamide to urea and belongs to the family of HD-domain metalloenzymes (named after conserved active-site metal-binding His and Asp residues). Here, we report the X-ray structure of yeast Ddi2 to 2.6 Å resolution, revealing that Ddi2 is a dimeric zinc metalloenzyme. We also confirm that Ddi2 shares structural similarity with other known HD-domain proteins. HD residues His-55, His-88, and Asp-89 coordinate the active-site zinc, and the fourth zinc ligand is a water/hydroxide molecule. Other HD domain enzymes have a second aspartate metal ligand, but in Ddi2 this residue (Thr-157) does not interact with the zinc ion. Several Ddi2 active-site point mutations exhibited reduced catalytic activity. We kinetically and structurally characterized H137N and T157V mutants of Ddi2. A cyanamide soak of the Ddi2-T157V enzyme revealed cyanamide bound directly to the Zn2+ ion, having displaced the zinc-bound water molecule. The mode of cyanamide binding to Ddi2 resembles cyanamide binding to the active-site zinc of carbonic anhydrase, a known cyanamide hydratase. Finally, we observed that the sensitivity of ddi2Δ ddi3Δ to cyanamide was not rescued by plasmids harboring ddi2-H137N or ddi2-TI57V variants, demonstrating that yeast cells require a functioning cyanamide hydratase to overcome cyanamide-induced growth defects.

Yeast chromatin remodeling complexes and their roles in transcription.

The nucleosome is a small unit of chromatin, which is dynamic in eukaryotes. Chromatin conformation and post-translational modifications affect nucleosome dynamics under certain conditions, playing an important role in the epigenetic regulation of transcription, replication and reprogramming. The Snf2 remodeling family is one of the crucial remodeling complexes that tightly regulate chromatin structure and affect nucleosome dynamics. This family alters nucleosome positioning, exchanges histone variants, and assembles and disassembles nucleosomes at certain locations. Moreover, the Snf2 family, in conjunction with other co-factors, regulates gene expression in Saccharomyces cerevisiae. Here we first review recent findings on the Snf2 family remodeling complexes and then use some examples to illustrate the cooperation between different members of Snf2 family, and the cooperation between Snf2 family and other co-factors in gene regulation especially during transcription initiation.

Involvement of budding yeast Rad5 in translesion DNA synthesis through physical interaction with Rev1.

DNA damage tolerance (DDT) is responsible for genomic stability and cell viability by bypassing the replication block. In Saccharomyces cerevisiae DDT employs two parallel branch pathways to bypass the DNA lesion, namely translesion DNA synthesis (TLS) and error-free lesion bypass, which are mediated by sequential modifications of PCNA. Rad5 has been placed in the error-free branch of DDT because it contains an E3 ligase domain required for PCNA polyubiquitination. Rad5 is a multi-functional protein and may also play a role in TLS, since it interacts with the TLS polymerase Rev1. In this study we mapped the Rev1-interaction domain in Rad5 to the amino acid resolution and demonstrated that Rad5 is indeed involved in TLS possibly through recruitment of Rev1. Genetic analyses show that the dual functions of Rad5 can be separated and reconstituted. Crystal structure analysis of the Rad5-Rev1 interaction reveals a consensus RFF motif in the Rad5 N-terminus that binds to a hydrophobic pocket within the C-terminal domain of Rev1 that is highly conserved in eukaryotes. This study indicates that Rad5 plays a critical role in pathway choice between TLS and error-free DDT.

Active Site Gate Dynamics Modulate the Catalytic Activity of the Ubiquitination Enzyme E2-25K.

The ubiquitin proteasome system (UPS) signals for degradation of proteins through attachment of K48-linked polyubiquitin chains, or alterations in protein-protein recognition through attachment of K63-linked chains. Target proteins are ubiquitinated in three sequential chemical steps by a three-component enzyme system. Ubiquitination, or E2 enzymes, catalyze the central step by facilitating reaction of a target protein lysine with the C-terminus of Ub that is attached to the active site cysteine of the E2 through a thioester bond. E2 reactivity is modulated by dynamics of an active site gate, whose central residue packs against the active site cysteine in a closed conformation. Interestingly, for the E2 Ubc13, which specifically catalyzes K63-linked ubiquitination, the central gate residue adopts an open conformation. We set out to determine if active site gate dynamics play a role in catalysis for E2-25K, which adopts the canonical, closed gate conformation, and which selectively synthesizes K48-linked ubiquitin chains. Gate dynamics were characterized using mutagenesis of key residues, combined with enzyme kinetics measurements, and main chain NMR relaxation. The experimental data were interpreted with all atom MD simulations. The data indicate that active site gate opening and closing rates for E2-25K are precisely balanced.

Utilization of a Strongly Inducible DDI2 Promoter to Control Gene Expression in Saccharomyces cerevisiae.

Regulating target gene expression is a common method in yeast research. In Saccharomyces cerevisiae, there are several widely used regulated expression systems, such as the GAL and Tet-off systems. However, all current expression systems possess some intrinsic deficiencies. We have previously reported that the DDI2 gene can be induced to very high levels upon cyanamide or methyl methanesulfonate treatment. Here we report the construction of gene expression systems based on the DDI2 promoter in both single- and multi-copy plasmids. Using GFP as a reporter gene, it was demonstrated that the target gene expression could be increased by up to 2,000-fold at the transcriptional level by utilizing the above systems. In addition, a DDI2-based construct was created for promoter shuffling in the budding yeast genome to control endogenous gene expression. Overall, this study offers a set of convenient and highly efficient experimental tools to control target gene expression in budding yeast.

Error-free DNA-damage tolerance in Saccharomyces cerevisiae.

DNA-damage tolerance (DDT) is an important mechanism for living cells to bypass replication blocks on the template strand. In Saccharomyces cerevisiae, DDT is mediated by the RAD6 epistasis group of genes, consisting of two parallel pathways: error-prone translesion DNA synthesis (TLS), and error-free lesion bypass. The two pathways are activated by sequential ubiquitination of PCNA on the Lys164 residue. When a replication fork is stalled at a lesion, PCNA is first monoubiquitinated by Rad6-Rad18, which leads to the TLS pathway. The subsequent ubiquitination by the Mms2-Ubc13-Rad5 complex on the monoubiquitinated PCNA is to form a Lys63-linked polyubiquitin chain that promotes error-free lesion bypass. While the TLS pathway has been extensively characterized, the molecular events leading to error-free lesion bypass by polyubiquitinated PCNA are largely obscure. Furthermore, PCNA can also be sumoylated at the same Lys164 residue, which helps to recruit Srs2, a helicase and anti-recombinase. This review summarizes recent advances in our understanding of error-free DDT and its interplay with Srs2 and homologous recombination.