isolating-interaction-nullimpaired-mutants-using-yeast-two-hybrid

Isolating Interaction-Null&#47;Impaired Mutants Using the Yeast Two-Hybrid Assay

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the ...

Kochi University of Technology

Initiation of chromosomal DNA replication in eukaryotes involves two steps: licensing and firing. In licensing, a core component of the replicative helicase, the Mcm2-7 complex, is loaded onto replication origins as an inactive double hexamer, which is activated in the firing step by firing factors. A reaction intermediate called the pre-initiation complex (pre-IC) has been proposed to assemble transiently during firing, but the existence of the pre-IC has not yet been confirmed. Here, we show, by systematic chromatin immunoprecipitation, that a distinct intermediate that fits the definition of the pre-IC assembles during firing in the budding yeast  Pre-IC assembly is observed in the absence of Mcm10, one of the firing factors, and is mutually dependent on all the firing factors whose association to replication origins is triggered by cyclin-dependent kinase. In the pre-IC, the Mcm2-7 double hexamer is separated into single hexamers, as in the active helicase. Our data indicate that pre-IC assembly functions as an all-or-nothing molecular switch that splits the Mcm2-7 double hexamer.

Pre-initiation complex assembly functions as a molecular switch that splits the Mcm2-7 double hexamer.

Post-replicative correction of replication errors by the mismatch repair (MMR) system is critical for suppression of mutations. Although the MMR system may need to handle nucleosomes at the site of chromatin replication, how MMR occurs in the chromatin environment remains unclear. Here, we show that nucleosomes are excluded from a >1-kb region surrounding a mismatched base pair in  egg extracts. The exclusion was dependent on the Msh2-Msh6 mismatch recognition complex but not the Mlh1-containing MutL homologs and counteracts both the HIRA- and CAF-1 (chromatin assembly factor 1)-mediated chromatin assembly pathways. We further found that the Smarcad1 chromatin remodeling ATPase is recruited to mismatch-carrying DNA in an Msh2-dependent but Mlh1-independent manner to assist nucleosome exclusion and that Smarcad1 facilitates the repair of mismatches when nucleosomes are preassembled on DNA. In budding yeast, deletion of , the homolog of Smarcad1, showed a synergistic increase of spontaneous mutations in combination with  or  deletion but no significant increase with  deletion. Genetic analyses also suggested that the function of Fun30 in MMR is to counteract CAF-1. Our study uncovers that the eukaryotic MMR system has an ability to exclude local nucleosomes and identifies Smarcad1/Fun30 as an accessory factor for the MMR reaction.

Nucleosomes around a mismatched base pair are excluded via an Msh2-dependent reaction with the aid of SNF2 family ATPase Smarcad1.

Protein-protein interactions are one of the most basic and critical processes underlying biological functions. Thus, identification of the interacting proteins of a protein of interest and further elucidation of the roles of the interactions is critical for understanding the related biological processes. The yeast two-hybrid (Y2H) method is a popular approach for identifying protein-protein interactions. Once interacting proteins are identified, a comparison of the phenotypes of mutants lacking the specific protein-protein interaction with those of the wild-type strain is a powerful tool for uncovering the former interactions' biological significance. However, isolation of such interaction-defective mutants is often laborious. Here, I describe a novel and efficient approach for isolating such mutants that uses the Y2H technique with a modified Y2H vector, and provide an example of how this approach can be used to screen interaction-null/impaired mutants. Because the strategy is simple and the modification of a pre-existing Y2H vector is sufficient for the screening purpose, the same strategy can be applied to any existing two-hybrid system.

An efficient method for the isolation of interaction-null/impaired mutants using the yeast two-hybrid technique.

Conditional mutants, such as temperature-sensitive (ts) mutants, are effective tools for the analysis of essential genes. However, such mutants are frequently leaky. To overcome this problem, it is helpful to isolate a "tight" conditional mutant of a gene of interest, e.g., by using ubiquitin-mediated protein degradation to eliminate the gene product. One such strategy is the auxin-inducible degron (AID) system, which is easy to use because the simple addition of auxin can induce the degradation of a target protein. Sometimes, however, elimination of the target protein is not sufficient, and an AID mutant exhibits a "leaky" phenotype. To address this issue, the improved AID (iAID) system was developed. In this approach, transcriptional repression by the "Tet-OFF" promoter is combined with proteolytic elimination of the target protein by the AID system, yielding a much tighter mutant. Because simple addition of tetracycline is sufficient to repress the Tet-OFF promoter, the combination of Tet-OFF and AID maintains the ease of use of the original AID system. In this manuscript, we describe how to construct and use iAID mutants in the budding yeast Saccharomyces cerevisiae.

Construction of Tight Conditional Mutants Using the Improved Auxin-Inducible Degron (iAID) Method in the Budding Yeast Saccharomyces cerevisiae.

DNA replication in eukaryotes is a multi-step process that consists of three main reactions: helicase loading (licensing), helicase activation (firing), and nascent DNA synthesis (elongation). Although the contributions of some chromatin regulatory factors in the licensing and elongation reaction have been determined, their functions in the firing reaction remain elusive. In the budding yeast Saccharomyces cerevisiae, Sld3, Sld7, and Cdc45 (3-7-45) are rate-limiting in the firing reaction and simultaneous overexpression of 3-7-45 causes untimely activation of late and dormant replication origins. Here, we found that 3-7-45 overexpression not only activated dormant origins in the silenced locus, HMLα, but also exerted an anti-silencing effect at this locus. For these, interaction between Sld3 and Esa1, a conserved histone acetyltransferase, was responsible. Moreover, the Sld3-Esa1 interaction was required for the untimely activation of late origins. These results reveal the Sld3-Esa1 interaction as a novel level of regulation in the firing reaction.

Interaction of replication factor Sld3 and histone acetyl transferase Esa1 alleviates gene silencing and promotes the activation of late and dormant replication origins.

Controlling the activity of the heterohexameric Mcm2-7 replicative helicase is crucial for regulation of replication origin activity in eukaryotes. Because bidirectional replication forks are generated from every replication origin, when origins are licensed for replication in the first step of DNA replication, two inactive Mcm2-7 heterohexiameric complexes are loaded around double stranded DNA as a head-to-head double hexamer. The helicases are subsequently activated via a 'firing' reaction, in which the Mcm2-7 double hexamer is converted into two active helicase units, the CMG complex, by firing factors. Dimerization of firing factors may contribute to this process by allowing simultaneous activation of two sets of helicases and thus efficient assembly of bidirectional replication forks. An example of this is dimerization of the firing factor Sld3/Treslin/Ticrr via its binding partner, Sld7/MTBP. In organisms in which no Sld7 ortholog has been identified, such as the fission yeast , Sld3 itself has a dimerization domain, and it has been suggested that this self-interaction is crucial for the firing reaction in this organism. Dimerization induces a conformational change in Sdl3 that appears to be critical for the firing reaction. Moreover, Mcm10 also seems to be regulated by self-interaction in yeasts. Although it is not yet clear to what extent dimerization of firing factors contributes to the firing reaction in eukaryotes, we discuss the possible roles of firing factor dimerization in simultaneous helicase activation.

Dimerization of Firing Factors for Replication Origin Activation in Eukaryotes: A Crucial Process for Simultaneous Assembly of Bidirectional Replication Forks?

Seiji Tanaka

Graduate School of Engineering,
School of Engineering Science

Kochi University of Technology

Isolating Interaction-Null/Impaired Mutants Using the Yeast Two-Hybrid Assay

Seiji Tanaka

Graduate School of Engineering,School of Engineering Science

Kochi University of Technology

Isolating Interaction-Null/Impaired Mutants Using the Yeast Two-Hybrid Assay

Graduate School of Engineering,
School of Engineering Science