Jachen A. Solinger

Rad54, a Swi2/Snf2-like Recombinational Repair Protein, Disassembles Rad51:dsDNA Filaments

Rad54 protein is a member of the Swi2/Snf2-like family of DNA-dependent/stimulated ATPases that dissociate and remodel protein complexes on dsDNA. Rad54 functions in the recombinational DNA repair (RAD52) pathway. Here we show that Rad54 protein dissociates Rad51 from nucleoprotein filaments formed on dsDNA. Addition of Rad54 protein overcomes inhibition of DNA strand exchange by Rad51 protein bound to substrate dsDNA. Species preference in the Rad51 dissociation and DNA strand exchange assays underlines the importance of specific Rad54-Rad51 protein interactions. Rad51 protein is unable to release dsDNA upon ATP hydrolysis, leaving it stuck on the heteroduplex DNA product after DNA strand exchange. We suggest that Rad54 protein is involved in the turnover of Rad51-dsDNA filaments.

Rad54 Protein Is Targeted to Pairing Loci by the Rad51 Nucleoprotein Filament

Rad51 and Rad54 proteins are important for the repair of double-stranded DNA (dsDNA) breaks by homologous recombination in eukaryotes. Rad51 assembles on single-stranded DNA (ssDNA) to form a helical nucleoprotein filament that performs homologous pairing with dsDNA; Rad54 stimulates this pairing substantially. Here, we demonstrate that Rad54 acts in concert with the mature Rad51-ssDNA filament. Enhancement of DNA pairing by Rad54 is greatest at an equimolar ratio relative to Rad51 within the filament. Reciprocally, the Rad51-ssDNA filament enhances both the dsDNA-dependent ATPase and the dsDNA unwinding activities of Rad54. We conclude that Rad54 participates in the DNA homology search as a component of the Rad51-nucleoprotein filament and that the filament delivers Rad54 to the dsDNA pairing locus, thereby linking the unwinding of potential target DNA with the homology search process.

Tethering complexes in the endocytic pathway: CORVET and HOPS

Endocytosis describes the processes by which proteins, peptides and solutes, and also pathogens, enter the cell. Endocytosed material progresses to endosomes. Genetic studies in yeast, worms, flies and mammals have identified a set of universally conserved proteins that are essential for early‐to‐late endosome transition and lysosome biogenesis, and for endolysosomal trafficking pathways, including autophagy. The two Vps‐C complexes CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting) perform diverse biochemical functions in endocytosis: they tether membranes, interact with Rab GTPases, activate and proof‐read SNARE assembly to drive membrane fusion, and possibly attach endosomes to the cytoskeleton. In addition, several of the CORVET and HOPS subunits have diversified in metazoans, and probably form additional specialized complexes to accomodate the higher complexity of trafficking pathways in these cells. Recent studies offer new insights into the complex relationships between CORVET and HOPS complexes and other factors of the endolysosomal pathway. Interactions with V‐ATPase, the ESCRT machinery, phosphoinositides, the cytoskeleton and the Rab switch suggest an intricate cooperative network for endosome maturation. Accumulating evidence supports the view that endosomal tethering complexes implement a regulatory logic that governs endomembrane identity and dynamics.

NADH-coupled microplate photometric assay for kinetic studies of ATP-hydrolyzing enzymes with low and high specific activities.

Genome database searches retrieve over 10,000 sequences that either are known to use NTP or contain motifs suggestive of that. Hence, ATP hydrolysis is a convenient tool for monitoring their biological activity. Among the most popular assays are measurements of the release of Pi, ADP, or Hþ [1]. For enzymes sensitive to the accumulation of ADP, an assay based on ADP recycling coupled to the oxidation of NADH represents a particular advantage [2,3]. The high molar absorption coefficient of NADH and the virtual absence of protein or DNA absorbance at this wavelength ensure reliable ATP hydrolysis measurements down to the micromolar concentration scale. A limitation of this technique is that it requires investment into relatively costly equipment with a limited capacity for parallel analysis. Microplate readers are devoid of these limitations, offering parallel analysis of many samples and great versatility. However, the lightpath of a microplate reader is variable and depends on the well fill volume, requiring proper calibration. Herein, we adapted the NADH-coupled ATPase assay for a 96-well microplate reader. A previous adaptation of this assay to the microplate format focused on P-glycoprotein, one particular ATPase [4]. Here, we elaborated general assay parameters to analyze a variety of ATPases with low and high specific activities. We show that beyond the numerous advantages of microplate readers (high throughput, low cost, ease of operation, use of disposable plasticware in place of quartz cells), the microplates ensure sufficiently high sensitivity and precision to measure ATP hydrolysis down to the submicromolar scale. We recommend practical tips for adapting the microplate-based assay for kinetic studies

Rad54 protein stimulates the postsynaptic phase of Rad51 protein-mediated DNA strand exchange

Rad54 and Rad51 are important proteins for the repair of double-stranded DNA breaks by homologous recombination in eukaryotes. As previously shown, Rad51 protein forms nucleoprotein filaments on single-stranded DNA, and Rad54 protein directly interacts with such filaments to enhance synapsis, the homologous pairing with a double-stranded DNA partner. Here we demonstrate that Saccharomyces cerevisiae Rad54 protein has an additional role in the postsynaptic phase of DNA strand exchange by stimulating heteroduplex DNA extension of established joint molecules in Rad51/Rpa-mediated DNA strand exchange. This function depended on the ATPase activity of Rad54 protein and on specific protein:protein interactions between the yeast Rad54 and Rad51 proteins.

The Caenorhabditis elegans Elongator Complex Regulates Neuronal α-tubulin Acetylation

Although acetylated α-tubulin is known to be a marker of stable microtubules in neurons, precise factors that regulate α-tubulin acetylation are, to date, largely unknown. Therefore, a genetic screen was employed in the nematode Caenorhabditis elegans that identified the Elongator complex as a possible regulator of α-tubulin acetylation. Detailed characterization of mutant animals revealed that the acetyltransferase activity of the Elongator is indeed required for correct acetylation of microtubules and for neuronal development. Moreover, the velocity of vesicles on microtubules was affected by mutations in Elongator. Elongator mutants also displayed defects in neurotransmitter levels. Furthermore, acetylation of α-tubulin was shown to act as a novel signal for the fine-tuning of microtubules dynamics by modulating α-tubulin turnover, which in turn affected neuronal shape. Given that mutations in the acetyltransferase subunit of the Elongator (Elp3) and in a scaffold subunit (Elp1) have previously been linked to human neurodegenerative diseases, namely Amyotrophic Lateral Sclerosis and Familial Dysautonomia respectively highlights the importance of this work and offers new insights to understand their etiology.

Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics

Rad51 and Rad54 are key proteins that collaborate during homologous recombination. Rad51 forms a presynaptic filament with ATP and ssDNA active in homology search and DNA strand exchange, but the precise role of its ATPase activity is poorly understood. Rad54 is an ATP-dependent dsDNA motor protein that can dissociate Rad51 from dsDNA, the product complex of DNA strand exchange. Kinetic analysis of the budding yeast proteins revealed that the catalytic efficiency of the Rad54 ATPase was stimulated by partial filaments of wild-type and Rad51-K191R mutant protein on dsDNA, unambiguously demonstrating that the Rad54 ATPase activity is stimulated under these conditions. Experiments with Rad51-K191R as well as with wild-type Rad51-dsDNA filaments formed in the presence of ATP, ADP or ATP-γ-S showed that efficient Rad51 turnover from dsDNA requires both the Rad51 ATPase and the Rad54 ATPase activities. The results with Rad51-K191R mutant protein also revealed an unexpected defect in binding to DNA. Once formed, Rad51-K191R-DNA filaments appeared normal upon electron microscopic inspection, but displayed significantly increased stability. These biochemical defects in the Rad51-K191R protein could lead to deficiencies in presynapsis (filament formation) and postsynapsis (filament disassembly) in vivo.

Active-Site Mutations in the Xrn1p Exoribonuclease of Saccharomyces cerevisiae Reveal a Specific Role in Meiosis

ABSTRACT Xrn1p of Saccharomyces cerevisiae is a major cytoplasmic RNA turnover exonuclease which is evolutionarily conserved from yeasts to mammals. Deletion of the XRN1 gene causes pleiotropic phenotypes, which have been interpreted as indirect consequences of the RNA turnover defect. By sequence comparisons, we have identified three loosely defined, common 5′-3′ exonuclease motifs. The significance of motif II has been confirmed by mutant analysis with Xrn1p. The amino acid changes D206A and D208A abolish singly or in combination the exonuclease activity in vivo. These mutations show separation of function. They cause identical phenotypes to that ofxrn1Δ in vegetative cells but do not exhibit the severe meiotic arrest and the spore lethality phenotype typical for the deletion. In addition, xrn1-D208A does not cause the severe reduction in meiotic popout recombination in a double mutant withdmc1 as does xrn1Δ. Biochemical analysis of the DNA binding, exonuclease, and homologous pairing activity of purified mutant enzyme demonstrated the specific loss of exonuclease activity. However, the mutant enzyme is competent to promote in vitro assembly of tubulin into microtubules. These results define a separable and specific function of Xrn1p in meiosis which appears unrelated to its RNA turnover function in vegetative cells.

Amino acid changes in Xrs2p, Dun1p, and Rfa2p that remove the preferred targets of the ATM family of protein kinases do not affect DNA repair or telomere length in Saccharomyces cerevisiae

In eukaryotes, mutations in a number of genes that affect DNA damage checkpoints or DNA replication also affect telomere length [Curr. Opin. Cell Biol. 13 (2001) 281]. Saccharomyces cerevisae strains with mutations in the TEL1 gene (encoding an ATM-like protein kinase) have very short telomeres, as do strains with mutations in XRS2, RAD50, or MRE11 (encoding members of a trimeric complex). Xrs2p and Mre11p are phosphorylated in a Tel1p-dependent manner in response to DNA damage [Genes Dev. 15 (2001) 2238; Mol. Cell 7 (2001) 1255]. We found that Xrs2p, but not Mre11p or Rad50p, is efficiently phosphorylated in vitro by immunopreciptated Tel1p. Strains with mutations eliminating all SQ and TQ motifs in Xrs2p (preferred targets of the ATM kinase family) had wild-type length telomeres and wild-type sensitivity to DNA damaging agents. We also showed that Rfa2p (a subunit of RPA) and the Dun1p checkpoint kinase, which are required for DNA damage repair and which are phosphorylated in response to DNA damage in vivo, are in vitro substrates of the Tel1p and Mec1p kinases. In addition, Dun1p substrates with no SQ or TQ motifs are phosphorylated by Mec1p in vitro very inefficiently, but retain most of their ability to be phosphorylated by Tel1p. We demonstrated that null alleles of DUN1 and certain mutant alleles of RFA2 result in short telomeres. As observed with Xrs2p, however, strains with mutations of DUN1 or RFA2 that eliminate SQ motifs have no effect on telomere length or DNA damage sensitivity.

Spontaneous and double-strand break-induced recombination, and gene conversion tract lengths, are differentially affected by overexpression of wild-type or ATPase-defective yeast Rad54

Rad54 plays key roles in homologous recombination (HR) and double-strand break (DSB) repair in yeast, along with Rad51, Rad52, Rad55 and Rad57. Rad54 belongs to the Swi2/Snf2 family of DNA-stimulated ATPases. Rad51 nucleoprotein filaments catalyze DNA strand exchange and Rad54 augments this activity of Rad51. Mutations in the Rad54 ATPase domain (ATPase(-)) impair Rad54 function in vitro, sensitize yeast to killing by methylmethane sulfonate and reduce spontaneous gene conversion. We found that overexpression of ATPase(-) Rad54 reduced spontaneous direct repeat gene conversion and increased both spontaneous direct repeat deletion and spontaneous allelic conversion. Overexpression of ATPase(-) Rad54 decreased DSB-induced allelic conversion, but increased chromosome loss and DSB-dependent lethality. Thus, ATP hydrolysis by Rad54 contributes to genome stability by promoting high-fidelity DSB repair and suppressing spontaneous deletions. Overexpression of wild-type Rad54 did not alter DSB-induced HR levels, but conversion tract lengths were reduced. Interestingly, ATPase(-) Rad54 decreased overall HR levels and increased tract lengths. These tract length changes provide new in vivo evidence that Rad54 functions in the post-synaptic phase during recombinational repair of DSBs.