Rad54: the Swiss Army knife of homologous recombination?

Homologous recombination (HR) is a ubiquitous cellular pathway that mediates transfer of genetic information between homologous or near homologous (homeologous) DNA sequences. During meiosis it ensures proper chromosome segregation in the first division. Moreover, HR is critical for the tolerance and repair of DNA damage, as well as in the recovery of stalled and broken replication forks. Together these functions preserve genomic stability and assure high fidelity transmission of the genetic material in the mitotic and meiotic cell divisions. This review will focus on the Rad54 protein, a member of the Snf2-family of SF2 helicases, which translocates on dsDNA but does not display strand displacement activity typical for a helicase. A wealth of genetic, cytological, biochemical and structural data suggests that Rad54 is a core factor of HR, possibly acting at multiple stages during HR in concert with the central homologous pairing protein Rad51

Molecular Characterization of the α-Glucosidase Gene (\u3ci\u3emalA\u3c/i\u3e) from the Hyperthermophilic Archaeon \u3ci\u3eSulfolobus solfataricus\u3c/i\u3e

Acidic hot springs are colonized by a diversity of hyperthermophilic organisms requiring extremes of temperature and pH for growth. To clarify how carbohydrates are consumed in such locations, the structural gene (malA) encoding the major soluble α-glucosidase (maltase) and flanking sequences from Sulfolobus solfataricus were cloned and characterized. This is the first report of an α-glucosidase gene from the archaeal domain. malA is 2,083 bp and encodes a protein of 693 amino acids with a calculated mass of 80.5 kDa. It is flanked on the 5’ side by an unusual 1-kb intergenic region. Northern blot analysis of the malA region identified transcripts for malA and an upstream open reading frame located 5’ to the 1-kb intergenic region. The malA transcription start site was located by primer extension analysis to a guanine residue 8 bp 5’ of the malA start codon. Gel mobility shift analysis of the malA promoter region suggests that sequences 3’ to position 233, including a consensus archaeal TATA box, play an essential role in malA expression. malA homologs were detected by Southern blot analysis in other S. solfataricus strains and in Sulfolobus shibatae, while no homologs were evident in Sulfolobus acidocaldarius, lending further support to the proposed revision of the genus Sulfolobus. Phylogenetic analyses indicate that the closest S. solfataricus α-glucosidase homologs are of mammalian origin. Characterization of the recombinant enzyme purified from Escherichia coli revealed differences from the natural enzyme in thermostability and electrophoretic behavior. Glycogen is a substrate for the recombinant enzyme. Unlike maltose hydrolysis, glycogen hydrolysis is optimal at the intracellular pH of the organism. These results indicate a unique role for the S. solfataricus α-glucosidase in carbohydrate metabolism

A truncated DNA-damage-signaling response is activated after DSB formation in the G1 phase of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the DNA damage response (DDR) is activated by the spatio-temporal colocalization of Mec1-Ddc2 kinase and the 9-1-1 clamp. In the absence of direct means to monitor Mec1 kinase activation in vivo, activation of the checkpoint kinase Rad53 has been taken as a proxy for DDR activation. Here, we identify serine 378 of the Rad55 recombination protein as a direct target site of Mec1. Rad55-S378 phosphorylation leads to an electrophoretic mobility shift of the protein and acts as a sentinel for Mec1 activation in vivo. A single double-stranded break (DSB) in G1-arrested cells causes phosphorylation of Rad55-S378, indicating activation of Mec1 kinase. However, Rad53 kinase is not detectably activated under these conditions. This response required Mec1-Ddc2 and loading of the 9-1-1 clamp by Rad24-RFC, but not Rad9 or Mrc1. In addition to Rad55–S378, two additional direct Mec1 kinase targets are phosphorylated, the middle subunit of the ssDNA-binding protein RPA, RPA2 and histone H2A (H2AX). These data suggest the existence of a truncated signaling pathway in response to a single DSB in G1-arrested cells that activates Mec1 without eliciting a full DDR involving the entire signaling pathway including the effector kinases

The alpha-glucosidase of Sulfolobus solfataricus: Characterization of the gene and its activity

Sulfolobus solfataricus is a prokaryotic thermoacidophile and is phylogenetically classified within the crenarchaeotal subdivision of the archaea. It has the ability to grow both lithoautotrophically utilizing sulfur and carbon dioxide as the energy and carbon source respectively or chemoheterotrophically on a wide variety of sugars as the sole carbon and energy source. The $\alpha$-glucosidase of S. solfataricus was chosen to be a model enzyme for an investigation of chemoheterotrophic growth in S. solfataricus. To accomplished this the $\alpha$-glucosidase was characterized and its gene, malA, was cloned and characterized. In the work described here the role of $\alpha$-glucosidase in the heterotrophic growth of S. solfataricus was examined. The first step in the investigation was to purify the $\alpha$-glucosidase. The $\alpha$-glucosidase from S. solfataricus is a homotetramer with an apparent subunit mass of 80 kDa. The enzyme liberates glucose from maltose and maltooligomers at a rate that decreases with increasing substrate length. Maximal activity of the purified enzyme was reached at 105\sp\circC, it exhibited half lives of 11 hours at 85\sp\circC, 3.0 hours at 95\sp\circC and 2.75 hours at 100\sp\circC, and a pH optima of 4.5. The enzyme exhibited novel chromatographic properties requiring pretreatment with 6 M guanidine hydrochloride to electrophorese as a monomer. At reduced temperatures, only the fully denatured form of the enzyme was protease sensitive. $\alpha$-Glucosidase inhibitors demonstrated carbon source specific inhibition of S. solfataricus growth indicative of a vital role for $\alpha$-glucosidase in the growth of S. solfatavicus on maltose and starch. The $\alpha$-glucosidase gene, malA, is 2083 bp and encodes a protein of 693 amino acids. It is flanked on the 5\sp\prime side by an unusual 1 kb intergenic region. Phylogenetic analysis indicate the closest S. solfaruricus $\alpha$-glucosidase homologs are of mammalian origin. Characterization of the recombinant enzyme purified from E. coli revealed that glycogen is a substrate for the recombinant enzyme. Unlike maltose, glycogen hydrolysis is optimal at the intracellular pH of the organism. These results implicate a unique role for the S. solfataricus $\alpha$-glucosidase in carbohydrate metabolism. The $\alpha$-glucosidase being responsible for the catabolism of both extracellular and intracellular glucose polymers

Molecular Characterization of the α-Glucosidase Gene (\u3ci\u3emalA\u3c/i\u3e) from the Hyperthermophilic Archaeon \u3ci\u3eSulfolobus solfataricus\u3c/i\u3e

Acidic hot springs are colonized by a diversity of hyperthermophilic organisms requiring extremes of temperature and pH for growth. To clarify how carbohydrates are consumed in such locations, the structural gene (malA) encoding the major soluble α-glucosidase (maltase) and flanking sequences from Sulfolobus solfataricus were cloned and characterized. This is the first report of an α-glucosidase gene from the archaeal domain. malA is 2,083 bp and encodes a protein of 693 amino acids with a calculated mass of 80.5 kDa. It is flanked on the 5’ side by an unusual 1-kb intergenic region. Northern blot analysis of the malA region identified transcripts for malA and an upstream open reading frame located 5’ to the 1-kb intergenic region. The malA transcription start site was located by primer extension analysis to a guanine residue 8 bp 5’ of the malA start codon. Gel mobility shift analysis of the malA promoter region suggests that sequences 3’ to position 233, including a consensus archaeal TATA box, play an essential role in malA expression. malA homologs were detected by Southern blot analysis in other S. solfataricus strains and in Sulfolobus shibatae, while no homologs were evident in Sulfolobus acidocaldarius, lending further support to the proposed revision of the genus Sulfolobus. Phylogenetic analyses indicate that the closest S. solfataricus α-glucosidase homologs are of mammalian origin. Characterization of the recombinant enzyme purified from Escherichia coli revealed differences from the natural enzyme in thermostability and electrophoretic behavior. Glycogen is a substrate for the recombinant enzyme. Unlike maltose hydrolysis, glycogen hydrolysis is optimal at the intracellular pH of the organism. These results indicate a unique role for the S. solfataricus α-glucosidase in carbohydrate metabolism

Repair of DNA Double-Strand Breaks following UV Damage in Three Sulfolobus solfataricus Strains

DNA damage repair mechanisms have been most thoroughly explored in the eubacterial and eukaryotic branches of life. The methods by which members of the archaeal branch repair DNA are significantly less well understood but have been gaining increasing attention. In particular, the approaches employed by hyperthermophilic archaea have been a general source of interest, since these organisms thrive under conditions that likely lead to constant chromosomal damage. In this work we have characterized the responses of three Sulfolobus solfataricus strains to UV-C irradiation, which often results in double-strand break formation. We examined S. solfataricus strain P2 obtained from two different sources and S. solfataricus strain 98/2, a popular strain for site-directed mutation by homologous recombination. Cellular recovery, as determined by survival curves and the ability to return to growth after irradiation, was found to be strain specific and differed depending on the dose applied. Chromosomal damage was directly visualized using pulsed-field gel electrophoresis and demonstrated repair rate variations among the strains following UV-C irradiation-induced double-strand breaks. Several genes involved in double-strand break repair were found to be significantly upregulated after UV-C irradiation. Transcript abundance levels and temporal expression patterns for double-strand break repair genes were also distinct for each strain, indicating that these Sulfolobus solfataricus strains have differential responses to UV-C-induced DNA double-strand break damage

An archaeal RadA paralog influences presynaptic filament formation

•The SsoRal1 paralog reduces recombinase ATPase activity.•SsoRal1 modulates SsoRadA presynaptic filament dynamics.•SsoRadA-mediated joint molecule formation is stimulated by SsoRal1.•SsoRal1 activities correlate with a mediator function in homologous recombination. Recombinases of the RecA family play vital roles in homologous recombination, a high-fidelity mechanism to repair DNA double-stranded breaks. These proteins catalyze strand invasion and exchange after forming dynamic nucleoprotein filaments on ssDNA. Increasing evidence suggests that stabilization of these dynamic filaments is a highly conserved function across diverse species. Here, we analyze the presynaptic filament formation and DNA binding characteristics of the Sulfolobus solfataricus recombinase SsoRadA in conjunction with the SsoRadA paralog SsoRal1. In addition to constraining SsoRadA ssDNA-dependent ATPase activity, the paralog also enhances SsoRadA ssDNA binding, effectively influencing activities necessary for presynaptic filament formation. These activities result in enhanced SsoRadA-mediated strand invasion in the presence of SsoRal1 and suggest a filament stabilization function for the SsoRal1 protein

Molecular Characterization of the α-Glucosidase Gene (malA) from the Hyperthermophilic Archaeon Sulfolobus solfataricus

Acidic hot springs are colonized by a diversity of hyperthermophilic organisms requiring extremes of temperature and pH for growth. To clarify how carbohydrates are consumed in such locations, the structural gene ( malA ) encoding the major soluble α-glucosidase (maltase) and flanking sequences from Sulfolobus solfataricus were cloned and characterized. This is the first report of an α-glucosidase gene from the archaeal domain. malA is 2,083 bp and encodes a protein of 693 amino acids with a calculated mass of 80.5 kDa. It is flanked on the 5′ side by an unusual 1-kb intergenic region. Northern blot analysis of the malA region identified transcripts for malA and an upstream open reading frame located 5′ to the 1-kb intergenic region. The malA transcription start site was located by primer extension analysis to a guanine residue 8 bp 5′ of the malA start codon. Gel mobility shift analysis of the malA promoter region suggests that sequences 3′ to position −33, including a consensus archaeal TATA box, play an essential role in malA expression. malA homologs were detected by Southern blot analysis in other S. solfataricus strains and in Sulfolobus shibatae , while no homologs were evident in Sulfolobus acidocaldarius , lending further support to the proposed revision of the genus Sulfolobus . Phylogenetic analyses indicate that the closest S. solfataricus α-glucosidase homologs are of mammalian origin. Characterization of the recombinant enzyme purified from Escherichia coli revealed differences from the natural enzyme in thermostability and electrophoretic behavior. Glycogen is a substrate for the recombinant enzyme. Unlike maltose hydrolysis, glycogen hydrolysis is optimal at the intracellular pH of the organism. These results indicate a unique role for the S. solfataricus α-glucosidase in carbohydrate metabolism