Pif1 family helicases are conserved from bacteria to humans. Here, we report a novel DNA patrolling activity which may underlie Pif1's diverse functions: monomer preferentially anchors itself 3'-tailed junction and periodically reel in the 3' tail with step size of one nucleotide, extruding loop. This periodic is used unfold an intramolecular G-quadruplex (G4) structure on every encounter, sufficient unwind RNA-DNA heteroduplex but not duplex DNA. Instead leaving after G4 unwinding, allowing...

The Mcm2-7 (minichromosome maintenance) complex is a toroidal AAA(+) ATPase and the putative eukaryotic replicative helicase. Unlike typical homohexameric helicase, contains six distinct, essential, evolutionarily conserved subunits. Precedence to other proteins suggests that Mcm active sites are formed combinatorially, with Walker A B motifs contributed by one subunit catalytically essential arginine (arginine finger) adjacent subunit. To test this prediction, we used copurification...

The heat stress response activates the transcription factor shock 1 (HSF1), which subsequently upregulates proteins to maintain integrity of proteome. HSF1 activation requires nuclear localization, trimerization, DNA binding, phosphorylation and gene transactivation. Phosphorylation at S326 is an important regulator transcriptional activity. mediated by AKT1, mTOR, p38, MEK1 DYRK2. Here, we observed AKT1 independently mTOR. AKT2 also phosphorylated but showed weak ability activate HSF1....

The MCM2-7 complex, a hexamer containing six distinct and essential subunits, is postulated to be the eukaryotic replicative DNA helicase. Although all subunits function at replication fork, only specific subcomplex consisting of MCM4, 6, 7 (MCM467) not complex exhibits helicase activity in vitro. To understand why lacks address possible MCM2, 3, 5 we have compared biochemical properties Saccharomyces cerevisiae MCM467 complexes. We demonstrate that both complexes are toroidal possess...

Acquisition of foreign DNA by natural transformation is an important mechanism adaptation and evolution in diverse microbial species. Here, we characterize the ComM, a broadly conserved AAA+ protein previously implicated homologous recombination transforming (tDNA) naturally competent Gram-negative bacterial In vivo, found that ComM was required for efficient comigration linked genetic markers Vibrio cholerae Acinetobacter baylyi, which consistent with role branch migration. Also,...

Single-stranded DNA (ssDNA) binding proteins (ssBPs) are essential in eukaryotes to protect telomeres from nuclease activity. In Saccharomyces cerevisiae , the ssBP Cdc13 is an protein that acts as a central regulator of telomere length homeostasis and chromosome end protection, both alone part Cdc13-Stn1-Ten1 (CST) complex. has high affinity for telomeric ssDNA, with very slow off-rate. Previously, we reported despite this tight ssDNA binding, rapidly exchanges between bound unbound...

The Mcm2-7 complex is the eukaryotic replicative helicase, a toroidal AAA+ molecular motor that uses adenosine triphosphate (ATP) binding and hydrolysis to separate duplex DNA strands during replication. This heterohexameric helicase contains six different essential subunits (Mcm2 through Mcm7), with corresponding dimer interfaces forming ATPase active sites from conserved motifs of adjacent subunits. As all other known hexameric helicases are formed identical subunits, function unique...

Pif1 family helicases, which are found in nearly all eukaryotes, have important roles both nuclear and mitochondrial genome maintenance. Recently, the increasing availability of sequences has revealed that helicases also widely diverse prokaryotes, but it is currently unknown what physiological function(s) prokaryotic might perform. This Perspective aims to briefly introduce reader well-studied eukaryotic speculate on such enzymes may play bacteria. On basis our hypotheses, we predict for...

Most currently available small molecule inhibitors of DNA replication lack enzymatic specificity, resulting in deleterious side effects during use cancer chemotherapy and limited experimental usefulness as mechanistic tools to study replication. Towards development targeted inhibitors, we have focused on Mcm2-7 (minichromosome maintenance protein 2–7), a highly conserved helicase key regulatory component eukaryotic Unexpectedly found that the fluoroquinolone antibiotic ciprofloxacin...

Human RecQ4 (hRecQ4) affects cancer and aging but is difficult to study because it a fusion between helicase an essential replication factor. Budding yeast Hrq1 homologous the disease-linked domain of and, like hRecQ4, robust 3'-5' helicase. Additionally, has unusual property forming heptameric rings. Cells lacking exhibited two DNA damage phenotypes: hypersensitivity interstrand crosslinks (ICLs) telomere addition breaks. Both activities are rare; their coexistence in single protein...

The five human RecQ helicases participate in multiple processes required to maintain genome integrity. Of these, the disease-linked RecQ4 is least studied because it poses many technical challenges. We previously demonstrated that yeast Hrq1 helicase displays similar functions vivo, and here, we report biochemical structural characterization of these enzymes. In vitro, are DNA-stimulated ATPases robust helicases. Further, activities were sensitive DNA sequence structure, with preferentially...

PIF1 family helicases are conserved from bacteria to man. With the exception of well-studied yeast (e.g., ScPif1 and ScRrm3), however, very little is known about how these enzymes help maintain genome stability. Indeed, we lack a basic understanding protein domains found N- C-terminal characteristic central helicase domain in proteins. Here, using chimeric constructs, show that ScRrm3 interchangeable N-terminus important for its function vivo. This suggests evolved functional modules fused...

Yeasts are ubiquitous microbes found in virtually all environments. Many yeast species can ferment sugar into ethanol and CO2, humans have taken advantage of these characteristics to produce fermented beverages for thousands years. As a naturally abundant source fermentable sugar, honey has had central role such fermentations since Neolithic times. However, as beverage fermentation become industrialized, the processes been streamlined, including narrow almost exclusive usage yeasts genus...

Telomere length homeostasis is vital for maintaining genomic stability and regulated by multiple factors, including telomerase activity DNA helicases. The Saccharomyces cerevisiae Pif1 helicase was the first discovered catalytic inhibitor of telomerase, but recent experimental evidence suggests that Hrq1, yeast homolog disease-linked human RecQ-like 4 (RECQL4), plays a similar role via an undefined mechanism. Using extracts enriched in vitro primer extension assay, here we determined effects...