MRE11 complex links RECQ5 helicase to sites of DNA damage

RECQ5 DNA helicase suppresses homologous recombination (HR) possibly through disruption of RAD51 filaments. Here, we show that RECQ5 is constitutively associated with the MRE11-RAD50-NBS1 (MRN) complex, a primary sensor of DNA double-strand breaks (DSBs) that promotes DSB repair and regulates DNA damage signaling via activation of the ATM kinase. Experiments with purified proteins indicated that RECQ5 interacts with the MRN complex through both MRE11 and NBS1. Functional assays revealed that RECQ5 specifically inhibited the 3′→5′ exonuclease activity of MRE11, while MRN had no effect on the helicase activity of RECQ5. At the cellular level, we observed that the MRN complex was required for the recruitment of RECQ5 to sites of DNA damage. Accumulation of RECQ5 at DSBs was neither dependent on MDC1 that mediates binding of MRN to DSB-flanking chromatin nor on CtIP that acts in conjunction with MRN to promote resection of DSBs for repair by HR. Collectively, these data suggest that the MRN complex recruits RECQ5 to sites of DNA damage to regulate DNA repai

First Staphylococcal Cassette Chromosome mec Containing a mecB-Carrying Gene Complex Independent of Transposon Tn6045 in a Macrococcus caseolyticus Isolate from a Canine Infection.

A methicillin-resistant mecB-positive Macrococcus caseolyticus (strain KM45013) was isolated from the nares of a dog with rhinitis. It contained a novel 39-kb transposon-defective complete mecB-carrying staphylococcal cassette chromosome mec element (SCCmecKM45013). SCCmecKM45013 contained 49 coding sequences (CDSs), was integrated at the 3' end of the chromosomal orfX gene, and was delimited at both ends by imperfect direct repeats functioning as integration site sequences (ISSs). SCCmecKM45013 presented two discontinuous regions of homology (SCCmec coverage of 35%) to the chromosomal and transposon Tn6045-associated SCCmec-like element of M. caseolyticus JCSC7096: (i) the mec gene complex (98.8% identity) and (ii) the ccr-carrying segment (91.8% identity). The mec gene complex, located at the right junction of the cassette, also carried the β-lactamase gene blaZm (mecRm-mecIm-mecB-blaZm). SCCmecKM45013 contained two cassette chromosome recombinase genes, ccrAm2 and ccrBm2, which shared 94.3% and 96.6% DNA identity with those of the SCCmec-like element of JCSC7096 but shared less than 52% DNA identity with the staphylococcal ccrAB and ccrC genes. Three distinct extrachromosomal circularized elements (the entire SCCmecKM45013, ΨSCCmecKM45013 lacking the ccr genes, and SCCKM45013 lacking mecB) flanked by one ISS copy, as well as the chromosomal regions remaining after excision, were detected. An unconventional circularized structure carrying the mecB gene complex was associated with two extensive direct repeat regions, which enclosed two open reading frames (ORFs) (ORF46 and ORF51) flanking the chromosomal mecB-carrying gene complex. This study revealed M. caseolyticus as a potential disease-associated bacterium in dogs and also unveiled an SCCmec element carrying mecB not associated with Tn6045 in the genus Macrococcus

Human RECQL5β stimulates flap endonuclease 1

Human RECQL5 is a member of the RecQ helicase family which is implicated in genome maintenance. Five human members of the family have been identified; three of them, BLM, WRN and RECQL4 are associated with elevated cancer risk. RECQL1 and RECQL5 have not been linked to any human disorder yet; cells devoid of RECQL1 and RECQL5 display increased chromosomal instability. Here, we report the physical and functional interaction of the large isomer of RECQL5, RECQL5β, with the human flap endonuclease 1, FEN1, which plays a critical role in DNA replication, recombination and repair. RECQL5β dramatically stimulates the rate of FEN1 cleavage of flap DNA substrates. Moreover, we show that RECQL5β and FEN1 interact physically and co-localize in the nucleus in response to DNA damage. Our findings, together with the previous literature on WRN, BLM and RECQL4’s stimulation of FEN1, suggests that the ability of RecQ helicases to stimulate FEN1 may be a general feature of this class of enzymes. This could indicate a common role for the RecQ helicases in the processing of oxidative DNA damage

MRE11 complex links RECQ5 helicase to sites of DNA damage

RECQ5 DNA helicase suppresses homologous recombination (HR) possibly through disruption of RAD51 filaments. Here, we show that RECQ5 is constitutively associated with the MRE11–RAD50–NBS1 (MRN) complex, a primary sensor of DNA double-strand breaks (DSBs) that promotes DSB repair and regulates DNA damage signaling via activation of the ATM kinase. Experiments with purified proteins indicated that RECQ5 interacts with the MRN complex through both MRE11 and NBS1. Functional assays revealed that RECQ5 specifically inhibited the 3′→5′ exonuclease activity of MRE11, while MRN had no effect on the helicase activity of RECQ5. At the cellular level, we observed that the MRN complex was required for the recruitment of RECQ5 to sites of DNA damage. Accumulation of RECQ5 at DSBs was neither dependent on MDC1 that mediates binding of MRN to DSB-flanking chromatin nor on CtIP that acts in conjunction with MRN to promote resection of DSBs for repair by HR. Collectively, these data suggest that the MRN complex recruits RECQ5 to sites of DNA damage to regulate DNA repair

Complete Circular Genome Sequence of a mecB- and mecD-Containing Strain of Macrococcus canis.

The complete genome sequence of Macrococcus canis strain 19/EPI0118, isolated from a veterinary clinic environment in Switzerland, was determined using hybrid assembly of Oxford Nanopore and Illumina reads. 19/EPI0118 harbored the methicillin resistance genes mecB and mecD on a staphylococcal cassette chromosome mec element and a Macrococcus chromosomal resistance island, respectively

Functional characterization of the human RECQ5 protein

RecQ DNA helicases are involved in processing of complex DNA structures arising during DNA metabolism to prevent aberrant mitotic recombination. Inherited defects in three of the five human RecQ helicases give rise to cancer predisposition syndromes. RECQ5 has not been associated with human disease. However, deletion of the Recq5 gene in mice results in cancer susceptibility. How RECQ5 could act as a tumor suppressor is not yet clear. Recent findings suggest that RECQ5 has a role in DNA replication and homologous recombination. RECQ5 associates with the replication machinery and accumulates at sites of stalled replication forks and DNA double-strand breaks. In addition, RECQ5 interacts physically with the RAD51 recombinase and possesses the ability to disrupt RAD51 presynaptic filaments. In a first study, we analyzed the activity of RECQ5 helicase on DNA structures that resemble stalled replication forks. RECQ5 was found to convert M13-based forked DNA substrates with a long leading-strand gap into four-way junctions as revealed by restriction-enzyme digestion of the reaction products. However, these structures were not sensitive to cleavage by the Holliday-junction resolvase RusA. These controversial findings and the observed low extent of RECQ5-promoted fork-regression reaction argue against a role for RECQ5 in the repair of stalled replication forks by template switching. In a second study, we explored the mechanism underlying RECQ5- mediated disruption of RAD51 presynaptic filaments. We investigated whether the observed physical interaction between RECQ5 and RAD51 is required for RECQ5-mediated displacement of RAD51 from ssDNA. To do so, we mapped precisely the RAD51-interaction site on RECQ5 and tested RECQ5 mutants that fail to interact with RAD51 for the ability to displace RAD51 from ssDNA in a topoisomerase-linked RAD51-trap assay. We found that direct RAD51 binding enhances the RAD51 filament-disruption activity of RECQ5 but it is not essential for it. In addition, we found that the helicase core fragment of RECQ5 was not able to displace RAD51 from ssDNA, suggesting a mechanistic difference between DNA unwinding and protein-DNA complex- disruption activity of RECQ5. ZUSAMMENFASSUNG RecQ DNA Helikasen sind in die Verarbeitung von komplexen DNA Strukturen involviert, die während dem DNA Metabolismus entstehen und verhindern unangemessene mitotische Rekombination. Vererbte Gendefekte in drei der fünf humanen RecQ Helikasen verursachen Krebsprädispositions-Syndrome. RECQ5 ist nicht assoziiert mit einer Erkrankung beim Menschen. Ein inaktiviertes Recq5 Gen in der Maus verursacht hingegen eine Disposition für Krebserkankungen. Wie RECQ5 als Tumorsuppressor agiern könnte, ist bis jetzt nicht klar. Neuere Daten weisen auf eine Funktion von RECQ5 in der DNA Replikation und homologer Rekombination hin. RECQ5 ist assoziiert mit der Replikationsmaschinerie und akkumuliert an blockierten Replikationsgabeln und DNA Doppelstrangbrüchen. Zusätzlich interagiert RECQ5 direkt mit der RAD51 Rekombinase und besitzt die Fähigkeit präsynaptische RAD51 Filamente zu zerlegen. Im ersten Teil der Studie untersuchten wir die RECQ5 Helikase Aktivität auf M13-basierten DNA Strukturen, die blockierten Replikationsgabeln glichen. Durch Restriktionsenzym-Verdau der Reaktionsprodukte wurde sichtbar gemacht, dass RECQ5 gabelförmige DNA Substrate mit einer grossen Lücke im Folgestrang in kreuzförmige DNA Strukturen umwandeln konnte. Diese Strukturen wurden aber nicht vom Holliday-Struktur spezifischen Enzym RusA geschnitten. Diese kontroversen Ergebnisse und das geringe Ausmass der Regressionsreaktion katalysiert durch RECQ5 sprechen gegen eine Funktion von RECQ5 in der Reparatur von blockierten Replikationsgabeln durch einen Matrizenwechsel-Mechanismus. Im zweiten Teil dieser Studie untersuchten wir den Mechanismus durch den RECQ5 präsynaptische RAD51 Filamente zerlegt. Wir untersuchten, ob die beobachtete direkte Interaktion zwischen RAD51 und RECQ5 dazu notwendig ist. Dafür kartierten wir die präzise Interaktionsstelle von RAD51 auf RECQ5. RECQ5 Mutanten, die nicht mit RAD51 interagieren konnten, wurden dann in einem Topoisomerase-basierten RAD51 Assay getestet auf ihre Fähigkeit RAD51 von einzelsträngiger DNA zu entfernen. Wir konnten zeigen, dass direkte RAD51-RECQ5 Interaktion stimulierend auf das Entfernen von RAD51 Filamenten durch RECQ5 wirkt, aber nicht notwendig dafür ist. Zusätzlich beobachteten wir, dass das Helikase-core Fragment von RECQ5 nicht in der Lage war RAD51 von einzelsträngiger DNA zu entfernen, was auf einen mechanistischen Unterschied zwischen dem Entwinden von DNA und dem Entfernen von an DNA gebundene Proteine durch RECQ5 hindeutet

The bla and mec families of β-lactam resistance genes in the genera Macrococcus, Mammaliicoccus and Staphylococcus: an in-depth analysis with emphasis on Macrococcus.

β-Lactamases (Bla) and low-affinity penicillin-binding proteins (PBP2A) are responsible for β-lactam resistance in the genera Macrococcus, Mammaliicoccus and Staphylococcus. These resistance mechanisms are in most species acquired through mobile genetic elements that carry a blaZ-like β-lactamase gene for penicillin resistance and/or a mec gene (mecA, mecB, mecC, mecD) encoding a PBP2A for resistance to virtually all classes of β-lactams. The mecA and mecC genes can be acquired through staphylococcal cassette chromosome mec (SCCmec) elements in Staphylococcus and Mammaliicoccus. The mecB and mecD genes are found in Macrococcus on SCCmec elements, as well as on unrelated mecD-carrying Macrococcus resistance islands (McRImecD) and large mecB-carrying plasmids. This review provides a phylogenetic overview of Macrococcus, Mammaliicoccus and Staphylococcus species and an in-depth analysis of the genetic structures carrying bla and mec genes in these genera. Native bla genes were detected in species belonging to the novobiocin-resistant Staphylococcus saprophyticus group and Mammaliicoccus. The evolutionary relatedness between Macrococcus and Mammaliicoccus is illustrated on the basis of a similar set of intrinsic PBPs, especially, the presence of a second class A PBP. The review further focuses on macrococcal elements carrying mecB and mecD, and compares them with structures present in Staphylococcus and Mammaliicoccus. It also discusses the different recombinases (ccr of SCCmec) and integrases (int of McRI) that contribute to the mobility of methicillin resistance genes, revealing Macrococcus as an important source for mobilization of antibiotic resistance genes within the family of Staphylococcaceae

New MLSB resistance gene erm(43) in Staphylococcus lentus

The search for a specific rRNA methylase motif led to the identification of the new macrolide, lincosamide, and streptogramin B resistance gene erm(43) in Staphylococcus lentus. An inducible resistance phenotype was demonstrated by cloning and expressing erm(43) and its regulatory region in Staphylococcus aureus. The erm(43) gene was detected in two different DNA fragments, of 6,230 bp and 1,559 bp, that were each integrated at the same location in the chromosome in several S. lentus isolates of human, dog, and chicken origin

New Transposon Tn6133 in Methicillin-Resistant Staphylococcus aureus ST398 Contains vga(E), a Novel Streptogramin A, Pleuromutilin, and Lincosamide Resistance Gene▿

A novel streptogramin A, pleuromutilin, and lincosamide resistance determinant, Vga(E), was identified in porcine methicillin-resistant Staphylococcus aureus (MRSA) ST398. The vga(E) gene encoded a 524-amino-acid protein belonging to the ABC transporter family. It was found on a multidrug resistance-conferring transposon, Tn6133, which was comprised of Tn554 with a stably integrated 4,787-bp DNA sequence harboring vga(E). Detection of Tn6133 in several porcine MRSA ST398 isolates and its ability to circularize suggest a potential for dissemination