Chromatin dynamics during DSB repair

We show that double strand breaks (DSBs) induced in chromatin of low as well as high density by exposure of human cells to gamma-rays are repaired in low-density chromatin. Extensive chromatin decondensation manifested in the vicinity of DSBs by decreased intensity of chromatin labelling, increased H4K5 acetylation, and decreased H3K9 dimethylation was observed already 15 min after irradiation. Only slight movement of sporadic DSB loci for short distances was noticed in living cells associated with chromatin decondensation around DSBs. This frequently resulted in their protrusion into the low-density chromatin domains. In these regions, the clustering (contact or fusion) of DSB foci was seen in vivo, and in situ after cell fixation. The majority of these clustered foci were repaired within 240 min, but some of them persisted in the nucleus for several days after irradiation, indicating damage that is not easily repaired. We propose that the repair of DSB in clustered foci might lead to misjoining of ends and, consequently, to exchange aberrations. On the other hand, the foci that persist for several days without being repaired could lead instead to cell death.     

Large propeptides of fungal beta-N-acetylhexosaminidases are novel enzyme regulators that must be intracellularly processed to control activity, dimerization, and secretion into the extracellular environment

Filamentous fungi produce and secrete beta-N-acetylhexosaminidases, Hex, as important components of the binary chitinolytic systems involved in the formation of septa and hyphenation. Enzyme reconstitution experiments published previously indicate that Hex can occur in the form of two molecular species containing either one or two molecules of the propeptide noncovalently associated with the enzyme dimer. Here, we describe a novel mechanism for the regulation of the activity of Hex based on the association of their catalytic subunits with the large N-terminal propeptides in vivo. We show that the enzyme precursor is processed early in the biosynthesis, shortly after the addition of N-glycans through the action of a dibasic peptidase, cleaving both before and after the dibasic sequence. The processing site for this unique dibasic peptidase, different from that of kexins, is conserved among the beta-N-acetylhexosaminidases from filamentous fungi, and inhibition of the dibasic peptidase abrogates enzyme folding and activation. Binding of the released propeptide to the catalytic subunit of Hex is essential for its activation. An examination of the kinetics of Hex activation and dimerization in vitro allowed us to understand the unusually high efficiency of the assembly of this enzyme. We also report that the fungus is able to actively regulate the concentration of the processed propeptide in endoplasmic reticulum and thus the specific activity of the produced Hex. This novel regulatory mechanism enables the control of the catalytic activity and architecture of the secreted enzyme according to the needs of the producing cell at various stages of its growth cycle.     

Staphylococcus petrasii sp. nov. including S. petrasii subsp. petrasii subsp. nov. and S. petrasii subsp. croceilyticus subsp. nov., isolated from human clinical specimens and human ear infections

Thirteen coagulase-negative, oxidase-negative, and novobiocin-susceptible staphylococci were isolated from human clinical specimens. The isolates were differentiated from known staphylococcal species on the basis of 16S rRNA, hsp60, rpoB, dnaJ, tuf, and gap gene sequencing, automated ribotyping, (GTG)₅-PCR fingerprinting, and MALDI-TOF MS analysis. Phylogenetic analysis based on the 16S rRNA gene sequence indicated phylogenetic relatedness of the analyzed strains to Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus devriesei, and Staphylococcus lugdunensis. DNA–DNA hybridization experiments between representative strains CCM 8418^T, CCM 8421^T, and the closest phylogenetic neighbors confirmed that the isolates represent novel Staphylococcus species, for which the name Staphylococcus petrasii sp. nov. is proposed. Genotypic and phenotypic analyses unambiguously split the strains into two closely related subclusters. Based on the results, two novel subspecies S. petrasii subsp. petrasii subsp. nov. and S. petrasii subsp. croceilyticus subsp. nov. are proposed, with type strains CCM 8418^T (=CCUG 62727^T) and CCM 8421^T (=CCUG 62728^T), respectively.     

Single-molecule visualization of human RECQ5 interactions with single-stranded DNA recombination intermediates

RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51–ssDNA filaments. RECQ5 interacts with RAD51 through protein–protein contacts, and disruption of this interface through a RECQ5–F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51–K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51–I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.