The N-Terminal Region of the RecU Holliday Junction Resolvase Is Essential for Homologous Recombination

The RecU Holliday junction (HJ)-resolving enzyme is highly conserved in the Firmicutes phylum of bacteria. In Bacillus subtilis, the recU gene has two putative initiation codons, at positions 1 and 33. In rec⁺ cells, only the full-length RecU polypeptide (206 residues, 23.9 kDa) was detected even after different stress treatments. To address the relevance of the flexible N-terminus, we constructed mutant variants. Experiments in vivo revealed that recUΔ1-32 (which initiates at Met33 and encodes RecUΔ1-32) and recU31 (the conserved Arg31 residue was substituted with alanine to give RecUR31A) are genuine RecU mutants, rendering cells impaired in DNA repair and chromosomal segregation. RecU has three activities: It (i) cleaves HJs, (ii) anneals complementary strands and (iii) modulates RecA activities. RecUR31A binds and cleaves HJ DNA in vitro as efficiently as wild-type RecU, but RuvB·ATPγS·Mg²⁺ fails to stimulate the RecUR31A cleavage reaction. In contrast, RecUΔ1-32 forms unstable complexes with DNA and fails to cleave HJs. RecU and its variants are capable of promoting DNA strand annealing and exert a negative effect on deoxy-ATP-dependent RecA-mediated DNA strand exchange. This study shows that the flexible N-terminus of RecU is essential for protein activity.     

Myocardial gene expression profiling of rewarming shock in a rodent model of accidental hypothermia

Background

Severe accidental hypothermia represents a cardiovascular emergency associated with high mortality and poor recovery of cardiac function. The biochemical changes occurring within the heart during the development of hypothermia and subsequent resuscitation are not known.

Methods

By mRNA expression profiling, we have characterized gene expression changes occurring within the myocardium in an intact rat model of accidental hypothermia during cooling to a core temperature of 15 °C and subsequent rewarming to 37 °C. During the rewarming phase, these animals develop a profound low-output cardiac failure.

Results

Hypothermia induces expression of known mediators of thermotolerance, including heat-shock protein 70 and several factors involved in protection against apoptotic cell death. Upregulation of genes involved in autophagy and increased abundance of autophagosomal vesicles suggest involvement of autophagic degeneration in the development of myocardial dysfunction occurring during rewarming from hypothermia. Rewarming from hypothermia also induces expression of several pro-inflammatory genes involved in the nuclear factor kappa B (NFκB) signaling cascade.

Conclusions

Our data demonstrate that rewarming from hypothermia is associated with the induction of a cellular stress–response, including upregulation of autophagy and activation of pro-inflammatory signaling cascades. These data provide a framework for understanding the molecular changes that occur during induction of and rewarming from severe hypothermia, and identifies potential targets for cardioprotective interventions in resuscitation of victims of hypothermia.     

Crystal structures of an O-like blue form and an anion-free yellow form of pharaonis halorhodopsin

Halorhodopsin from Natronomonas pharaonis (pHR) was previously crystallized into a monoclinic space group C2, and the structure of the chloride-bound purple form was determined. Here, we report the crystal structures of two chloride-free forms of pHR, that is, an O-like blue form and an M-like yellow form. When the C2 crystal was soaked in a chloride-free alkaline solution, the protein packing was largely altered and the yellow form containing all-trans retinal was generated. Upon neutralization, this yellow form was converted into the blue form. From structural comparison of the different forms of pHR, it was shown that the removal of a chloride ion from the primary binding site (site I), which is located between the retinal Schiff base and Thr126, is accompanied by such a deformation of helix C that the side chain of Thr126 moves toward helix G, leading to a significant shrinkage of site I. A large structural change is also induced in the chloride uptake pathway, where a flip motion of the side chain of Glu234 is accompanied by large movements of the surrounding aromatic residues. Irrespective of different charge distributions at the active site, there was no large difference in the structures of the yellow form and the blue form. It is shown that the yellow-to-purple transition is initiated by the entrance of one water and one HCl to the active site, where the proton and the chloride ion in HCl are transferred to the Schiff base and site I, respectively.     

Effects of Chloride Ion Binding on the Photochemical Properties of Salinibacter Sensory Rhodopsin I

Microbial organisms utilize light not only as energy sources but also as signals by which rhodopsins (containing retinal as a chromophore) work as photoreceptors. Sensory rhodopsin I (SRI) is a dual photoreceptor that regulates both negative and positive phototaxis in microbial organisms, such as the archaeon Halobacterium salinarum and the eubacterium Salinibacter ruber. These organisms live in highly halophilic environments, suggesting the possibility of the effects of salts on the function of SRI. However, such effects remain unclear because SRI proteins from H. salinarum (HsSRI) are unstable in dilute salt solutions. Recently, we characterized a new SRI protein (SrSRI) that is stable even in the absence of salts, thus allowing us to investigate the effects of salts on the photochemical properties of SRI. In this study, we report that the absorption maximum of SrSRI is shifted from 542 to 556 nm in a Cl⁻-dependent manner with a K_m of 307 ± 56 mM, showing that Cl⁻-binding sites exist in SRI. The bathochromic shift was caused not only by NaCl but also by other salts (NaI, NaBr, and NaNO₃), implying that I⁻, Br⁻, and NO₃⁻ can also bind to SrSRI. In addition, the photochemical properties during the photocycle are also affected by chloride ion binding. Mutagenesis studies strongly suggested that a conserved residue, His131, is involved in the Cl⁻-binding site. In light of these results, we discuss the effects of the Cl⁻ binding to SRI and the roles of Cl⁻ binding in its function.     

Temporal squama shape in fossil hominins: relationships to cranial shape and a determination of character polarity

In 1943, Weidenreich described the squamosal suture of Homo erectus as long, low, and simian in character and suggested that this morphology was dependent upon the correlation between the size of the calvarium and the face. Many researchers now consider this character to be diagnostic of H. erectus. The relationship between cranial size and shape and temporal squama morphology, however, is unclear, and several authors have called for detailed measurements of squamosal variation to be collected before any conclusions are drawn regarding the nature of the morphology observed in H. erectus. Thirteen fossil and extant taxa were examined to address two questions: 1) Are size and shape of the temporal squama correlated with cranial vault morphology? and 2) Is the H. erectus condition plesiomorphic? To answer these questions, measurements were collected and indices were calculated for squamosal suture height, length, and area in relation to metric variables describing cranial size and shape. A two‐dimensional morphometric study was also completed using High Resolution‐Polynomial Curve Fitting (HR‐PCF) to investigate correlations between curvature of the squamosal suture and curvature of the cranial vault. Results of both analyses indicate that squamosal suture form is related to cranial size and shape. Furthermore, the plesiomorphic condition of the squamosal suture for hominins was identified as high and moderately arched; this condition is retained in H. erectus and is distinct from the great ape condition. It is suggested that this similarity is the result of increased cranial length without a corresponding increase in cranial height. Am J Phys Anthropol, 2008. © 2008 Wiley‐Liss, Inc.     

DNA-damage response in chromatin of ribosomal genes and the surrounding genome

DNA repair events have functional significance especially for genome stability. Although the DNA damage response within the whole genome has been extensively studied, the region-specific characteristics of nuclear sub-compartments such as the nucleolus or fragile sites have not been fully elucidated. Here, we show that the heterochromatin protein HP1 and PML protein recognize spontaneously occurring 53BP1- or γ-H2AX-positive DNA lesions throughout the genome. Moreover, 53BP1 nuclear bodies, which co-localize with PML bodies, also occur within the nucleoli compartments. Irradiation of the human osteosarcoma cell line U2OS with γ-rays increases the degree of co-localization between 53BP1 and PML bodies throughout the genome; however, the 53BP1 protein is less abundant in chromatin of ribosomal genes and fragile sites (FRA3B and FRA16D) in γ-irradiated cells. Most epigenomic marks on ribosomal genes and fragile sites are relatively stable in both non-irradiated and γ-irradiated cells. However, H3K4me2, H3K9me3, H3K27me3 and H3K79me1 were significantly changed in promoter and coding regions of ribosomal genes after exposure of cells to γ-rays. In fragile sites, γ-irradiation induces a decrease in H3K4me3, changes the levels of HP1β, and modifies the levels of H3K9 acetylation, while the level of H3K9me3 was relatively stable. In these studies, we confirm a specific DNA-damage response that differs between the ribosomal genes and fragile sites, which indicates the region-specificity of DNA repair.