Solution structure and characterization of the DNA-binding activity of the B3BP-Smr domain

The MutS1 protein recognizes unpaired bases and initiates mismatch repair, which are essential for high-fidelity DNA replication. The homologous MutS2 protein does not contribute to mismatch repair, but suppresses homologous recombination. MutS2 lacks the damage-recognition domain of MutS1, but contains an additional C-terminal extension: the small MutS-related (Smr) domain. This domain, which is present in both prokaryotes and eukaryotes, has previously been reported to bind to DNA and to possess nicking endonuclease activity. We determine here the solution structure of the functionally active Smr domain of the Bcl3-binding protein (also known as Nedd4-binding protein 2), a protein with unknown function that lacks other domains present in MutS proteins. The Smr domain adopts a two-layer α-β sandwich fold, which has a structural similarity to the C-terminal domain of IF3, the R3H domain, and the N-terminal domain of DNase I. The most conserved residues are located in three loops that form a contiguous, exposed, and positively charged surface with distinct sequence identity for prokaryotic and eukaryotic Smr domains. NMR titration experiments and DNA binding studies using Bcl3-binding protein-Smr domain mutants suggested that these most conserved loop regions participate in DNA binding to single-stranded/double-stranded DNA junctions. Based on the observed DNA-binding-induced multimerization, the structural similarity with both subdomains of DNase I, and the experimentally identified DNA-binding surface, we propose a model for DNA recognition by the Smr domain.     

The high-resolution NMR structure of the R21A Spc-SH3:P41 complex: Understanding the determinants of binding affinity by comparison with Abl-SH3

Background  

SH3 domains are small protein modules of 60–85 amino acids that bind to short proline-rich sequences with moderate-to-low affinity and specificity. Interactions with SH3 domains play a crucial role in regulation of many cellular processes (some are related to cancer and AIDS) and have thus been interesting targets in drug design. The decapeptide APSYSPPPPP (p41) binds with relatively high affinity to the SH3 domain of the Abl tyrosine kinase (Abl-SH3), while it has a 100 times lower affinity for the α-spectrin SH3 domain (Spc-SH3).     

Oxidative damage to DNA and single strand break repair capacity: relationship to other measures of oxidative stress in a population cohort

It is well accepted that oxidative DNA repair capacity, oxidative damage to DNA and oxidative stress play central roles in aging and disease development. However, the correlation between oxidative damage to DNA, markers of oxidant stress and DNA repair capacity is unclear. In addition, there is no universally accepted panel of markers to assess oxidative stress in humans. Our interest is oxidative damage to DNA and its correlation with DNA repair capacity and other markers of oxidative stress. We present preliminary data from a small comet study that attempts to correlate single strand break (SSB) level with single strand break repair capacity (SSB-RC) and markers of oxidant stress and inflammation. In this limited study of four very small age-matched 24-individual groups of male and female whites and African-Americans aged 30-64 years, we found that females have higher single strand break (SSB) levels than males (p=0.013). There was a significant negative correlation between SSB-RC and SSB level (p=0.041). There was a positive correlation between SSBs in African American males with both heme degradation products (p=0.008) and high-sensitivity C-reactive protein (hs-CRP) (p=0.022). We found a significant interaction between hs-CRP and sex in their effect on residual DNA damage (p=0.002). Red blood cell reduced glutathione concentration was positively correlated with the levels of oxidized bases detected by endonuclease III (p=0.047), heme degradation products (p=0.015) and hs-CRP (p=0.020). However, plasma carbonyl levels showed no significant correlation with other markers. The data from the literature and from our very limited study suggest a complex relationship between measures of oxidative stress and frequently used clinical parameters believed to reflect inflammation or oxidative stress.     

Structure of the DNA-bound BRCA1 C-terminal Region from Human Replication Factor C p140 and Model of the Protein-DNA Complex

BRCA1 C-terminal domain (BRCT)-containing proteins are found widely throughout the animal and bacteria kingdoms where they are exclusively involved in cell cycle regulation and DNA metabolism. Whereas most BRCT domains are involved in protein-protein interactions, a small subset has bona fide DNA binding activity. Here, we present the solution structure of the BRCT region of the large subunit of replication factor C bound to DNA and a model of the structure-specific complex with 5′-phosphorylated double-stranded DNA. The replication factor C BRCT domain possesses a large basic patch on one face, which includes residues that are structurally conserved and ligate the phosphate in phosphopeptide binding BRCT domains. An extra α-helix at the N terminus, which is required for DNA binding, inserts into the major groove and makes extensive contacts to the DNA backbone. The model of the protein-DNA complex suggests 5′-phosphate recognition by the BRCT domains of bacterial NAD⁺-dependent ligases and a nonclamp loading role for the replication factor C complex in DNA transactions.     

Identifying potential survival strategies of HIV-1 through virus-host protein interaction networks

Background  

The National Institute of Allergy and Infectious Diseases has launched the HIV-1 Human Protein Interaction Database in an effort to catalogue all published interactions between HIV-1 and human proteins. In order to systematically investigate these interactions functionally and dynamically, we have constructed an HIV-1 human protein interaction network. This network was analyzed for important proteins and processes that are specific for the HIV life-cycle. In order to expose viral strategies, network motif analysis was carried out showing reoccurring patterns in virus-host dynamics.     

Functional consequences of DECTIN-1 early stop codon polymorphism Y238X in rheumatoid arthritis

Introduction

We have previously demonstrated that ex vivo inhibition of costimulatory molecules on antigen-pulsed dendritic cells (DCs) can be useful for induction of antigen-specific immune deviation and suppression of autoimmune arthritis in the collagen induced arthritis (CIA) model. The current study evaluated a practical method of immune modulation through temporary systemic inhibition of the costimulatory molecule CD40.

Methods

Mice with collagen II (CII)-induced arthritis (CIA) were administered siRNA targeting the CD40 molecule. Therapeutic effects were evaluated by clinical symptoms, histopathology, Ag-specific T cell and B cell immune responses.

Results

Systemic administration of CD40-targeting siRNA can inhibit antigen-specific T cell response to collagen II, as well as prevent pathogenesis of disease in both a pre- and post-immunization manner in the CIA model. Disease amelioration was associated with suppression of Th1 cytokines, attenuation of antibody production, and upregulation of T regulatory cells.

Conclusions

These studies support the feasibility of transient gene silencing at a systemic level as a mechanism of resetting autoreactive immunity.     

Characterization of a polymorphism in the 3′ part of the chicken vitellogenin gene

Summary An allele giving rise to a polymorphism within the 3 part of the chicken vitellogenin gene was cloned, sequenced, and compared to the previously cloned allele. The polymorphism is formed by a perfect copy of 343 bp from intron 32 in tandem array with a perfect copy of 244 bp from intron 33; this 587-bp element is inserted in a head-to-tail arrangement in intron 33. We propose a mechanism in which an unequal crossing-over resulted in a vitellogenin gene with two exons 33, one of which was subsequently deleted. Thus, intron 33 was enlarged by the tandem repeats without affecting the protein-encoding sequence of the gene. At the boundaries of the repeated elements, two short direct repeats are found that resemble the recombination signals of immunoglobulin genes. They may have had a key role in the formation of the new allele.     

Different incorporation of glucocorticoid hormone into diploid and tetraploid liver nuclei

Tetraploid parenchymal rat liver nuclei incorporate about twice as much (³H)dexamethasone as diploid parenchymal nuclei both in vivo and in vitro. This suggests that the ability of hepatic nuclei to incorporate glucocorticoid hormone is influenced by the number of copies of the genome in these nuclei.     

A mutation in the RNA polymerase β′ subunit causing depressed ribosomal RNA synthesis in Escherichia coli

Molecular Genetics and Genomics - Macromolecular synthesis in an Escherichia coli mutant with a temperature-sensitive β′ subunit of RNA polymerase was analysed. At the non-permissive...