Replication Fork Collapse and Genome Instability in a Deoxycytidylate Deaminase Mutant

Ribonucleotide reductase (RNR) and deoxycytidylate deaminase (dCMP deaminase) are pivotal allosteric enzymes required to maintain adequate pools of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis and repair. Whereas RNR inhibition slows DNA replication and activates checkpoint responses, the effect of dCMP deaminase deficiency is largely unknown. Here, we report that deleting the Schizosaccharomyces pombe dcd1⁺ dCMP deaminase gene (SPBC2G2.13c) increases dCTP ∼30-fold and decreases dTTP ∼4-fold. In contrast to the robust growth of a Saccharomyces cerevisiae dcd1Δ mutant, fission yeast dcd1Δ cells delay cell cycle progression in early S phase and are sensitive to multiple DNA-damaging agents, indicating impaired DNA replication and repair. DNA content profiling of dcd1Δ cells differs from an RNR-deficient mutant. Dcd1 deficiency activates genome integrity checkpoints enforced by Rad3 (ATR), Cds1 (Chk2), and Chk1 and creates critical requirements for proteins involved in recovery from replication fork collapse, including the γH2AX-binding protein Brc1 and Mus81 Holliday junction resolvase. These effects correlate with increased nuclear foci of the single-stranded DNA binding protein RPA and the homologous recombination repair protein Rad52. Moreover, Brc1 suppresses spontaneous mutagenesis in dcd1Δ cells. We propose that replication forks stall and collapse in dcd1Δ cells, burdening DNA damage and checkpoint responses to maintain genome integrity.     

Histological evidences of reparative and regenerative effects of beta-adrenoceptor agonists, clenbuterol and isoproterenol, in denervated rat skeletal muscle

The aim of this study was to determine the contribution of beta-adrenoceptor activation in the reconstruction of the structural and functional organization of denervated skeletal muscle. beta-agonists, clenbuterol (1.2 mg/kg body weight) and isoproterenol (2 mg/kg body weight), administration (daily oral administration; maximum 7 days) to normal innervated rats as well as denervated animals caused muscle hypertrophy. An increase in mean fiber diameter confirmed this stimulated growth both in normal innervated and denervated rat gastrocnemius muscle. Examination of muscle nuclei from treated but normal innervated rat gastrocnemius exhibited features like large size, active nucleoplasm and an increase in their number per fiber cross section and per mm mean fiber length indicating towards an elevated biosynthetic activity in tissue in the presence of beta adrenoceptor agonists. Administration of drugs to normal innervated animals resulted in an emergence of central muscle nuclei. The hyperactive and enlarged muscle nuclei ultimately organized themselves into unusually elongated nuclear streaks. beta agonist treatment to denervated rats resulted in amelioration of atrophic state of tissue characterized by hypertrophy of muscle fibers thus lending to a restoration of structural organization of tissue. Bizarre shapes of nuclei in denervated muscle tend to recover to that characteristic to normal innervated muscle in presence of clenbuterol and isoproterenol hydrochloride. All observations were confirmed by administering butoxamine, a beta-adrenoceptor antagonist along with beta-agonists. The results suggests that both clenbuterol and isoproterenol hydrochloride are capable of mimicking normal innervation functions in skeletal muscle and thus play important role in the structural and functional reorganization of tissue. Amelioration of denervation atrophy in rat gastrocnemius in the presence of beta-agonists supports this.     

Calreticulin transacylase: Genesis,mechanism of action and biological applications

Our earlier investigations have identified a unique enzyme in the endoplasmic reticulum (ER) termed Acetoxy Drug: Protein Transacetylase (TAase) catalyzing the transfer of acetyl group from polyphenolic acetates (PA) to certain receptor proteins (RP). An elegant assay procedure for TAase was developed based on the inhibition of glutathione S-transferase (GST) due to acetylation by a model acetoxycoumarin, 7, 8-Diacetoxy-4-methylcoumarin (DAMC). TAase purified from various mammalian tissue microsomes to homogeneity exhibited a molecular weight (M.wt) of 55 kDa. Further, by N-terminal sequencing TAase was identified as Calreticulin (CR), a multifunctional Ca²⁺-binding protein in ER lumen. The identity of TAase with CR was evidenced by proteomics studies such as immunoreactivity with anti-CR antibody and mass spectrometry. This function of CR was termed Calreticulin transacetylase (CRTAase). CRTAase was also found to mediate the transfer of acetyl group from DAMC to RP such as NADPH Cytochrome c Reductase (CYPR) and Nitric Oxide Synthase (NOS). The autoacetylation of purified human placental CRTAase concomitant with the acetylation of RP by DAMC was observed. CRTAase activity was found to be inhibited by Ca²⁺. Our investigations on the individual domains (N, P and C) of CR from a nematode Haemonchus contortus revealed that the P-domain alone was found to possess CRTAase activity. Based on the observation that the autoacetylated CR was a stable intermediate in the CRTAase catalyzed protein acetylation by PA, a putative mechanism was proposed. Further, CRTAase was also found capable of transferring propionyl group from a propoxy derivative of polyphenol, 7,8-Dipropoxy-4-methylcoumarin (DPMC) to RP and concomitant autopropionylation of CR was encountered. Hence, CRTAase was assigned the general term Calreticulin Transacylase. Also, CRTAase was found to act upon the biological acyl group donors, acetyl CoA and propionyl CoA. CRTAase mediated modulation of specific functional proteins by way of acylation was exploited to elicit the biological applications of PA.     

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.Download video file.^{(44M, mov)}     

Acidic pH enhances structure and structural stability of the capsid protein of hepatitis E virus

Hepatitis E virus (HEV) is enterically transmitted and endemic to tropical areas of the world. The major capsid protein of HEV is pORF2 ( approximately 74 kDa), encoded by open reading frame 2 (ORF2). When expressed in insect cells, it is processed into a approximately 55 kDa form (n-pORF2). We also generated a mutant, m-pORF2, lacking a C-terminal hydrophobic region shown earlier to be required for its homo-oligomerization. Circular dichroism was used to measure the secondary structure and stability of these proteins as a function of pH and temperature. With decreasing pH both proteins acquired increasing alpha-helicity and thermal stability in terms of midpoint of denaturation and the Gibbs energy change.     

Brugia malayi and Wuchereria bancrofti: gene comparison and recombinant expression of pi-class related glutathione S-transferases

The nucleotide sequences for Brugia malayi and Wuchereria bancrofti GST have been submitted to EMBL, GenBank, and DDBJ Nucleotide Sequence Databases under Accession Nos. Y12788 and AY195867.     

Identification of an inhibitor binding site of poly(ADP-ribose) glycohydrolase

Polymers of ADP-ribose involved in the maintenance of genomic integrity are converted to free ADP-ribose by the action of poly(ADP-ribose) glycohydrolase (PARG). As an approach to mapping functions of PARG onto the amino acid sequence of the protein, we report here experiments that identify an amino acid residue involved in the binding of potent PARG inhibitors. A photoreactive inhibitor, [alpha-(32)P]-8-azidoadenosine diphosphate (hydroxymethyl)pyrrolidinediol (8-N(3)-ADP-HPD), was used to photolabel a recombinant bovine PARG catalytic fragment (rPARG-CF). N-Terminal sequencing of tryptic and subtilitic peptides of photoderivatized rPARG-CF identified tyrosine 796 (Y796), a residue conserved in PARG across a wide range of organisms, as a site of photoderivatization. Site-directed mutants where this tyrosine residue was replaced with an alanine residue (Y796A) had a nearly 8-fold decrease in catalytic efficiency (k(cat)/K(M)), while replacement with a tryptophan residue (Y796W) had little effect on catalytic efficiency. Surface plasmon resonance spectroscopy using the PARG inhibitor 8-(aminohexyl)amino-ADP-HPD demonstrated that the binding constant of the inhibitor for Y796A was 21-fold lower (K(D) = 170 nM) than that of wild-type PARG (K(D) = 8.2 nM), while Y796W displayed a binding affinity similar to that of the wild-type enzyme. Our results indicate that Y796 is involved in inhibitor binding to PARG via a ring stacking interaction and identify a highly conserved region of the protein that putatively contains other residues involved in catalytic activity and/or substrate recognition.