Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors

Beta human papillomavirus (β-HPV) are hypothesized to make DNA damage more mutagenic and potentially more carcinogenic. Double strand breaks (DSBs) are the most deleterious DNA lesion. They are typically repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs after DNA replication while NHEJ can occur at any point in the cell cycle. HR and NHEJ are not thought to occur in the same cell at the same time. HR is restricted to cells in phases of the cell cycle where homologous templates are available, while NHEJ occurs primarily during G1. β-HPV type 8 protein E6 (8E6) attenuates both repair pathways. We use a series of immunofluorescence microscopy and flow cytometry experiments to better define the impact of this attenuation. We found that 8E6 causes colocalization of HR factors (RPA70 and RAD51) with an NHEJ factor (activated DNA-PKcs or pDNA-PKcs) at persistent DSBs. 8E6 also causes RAD51 foci to form during G1. The initiation of NHEJ and HR at the same lesion could lead to antagonistic DNA end processing. Further, HR cannot be readily completed in an error-free manner during G1. Both aberrant repair events would cause deletions. To determine if these mutations were occurring, we used next generation sequencing of the 200kb surrounding a CAS9-induced DSB. 8E6 caused a 21-fold increase in deletions. Chemical and genetic inhibition of p300 as well as an 8E6 mutant that is incapable of destabilizing p300 demonstrates that 8E6 is acting via p300 destabilization. More specific chemical inhibitors of DNA repair provided mechanistic insight by mimicking 8E6-induced dysregulation of DNA repair in a virus-free system. Specifically, inhibition of NHEJ causes RAD51 foci to form in G1 and colocalization of RAD51 with pDNA-PKcs.     

Digestion of haemoglobin by schistosomes: 35 years on

Adult schistosomes obtain nutrients by digesting haemoglobin, which they obtain from ingested host red blood cells. Here John Dalton, Angela Smith, Karen Clough and Paul Brindley argue that a cathepsin L proteinase recently identified in their laboratory as the predominant cysteine proteinase activity of Schistosoma mansoni may play the leading role in haemoglobin digestion. They discuss the importance of elucidating the roles of both cathepsin B and cathepsin L in the digestion of haemoglobin, since both should be considered important targets to which novel schistosomicidal agents could be directed.     

Dilated cardiomyopathy caused by tissue-specific ablation of SC35 in the heart

Many genetic diseases are caused by mutations in cis-acting splicing signals, but few are triggered by defective trans-acting splicing factors. Here we report that tissue-specific ablation of the splicing factor SC35 in the heart causes dilated cardiomyopathy (DCM). Although SC35 was deleted early in cardiogenesis by using the MLC-2v-Cre transgenic mouse, heart development appeared largely unaffected, with the DCM phenotype developing 3-5 weeks after birth and the mutant animals having a normal life span. This nonlethal phenotype allowed the identification of downregulated genes by microarray, one of which was the cardiac-specific ryanodine receptor 2. We showed that downregulation of this critical Ca2+ release channel preceded disease symptoms and that the mutant cardiomyocytes exhibited frequency-dependent excitation-contraction coupling defects. The implication of SC35 in heart disease agrees with a recently documented link of SC35 expression to heart failure and interference of splicing regulation during infection by myocarditis-causing viruses. These studies raise a new paradigm for the etiology of certain human heart diseases of genetic or environmental origin that may be triggered by dysfunction in RNA processing.     

Genetically altered mice to evaluate glutathione homeostasis in health and disease

The tripeptide glutathione (GSH) is part of an integrated antioxidant system that protects cells and tissues from oxidative damage. Oxidative stress can result from exposure to excessive amounts of endogenous and exogenous electrophiles. Until recently, animal and cell model systems used to investigate the role of GSH in disease processes had employed chemical agents that deplete cellular GSH by inhibiting GSH synthesis or by reacting chemically with GSH. Such models have proven useful, but questions concerning nonspecific effects of such chemicals remain. Recently, our laboratories and others have developed mouse models with genetic deficiencies in enzymes of the GSH biosynthetic pathway. This review focuses on the regulation of GSH homeostasis and, specifically, the new GSH-deficient mouse models that have been developed. These models will improve our understanding of the role of GSH in animal and human diseases.     

Psychological impact of genetic testing for breast cancer susceptibility in women of Ashkenazi Jewish background: a prospective study

The recognition that the prevalence of three founder mutations in the BRCA1 and BRCA2 genes is over 2% in Ashkenazi Jews has resulted in numerous epidemiological research studies of this ethno-religious group. To determine the effects of incorporating research into clinical practice, a psychological impact study of women participating in an epidemiological study was conducted. Sixty women of Ashkenazi Jewish background who underwent genetic testing for founder mutations were assessed using mailed, self-administered questionnaires with validated measures of psychological outcome. Forty-three women elected to learn their results and 17 women declined to do so. Women who elected to learn their results were also assessed 7-10 days, 4 months, and 12 months after results disclosure. Women who chose to learn their results had significantly higher baseline breast cancer anxiety, compared to those who elected not to learn their results (z = -2.27; p = 0.023). Unaffected women who elected to learn their results showed a significant decrease in breast cancer anxiety 4 months (z = -2.37, p = 0.018) and 12 months (z = -3.06, p = 0.002) post-notification compared to baseline. Genetic testing for mutations common in Ashkenazi Jewish women with result disclosure does not lead to adverse psychological outcomes.     

Pericardial and pericardioperitoneal canal relationships to cardiac function in the white sturgeon (Acipenser transmontanus)

Sturgeons are primitive bony fishes and their hearts have structural features found in other primitive fishes. Sturgeons have a pericardioperitoneal canal (PPC), a one-way conduit into the peritoneum. A PPC also occurs in elasmobranchs (sharks and rays) and studies with that group demonstrate that pericardial pressure and pericardial fluid loss via the PPC affect stroke volume. A study of white sturgeon (Acipenser transmontanus) heart function was conducted to test for a comparable PPC and pericardial effects. White sturgeon-elasmobranch heart-function similarities include biphasic ventricular filling, a comparable operational pericardial pressure (-0.03 kPa), and a strongly negative pressure (-0.2 to -0.6 kPa) with complete pericardial fluid withdrawal. Differences include the white sturgeon's relatively smaller atrium and ventricle but a larger conus arteriosus. Although white sturgeon heart size is also smaller, its pericardial volume is disproportionately less (2.4 to 2.7 vs. 3.5 to 5.4 ml kg(-1) in elasmobranchs), meaning it has less scope for increasing stroke volume upon PPC fluid release. These differences may reflect the phylogenetic progression from the less complex operation of the elasmobranch heart, which lacks sympathetic innervation and has a mechanically mediated (PPC) stroke volume, to the condition in the more derived bony fishes which have sympathetic and parasympathetic regulation of both stroke volume and heart rate.     

Comparative analysis detects dependencies among the 5' splice-site positions

Human-mouse comparative genomics is an informative tool to assess sequence functionality as inferred from its conservation level. We used this approach to examine dependency among different positions of the 5' splice site. We compiled a data set of 50,493 homologous human-mouse internal exons and analyzed the frequency of changes among different positions of homologous human-mouse 5' splice-site pairs. We found mutual relationships between positions +4 and +5, +5 and +6, -2 and +5, and -1 and +5. We also demonstrated the association between the exonic and the intronic positions of the 5' splice site, in which a stronger interaction of U1 snRNA and the intronic portion of the 5' splice site compensates for weak interaction of U1 snRNA and the exonic portion of the 5' splice site, and vice versa. By using an ex vivo system that mimics the effect of mutation in the 5' splice site leading to familial dysautonomia, we demonstrated that U1 snRNA base-pairing with positions +6 and -1 is the only functional requirement for mRNA splicing of this 5' splice site. Our findings indicate the importance of U1 snRNA base-pairing to the exonic portion of the 5' splice site.     

Crystal structure of the BstDEAD N-terminal domain: a novel DEAD protein from Bacillus stearothermophilus

Most cellular processes requiring RNA structure rearrangement necessitate the action of Asp-Glu-Ala-Asp (DEAD) proteins. Members of the family, named originally for the conserved DEAD amino acid sequence, are thought to disrupt RNA structure and facilitate its rearrangement by unwinding short stretches of duplex RNA. BstDEAD is a novel 436 amino acid representative of the DEAD protein family from Bacillus stearothermophilus that contains all eight conserved motifs found in DEAD proteins and is homologous with other members of the family. Here, we describe the 1.85 A resolution structure of the N-terminal domain (residues 1-211) of BstDEAD (BstDEAD-NT). Similar to the corresponding domains of related helicases, BstDEAD-NT adopts a parallel alpha/beta structure with RecA-like topology. In general, the conserved motifs superimpose on closely related DEAD proteins and on more distantly related helicases such as RecA. This affirms the current belief that the core helicase domains, responsible for mechanistic activity, are structurally similar in DEAD proteins. In contrast, however, the so-called Walker A P-loop, which binds the beta- and gamma-phosphates of ATP, adopts a rarely seen "closed" conformation that would sterically block ATP binding. The closed conformation may be indicative of a general regulatory feature among DEAD proteins (and RNA helicases) that differs from that used by DNA helicases. BstDEAD also contains a unique extension of approximately 60 residues at the C terminus that is highly basic, suggesting that it might bind nucleic acids and, in so doing, confer specificity to the helicase activity of the core region.     

Myotonic dystrophy type 2: human founder haplotype and evolutionary conservation of the repeat tract

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, can be caused by a mutation on either chromosome 19 (DM1) or 3 (DM2). In 2001, we demonstrated that DM2 is caused by a CCTG expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene. To investigate the ancestral origins of the DM2 expansion, we compared haplotypes for 71 families with genetically confirmed DM2, using 19 short tandem repeat markers that we developed that flank the repeat tract. All of the families are white, with the majority of Northern European/German descent and a single family from Afghanistan. Several conserved haplotypes spanning >700 kb appear to converge into a single haplotype near the repeat tract. The common interval that is shared by all families with DM2 immediately flanks the repeat, extending up to 216 kb telomeric and 119 kb centromeric of the CCTG expansion. The DM2 repeat tract contains the complex repeat motif (TG)(n)(TCTG)(n)(CCTG)(n). The CCTG portion of the repeat tract is interrupted on normal alleles, but, as in other expansion disorders, these interruptions are lost on affected alleles. We examined haplotypes of 228 control chromosomes and identified a potential premutation allele with an uninterrupted (CCTG)(20) on a haplotype that was identical to the most common affected haplotype. Our data suggest that the predominant Northern European ancestry of families with DM2 resulted from a common founder and that the loss of interruptions within the CCTG portion of the repeat tract may predispose alleles to further expansion. To gain insight into possible function of the repeat tract, we looked for evolutionary conservation. The complex repeat motif and flanking sequences within intron 1 are conserved among human, chimpanzee, gorilla, mouse, and rat, suggesting a conserved biological function.