Lysine Residue 185 of Rad1 Is a Topological but Not a Functional Counterpart of Lysine Residue 164 of PCNA

Monoubiquitylation of the homotrimeric DNA sliding clamp PCNA at lysine residue 164 (PCNAK164) is a highly conserved, DNA damage-inducible process that is mediated by the E2/E3 complex Rad6/Rad18. This ubiquitylation event recruits translesion synthesis (TLS) polymerases capable of replicating across damaged DNA templates. Besides PCNA, the Rad6/Rad18 complex was recently shown in yeast to ubiquitylate also 9-1-1, a heterotrimeric DNA sliding clamp composed of Rad9, Rad1, and Hus1 in a DNA damage-inducible manner. Based on the highly similar crystal structures of PCNA and 9-1-1, K185 of Rad1 (Rad1K185) was identified as the only topological equivalent of PCNAK164. To investigate a potential role of posttranslational modifications of Rad1K185 in DNA damage management, we here generated a mouse model with a conditional deletable Rad1K185R allele. The Rad1K185 residue was found to be dispensable for Chk1 activation, DNA damage survival, and class switch recombination of immunoglobulin genes as well as recruitment of TLS polymerases during somatic hypermutation of immunoglobulin genes. Our data indicate that Rad1K185 is not a functional counterpart of PCNAK164

The Fanconi Anemia Core Complex Is Dispensable during Somatic Hypermutation and Class Switch Recombination

To generate high affinity antibodies during an immune response, B cells undergo somatic hypermutation (SHM) of their immunoglobulin genes. Error-prone translesion synthesis (TLS) DNA polymerases have been reported to be responsible for all mutations at template A/T and at least a fraction of G/C transversions. In contrast to A/T mutations which depend on PCNA ubiquitination, it remains unclear how G/C transversions are regulated during SHM. Several lines of evidence indicate a mechanistic link between the Fanconi Anemia (FA) pathway and TLS. To investigate the contribution of the FA pathway in SHM we analyzed FancG-deficient B cells. B cells deficient for FancG, an essential member of the FA core complex, were hypersensitive to treatment with cross-linking agents. However, the frequencies and nucleotide exchange spectra of SHM remained comparable between wild-type and FancG-deficient B cells. These data indicate that the FA pathway is not involved in regulating the outcome of SHM in mammals. In addition, the FA pathway appears dispensable for class switch recombination

Ernst Freund as Precursor of the Rational Study of Corporate Law

Gindis, David, Ernst Freund as Precursor of the Rational Study of Corporate Law (October 27, 2017). Journal of Institutional Economics, Forthcoming. Available at SSRN: https://ssrn.com/abstract=2905547, doi: https://dx.doi.org/10.2139/ssrn.2905547The rise of large business corporations in the late 19th century compelled many American observers to admit that the nature of the corporation had yet to be understood. Published in this context, Ernst Freund's little-known The Legal Nature of Corporations (1897) was an original attempt to come to terms with a new legal and economic reality. But it can also be described, to paraphrase Oliver Wendell Holmes, as the earliest example of the rational study of corporate law. The paper shows that Freund had the intuitions of an institutional economist, and engaged in what today would be called comparative institutional analysis. Remarkably, his argument that the corporate form secures property against insider defection and against outsiders anticipated recent work on entity shielding and capital lock-in, and can be read as an early contribution to what today would be called the theory of the firm.Peer reviewe

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Mutated JH4 intronic regions from wild-type and FancG-deficient GC B cells.

<p>Mutated JH4 intronic regions from wild-type and FancG-deficient GC B cells.</p

<i>Rad1</i>^K185R B cells do not display sensitivity to various DNA damaging agents.

<p>WT (blue) and <i>Rad1</i>^K185R (Red) B cells were stimulated with LPS and exposed to increasing amounts of UV-C (A), MMS (B), CisPt (C) and γ-irradiation (D). The percentage of survival is shown on the y-axis after four days of culture. Data represent the mean and SD of individual cultures (n = 3). The results are representatives of two independent experiments.</p

CSR is not altered in <i>Rad1</i>^K185R B cells.

<p>WT (gray bars) and <i>Rad1</i>^K185R (black bars) B cells were tested for their ability to switch to either IgG3 or IgG1 by stimulation with LPS or LPS and IL-4, respectively. Data represent the mean and SD of individual B cell cultures from three independent mice. The results are representatives of two independent experiments.</p

Normal SHM in FancG-deficient B cells.

<p>A.) Unaltered accumulation of somatic mutations in germinal center B cells of FancG-deficient mice. The frequency of SHM (% of mutations) as determined from 9 individual mice are shown per genotype (paired Student's t-test, p = 0.3). The mean values and SD are indicated. B.) Normal nucleotide exchange pattern in hypermutated Ig genes of FancG-deficient germinal center B cells. Values are expressed as the total number of mutations (left panel) and percentage of total mutations (right panel). Chi square testing did not reveal any significant changes in the pattern (<i>p</i><0.01).</p

<i>Rad1</i>^K185R MEFs have normal Chk1 activation.

<p>WT and <i>Rad1</i>^K185R MEFs were irradiated with 100 J/m² UV-C and harvested after 10, 40 and 70 minutes after irradiation. Subsequently, the Chk1 phosphorylation status at S345 (pChk1 S345) was investigated by Western blotting using pChk1 S345-specific antibodies. The results are representatives of two independent experiments.</p

FancG-deficient B cells are sensitive to DNA cross-linking agents.

<p>FancG-deficient (red) and wild-type (blue) B cells were stimulated with LPS and exposed to increasing doses of either UV-C (left panel) or Cisplatin (right panel). The percentage of survival after four days of culture is shown.</p