Structure of Human DNA Polymerase κ Inserting dATP Opposite an 8-OxoG DNA Lesion

Background: Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA and 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major lesions formed. It is amongst the most mutagenic lesions in cells because of its dual coding potential, wherein 8-oxoG(syn) can pair with an A in addition to normal base pairing of 8-oxoG(anti) with a C. Human DNA polymerase κ (Polκ) is a member of the newly discovered Y-family of DNA polymerases that possess the ability to replicate through DNA lesions. To understand the basis of Polκ\u27s preference for insertion of an A opposite 8-oxoG lesion, we have solved the structure of Polκ in ternary complex with a template-primer presenting 8-oxoG in the active site and with dATP as the incoming nucleotide. Methodology and Principal Findings: We show that the Polκ active site is well-adapted to accommodate 8-oxoG in the syn conformation. That is, the polymerase and the bound template-primer are almost identical in their conformations to that in the ternary complex with undamaged DNA. There is no steric hindrance to accommodating 8-oxoG in the syn conformation for Hoogsteen base-paring with incoming dATP. Conclusions and Significance: The structure we present here is the first for a eukaryotic translesion synthesis (TLS) DNA polymerase with an 8-oxoG:A base pair in the active site. The structure shows why Polκ is more efficient at inserting an A opposite the 8-oxoG lesion than a C. The structure also provides a basis for why Polκ is more efficient at inserting an A opposite the lesion than other Y-family DNA polymerases

Imitation of β-lactam binding enables broad-spectrum metallo-β-lactamase inhibitors

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-beta-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential beta-lactamase stable beta-lactam mimics. Subsequent structure-activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL-carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.Peer reviewe

The trouble with embryos

In an effort to quell ongoing debate about the ethics of human embryonic stem cell (hESC) research, there have been concerted efforts to develop ethical standards for both embryo and hESC research and to entrench these standards in law. Surprisingly these efforts have not included efforts at standardizing the meaning of the pivotal term ‘embryo’. This paper reviews the legal framework for embryo research in the United Kingdom, the United States and Germany and highlights the absence of any agreed upon standard for what counts as a human embryo. This is an important lacuna, especially in light of the most recent advances in stem cell research involving the reprogramming of human somatic cell nuclei to generate human induced pluripotent stem (iPS) cells

Radiological Impacts and Regulation of Rare Earth Elements in Non-Nuclear Energy Production

Energy industries account for a significant portion of total rare earth usage, both in the US and worldwide. Rare earth minerals are frequently collocated with naturally occurring radioactive material, imparting an occupational radiological dose during recovery. This paper explores the extent to which rare earths are used by various non-nuclear energy industries and estimates the radiological dose which can be attributed to these industries on absolute and normalized scales. It was determined that typical rare earth mining results in an occupational collective dose of approximately 0.0061 person-mSv/t rare earth elements, amounting to a total of 330 person-mSv/year across all non-nuclear energy industries (about 60% of the annual collective dose from one pressurized water reactor operated in the US, although for rare earth mining the impact is spread out over many more workers). About half of the collective dose from non-nuclear energy production results from use of fuel cracking catalysts for oil refining, although given the extent of the oil industry, it is a small dose when normalized to the energy equivalent of the oil that is used annually. Another factor in energy industries’ reliance on rare earths is the complicated state of the regulation of naturally occurring radiological materials; correspondingly, this paper also explores regulatory and management implications

The Crystal Structure of the NHL Domain in Complex with RNA Reveals the Molecular Basis of Drosophila Brain-Tumor-Mediated Gene Regulation

TRIM-NHL proteins are conserved among metazoans and control cell fate decisions in various stem cell linages. The Drosophila TRIM-NHL protein Brain tumor (Brat) directs differentiation of neuronal stem cells by suppressing self-renewal factors. Brat is an RNA-binding protein and functions as a translational repressor. However, it is unknown which RNAs Brat regulates and how RNA-binding specificity is achieved. Using RNA immunoprecipitation and RNAcompete, we identify Brat-bound mRNAs in Drosophila embryos and define consensus binding motifs for Brat as well as a number of additional TRIM-NHL proteins, indicating that TRIM-NHL proteins are conserved, sequence-specific RNA-binding proteins. We demonstrate that Brat-mediated repression and direct RNA-binding depend on the identified motif and show that binding of the localization factor Miranda to the Brat-NHL domain inhibits Brat activity. Finally, to unravel the sequence specificity of the NHL domain, we crystallize the Brat-NHL domain in complex with RNA and present a high-resolution protein-RNA structure of this fold