Properties of two DNA helicases of human cells.

DNA helicases are ubiquitous, non-specific dsDNA unwinding enzymes involved in all aspects of DNA metabolism. In the present work, I describe the properties of two DNA helicases of HeLa cells: those of a novel enzyme, human DNA helicase VH (HDH VII), and those of the separate subunits of human DNA helicase II. HDH VII possesses the highest specific activity among all the helicases, so far, extracted and characterized from HeLa cells, and this activity is further stimulated nearly a hundred-fold by hRPA. However, its abundance in the cell is very low since only 1 lOpg of pure protein could be recovered from 150 grams of cultured cells. The estimated molecular weight of HDH YD in its native form is close to 90-kDa whereas SDS-PAGE analysis reveals two unequal, closely-migrating bands in the region of 32-kDa, suggesting that the protein may have a hetero-trimeric conformation. The enzyme exhibits a double polarity of translocation: a property that distinguishes it from all characterized eukaryotic DNA helicases. In spite of its high specific activity, HDH VH shows a relatively low processivity even in the presence of hRPA and cannot unwind duplexes longer than 17 base pairs. The Ku antigen consists of two subunits of 70 and 83 kDa, separable from each other only by electrophoresis under denaturing conditions, and is endowed with both duplex DNA end-binding capacity and helicase activity. I have dissected the in vitro activities of the Ku molecule, and I report that whereas the DNA end-binding property remains a prerogative of the heterodimer, the helicase activity of the Ku molecule resides exclusively in the 70 kDa subunit, and that the helicase activity of the Ku heterodimer is stimulated upon phosphorylation by the DNA-dependent protein kinase DNA-PK

In vitro protein-DNA interactions at the human lamin B2 replication origin.

The complexity of mammalian origins of DNA replication has prevented, so far, the in vitro studies of the modalities of initiator protein binding and origin selection. We approached this problem by utilizing the human lamin B2 origin, wherein the precise start sites of replication initiation have been identified and known to be bound in vivo by the origin recognition complex (ORC). In order to analyze the in vitro interactions occurring at this origin, we have compared the DNA binding requirements and patterns of the human recombinant Orc4 with those of preparations of HeLa nuclear proteins containing the ORC complex. Here we show that both HsOrc4 alone and HeLa nuclear proteins recognize multiple sites within a 241-bp DNA sequence encompassing the lamin B2 origin. The DNA binding activity of HeLa cells requires the presence of ORC and can be reproduced in the absence of all the other proteins known to be recruited to origins by ORC. Both HsOrc4 alone and HeLa nuclear proteins exhibit cooperative and ATP-independent binding. This binding covers nucleotides 3853-3953 and then spreads outward. Because this region contains the start sites of DNA synthesis as well as the area protected in vivo and preserves protein binding capacity in vitro after removal of a fraction of the protected region, we suggest that it could contain the primary binding site. Thus the in vitro approach points to the sequence requirements for ORC binding as a key element for origin recognition

Malaria vaccine: between hopes and fears

By all accounts and standards malaria, the hemolytic disease caused by a mosquito¬vectored parasite, appears the most intractable health menace of mankind as it affects several millions of people worldwide and claims over a million lives annually especially in developing countries. Ironically though, the etiologic microorganism responsible for malaria appears also to be one of the most studied among all disease-causing pathogens. In fact, apart from a few sporadic episodes and very isolated cases recorded in the past few decades, malaria has essentially been successfully eradicated in all industrialized and rich countries. In this nations, a fine tuned combination of environmental sanitation, preventive surveillance, prompt treatment of all suspect cases and adequate national healthcare systems has successfully kept any dangers of disease resurgence at arms length. As a consequence, these countries have been declared malaria-free by the World Health Organization (WHO). Conversely, for many developing countries inadequate healthcare systems coupled with lack of basic infrastructure has rendered epidemiological studies and disease outbreak surveillance essentially impracticable, rendered preventive measures impossible or ineffective and reduced to nothingness the haphazard attempts at therapy. This paper offers a brief overview of problems that bedevilled earlier attempts at vaccine development, the current efforts and the contribution of modern biotechnology to the development of a malaria vaccine and looks at the potential dangers that could accompany a future vaccine against this long-lasting plague of Africa. Nigerian Medical Practitioner Vol. 49(5) 2006: 120-12

Inhibition of DNA unwinding and ATPase activities of human DNA helicase II by Chemotherapeutic agents

DNA helicases catalyze the unwinding of duplex DNA and thus play important roles in the processing of DNA, little is known about the effects of various cytotoxic or antitumor chemotherapeutic agents on purified human DNA helicases. We have determined the effect of actinomycin C1, VP-16, camptothecin, ethidium bromide, ellipticine, nogalamycin, novobiocin, genistein, m-AMSA, aphidicolin and daunorubicin on the enzymatic activities of purified human DNA helicase II which was identified as Ku autoantigen. Ku contains DNA helicase, ATPase and DNA end binding activities. Our data have shown that out of several chemotherapeutic agents tested ethidium bromide, actinomycin C1, daunorubicin and nogalamycin were inhibitors of DNA unwinding activity of human DNA helicase II with ID50 values of 8.44 μM, 11.68 μM, 6.23 μM and 0.42 μM respectively. These inhibitors also inhibited the ATPase activity but not the DNA binding activity of this helicase. This inhibition could be due to binding of these drugs to DNA, thereby impeding the movement of the helicase for unwinding action which may be their most important pharmacological function against cancer cells

Localization of proteins bound to a replication origin of human DNA along the cell cycle

The proteins bound in vivo at the human lamin B2 DNA replication origin and their precise sites of binding were investigated along the cell cycle utilizing two novel procedures based on immunoprecipitation following UV irradiation with a pulsed laser light source. In G(1), the pre-replicative complex contains CDC6, MCM3, ORC1 and ORC2 proteins; of these, the post-replicative complex in S phase contains only ORC2; in M phase none of them are bound. The precise nucleotide of binding was identified for the two ORC and the CDC6 proteins near the start sites for leading-strand synthesis; the transition from the pre- to the post-replicative complex is accompanied by a 17 bp displacement of the ORC2 protein towards the start site

Analysis of pan-African Centres of excellence in health innovation highlights opportunities and challenges for local innovation and financing in the continent

<p>Abstract</p> <p>A pool of 38 pan-African Centres of Excellence (CoEs) in health innovation has been selected and recognized by the African Network for Drugs and Diagnostics Innovation (ANDI), through a competitive criteria based process. The process identified a number of opportunities and challenges for health R&D and innovation in the continent: i) it provides a direct evidence for the existence of innovation capability that can be leveraged to fill specific gaps in the continent; ii) it revealed a research and financing pattern that is largely fragmented and uncoordinated, and iii) it highlights the most frequent funders of health research in the continent. The CoEs are envisioned as an innovative network of public and private institutions with a critical mass of expertise and resources to support projects and a variety of activities for capacity building and scientific exchange, including hosting fellows, trainees, scientists on sabbaticals and exchange with other African and non-African institutions.</p