Lynne S. Cox

Homologous regions of Fen1 and p21Cip1 compete for binding to the same site on PCNA : a potential mechanism to co-ordinate DNA replication and repair

Following genomic damage, the cessation of DNA replication is co-ordinated with onset of DNA repair; this co-ordination is essential to avoid mutation and genomic instability. To investigate these phenomena, we have analysed proteins that interact with PCNA, which is required for both DNA replication and repair. One such protein is p21Cip1, which inhibits DNA replication through its interaction with PCNA, while allowing repair to continue. We have identified an interaction between PCNA and the structure specific nuclease, Fen1, which is involved in DNA replication. Deletion analysis suggests that p21Cip1 and Fen1 bind to the same region of PCNA. Within Fen1 and its homologues a small region (10 amino acids) is sufficient for PCNA binding, which contains an 8 amino acid conserved PCNA-binding motif. This motif shares critical residues with the PCNA-binding region of p21Cip1. A PCNA binding peptide from p21Cip1 competes with Fen1 peptides for binding to PCNA, disrupts the Fen1-PCNA complex in replicating cell extracts, and concomitantly inhibits DNA synthesis. Competition between homologous regions of Fen1 and p21Cip1 for binding to the same site on PCNA may provide a mechanism to co-ordinate the functions of PCNA in DNA replication and repair.

MDM2 and MDMX bind and stabilize the p53-related protein p73

The p53 gene encodes one of the most important tumor suppressors in human cells and undergoes frequent mutational inactivation in cancers. MDM2, a transcriptional target of p53, binds p53 and can both inhibit p53-mediated transcription [1] [2] and target p53 for proteasome-mediated proteolysis [3] [4]. A close relative of p53, p73, has recently been identified [5] [6]. Here, we report that, like p53, p73alpha and the alternative transcription product p73beta also bind MDM2. Interaction between MDM2 and p53 represents a key step in the regulation of p53, as MDM2 promotes the degradation of p53. In striking contrast to p53, the half-life of p73 was found to be increased by binding to MDM2. Like MDM2, the MDM2-related protein MDMX also bound p73 and stabilized the level of p73. Moreover, the growth suppression functions of p73 and the induction of endogenous p21, a major mediator of the p53-dependent growth arrest pathway, were enhanced in the presence of MDM2. These differences between the regulation of p53 and p73 by MDM2/MDMX may highlight a physiological difference in their action.

Multiple pathways control cell growth and transformation: overlapping and independent activities of p53 and p21Cip1/WAF1/Sdi1

Many tumour therapies act by inducing a cellular damage response pathway mediated by the tumour suppressor protein p53. Alternative outcomes of p53 induction include apoptosis or transient cell‐cycle arrest, both thought to require the transcriptional activity of wild‐type p53. Current research highlights the action of a p53‐activated gene, p21Cip1/WAF1/Sdi1, which encodes a cyclin‐kinase inhibitor important in mediating p53‐dependent cell‐cycle arrest, while programmed cell death in response to DNA damage requires transcriptionally active p53 but not activation of p21Cip1/WAF1/Sdi1. This review examines the roles of p53 and p21Cip1/WAF1/Sdi1 in controlling cell proliferation, in the light of a new study on expression of p53 and p21 Cip1/WAF1/Sdi1 in squamous cell carcinoma of the larynx.

Asymmetry of DNA replication fork progression in Werner"s syndrome.

Human aging is associated with accumulation of cells that have undergone replicative senescence. The rare premature aging Werners syndrome (WS) provides a phenocopy of normal human aging and WS patient cells recapitulate the aging phenotype in culture as they rapidly lose the ability to proliferate or replicate their DNA. WS is associated with loss of functional WRN protein. Although the biochemical properties of WRN protein, which possesses both helicase and exonuclease activities, suggest an involvement in DNA metabolism, its action in cells is not clear. Here, we provide experimental evidence for a role of the WRN protein in DNA replication in normally proliferating cells. Most importantly, we demonstrate that in the absence of functional WRN protein, replication forks from origins of bidirectional replication fail to progress normally, resulting in marked asymmetry of bidirectional forks. We propose that WRN acts in normal DNA replication to prevent collapse of replication forks or to resolve DNA junctions at stalled replication forks, and that loss of this capacity may be a contributory factor in premature aging.

Who binds wins: Competition for PCNA rings out cell-cycle changes

PCNA, proliferating cell nuclear antigen, is a pivotal protein in DNA replication, DNA repair and possibly cell-cycle control. The protein has a trimeric ring structure that might slide along duplex DNA and form a platform for association with a variety of proteins, in particular holding the DNA polymerases in close association with their template. This article reviews evidence suggesting that the activity of PCNA in replication and repair is coordinated within the cell cycle by cooperative and competitive interactions with an extensive network of enzymes and regulatory proteins.

Sexual stage-specific expression of a third calcium-dependent protein kinase from Plasmodium falciparum.

A third calcium-dependent protein kinase (CDPK) gene has been isolated from the human malaria parasite Plasmodium falciparum by vectorette technology. The gene consists of five exons and four introns. The open reading frame resulting from removal of the four introns encodes a protein of 562 amino acid residues with a predicted molecular mass of 65.3 kDa. The encoded protein, termed PfCDPK3, consists of four distinct domains characteristic of a member of the CDPK family and displays the highest homology (46% identity and 69% similarity) to PfCDPK2, the second CDPK of P. falciparum. The N-terminal variable domain is rich in serine/threonine and lysine and contains multiple consensus phosphorylation sites for a range of protein kinases. The catalytic domain possesses all conserved motifs of the protein kinase family except for the highly conserved glutamic acid residue in subdomain VIII, which is replaced by a glutamine residue. The sequence of the junction domain comprising 31 amino acid residues is less conserved. The calmodulin-like regulatory domain contains four EF-hand calcium-binding motifs, each consisting of a loop of 12 amino acid residues which is flanked by two alpha-helices. Southern blotting of genomic DNA digests showed that the Pfcdpk3 gene is present as a single copy per haploid genome. A 2900 nucleotide transcript of this gene is expressed specifically in the sexual erythrocytic stage, indicating that PfCDPK3 is involved in sexual stage-specific events. It is proposed that PfCDPK3 may serve as a link between calcium and gametogenesis of P. falciparum.

Characterisation of the interaction between WRN, the helicase/exonuclease defective in progeroid Werner's syndrome, and an essential replication factor, PCNA.

Ageing is linked to the accumulation of replicatively senescent cells. The best model system to date for studying human cellular ageing is the progeroid Werners syndrome (WS), caused by a defect in WRN, a recQ-like helicase that also possesses exonuclease activity. In this paper, we characterise the interaction between WRN and an essential replication factor, PCNA. We show that wild-type WRN protein physically associates with PCNA at physiological protein concentrations in normal cells, while no association is seen in cells from patients with WS. We demonstrate co-localisation of WRN and PCNA at replication factories, show that PCNA binds to two distinct functional sites on WRN, and suggest a mechanism by which association between WRN and PCNA may be regulated in cells on DNA damage and during DNA replication.

Isolation and characterisation of a cAMP-dependent protein kinase catalytic subunit gene from Plasmodium falciparum☆

The cAMP-dependent protein kinase (PKA) is a key element of the signal transduction pathway by means of the second messenger cAMP. cAMP, generated as a result of activation of membranebound adenylyl cyclases by G protein-coupled surface receptors, exerts nearly all of its effects by activation of PKA [1]. In most organisms, PKA is a heterotetramer consisting of two catalytic and two regulatory subunits [1]. In Dictyostelium, however, PKA is a heterodimer composed of a catalytic and a regulatory subunit [2]. Kinase activity of the catalytic subunit is inhibited by the regulatory subunit. Binding of cAMP to the regulatory subunit alters its affinity for the catalytic subunit, and under physiological conditions, the catalytic subunit dissociates from the regulatory subunit and subsequently phosphorylates many substrate proteins. In mammalian cells, there are three isoforms of the catalytic subunits (Ca, Cb and Cg) and four isoforms of the regulatory subunits (RIa, RIb, RIIa and RIIb) [3] and the tissue-specific expression and assembly of these kinase isoforms are postulated to result in the diverse cellular responses to cAMP [1]. Apart from the regulatory subunits, the heat-stable protein kinase inhibitors (PKIs) are also able to bind the catalytic subunit with a high affinity and subsequently inhibit the kinase activity [1]. Plasmodium falciparum has a complex life cycle involving two different hosts and interactions with multiple cell types. The molecular and cellular mechanisms involved in regulation of proliferation and development of P. falciparum are unAbbre6iations: PKA, cAMP-dependent protein kinase; PfPKAc, Plasmodium falciparum cAMP-dependent protein kinase catalytic subunit; PKIs, the heat-stable protein kinase inhibitors; PfPP-a, Plasmodium falciparum protein serine/ threonine phosphatase a; PfPP-b, Plasmodium falciparum protein serine/threonine phosphatase b; PFGE, pulse-field gel electrophoresis. Note: Nucleotide sequence data reported in this paper are available in the GenBankTM, EMBL and DDJB databases under the accession number AF126719. * Corresponding author. Present address: Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK. Tel.: +44-1865-222419; fax: +44-1865-222431. E-mail address: lij@icrf.icnet.uk (J.-L. Li).

Increasing longevity through caloric restriction or rapamycin feeding in mammals: common mechanisms for common outcomes?

Significant extension of lifespan in important mammalian species is bound to attract the attention not only of the aging research community, but also the media and the wider public. Two recent papers published by Harrison et al. (2009) in Nature and by Colman et al. (2009) in Science report increased longevity of mice fed with rapamycin and of rhesus monkeys undergoing caloric restriction, respectively. These papers have generated considerable debate in the aging community. Here we assess what is new about these findings, how they fit with our knowledge of lifespan extension from other studies and what prospects this new work holds out for improvements in human longevity and human health span.

From old organisms to new molecules: integrative biology and therapeutic targets in accelerated human ageing

Abstract.Understanding the basic biology of human ageing is a key milestone in attempting to ameliorate the deleterious consequences of old age. This is an urgent research priority given the global demographic shift towards an ageing population. Although some molecular pathways that have been proposed to contribute to ageing have been discovered using classical biochemistry and genetics, the complex, polygenic and stochastic nature of ageing is such that the process as a whole is not immediately amenable to biochemical analysis. Thus, attempts have been made to elucidate the causes of monogenic progeroid disorders that recapitulate some, if not all, features of normal ageing in the hope that this may contribute to our understanding of normal human ageing. Two canonical progeroid disorders are Werner’s syndrome and Hutchinson-Gilford progeroid syndrome (also known as progeria). Because such disorders are essentially phenocopies of ageing, rather than ageing itself, advances made in understanding their pathogenesis must always be contextualised within theories proposed to help explain how the normal process operates. One such possible ageing mechanism is described by the cell senescence hypothesis of ageing. Here, we discuss this hypothesis and demonstrate that it provides a plausible explanation for many of the ageing phenotypes seen in Werner’s syndrome and Hutchinson-Gilford progeriod syndrome. The recent exciting advances made in potential therapies for these two syndromes are also reviewed.