Inhibition of the Bloom's and Werner's syndrome helicases by G-quadruplex interacting ligands.

G-Quadruplex DNAs are folded, non-Watson-Crick structures that can form within guanine-rich DNA sequences such as telomeric repeats. Previous studies have identified a series of trisubstituted acridine derivatives that are potent and selective ligands for G-quadruplex DNA. These ligands have been shown previously to inhibit the activity of telomerase, the specialized reverse transcriptase that regulates telomere length. The RecQ family of DNA helicases, which includes the Bloom's (BLM) and Werner's (WRN) syndrome gene products, are apparently unique among cellular helicases in their ability to efficiently disrupt G-quadruplex DNA. This property may be relevant to telomere maintenance, since it is known that the sole budding yeast RecQ helicase, Sgs1p, is required for a telomerase-independent telomere lengthening pathway reminiscent of the "ALT" pathway in human cells. Here, we show that trisubstituted acridine ligands are potent inhibitors of the helicase activity of the BLM and WRN proteins on both G-quadruplex and B-form DNA substrates. Inhibition of helicase activity is associated with both a reduction in the level of binding of the helicase to G-quadruplex DNA and a reduction in the degree to which the G-quadruplex DNA can support DNA-dependent ATPase activity. We discuss these results in the context of the possible utility of trisubstituted acridines as antitumor agents for the disruption of both telomerase-dependent and telomerase-independent telomere maintenance

Sister chromatid cohesion establishment occurs in concert with lagging strand synthesis

Cohesion establishment is central to sister chromatid tethering reactions and requires Ctf7/Eco1-dependent acetylation of the cohesin subunit Smc3. Ctf7/Eco1 is essential during S phase, and a number of replication proteins (RFC complexes, PCNA and the DNA helicase Chl1) all play individual roles in sister chromatid cohesion. While the mechanism of cohesion establishment is largely unknown, a popular model is that Ctf7/Eco1 acetylates cohesins encountered by and located in front of the fork. In turn, acetylation is posited both to allow fork passage past cohesin barriers and convert cohesins to a state competent to capture subsequent production of sister chromatids. Here, we report evidence that challenges this pre-replicative cohesion establishment model. Our genetic and biochemical studies link Ctf7/Eco1 to the Okazaki fragment flap endonuclease, Fen1. We further report genetic and biochemical interactions between Fen1 and the cohesion-associated DNA helicase, Chl1. These results raise a new model wherein cohesin deposition and establishment occur in concert with lagging strand-processing events and in the presence of both sister chromatids

Mechanism of Werner DNA Helicase: POT1 and RPA Stimulates WRN to Unwind beyond Gaps in the Translocating Strand

WRN belongs to the RecQ family of DNA helicases and it plays a role in recombination, replication, telomere maintenance and long-patch base excision repair. Here, we demonstrate that WRN efficiently unwinds DNA substrates containing a 1-nucleotide gap in the translocating DNA strand, but when the gap size is increased to 3-nucleotides unwinding activity significantly declines. In contrast, E. coli UvrD (3′→5′ helicase), which recognizes nicks in DNA to initiate unwinding, does not unwind past a 1-nucleotide gap. This unique ability of WRN to bypass gaps supports its involvement in DNA replication and LP-BER where such gaps can be produced by glycosylases and the apurinic/apyrimidinic endonuclease 1 (APE1). Furthermore, we tested telomere repeat binding factor 2 (TRF2), both variants 1 and 2 of protector of telomeres 1 (POT1v1 and POT1v2) and RPA on telomeric DNA substrates containing much bigger gaps than 3-nucleotides in order to determine whether unwinding could be facilitated through WRN-protein interaction. Interestingly, POT1v1 and RPA are capable of stimulating WRN helicase on gapped DNA and 5′-overhang substrates, respectively

BLM and RMI1 alleviate RPA inhibition of topoIIIα decatenase activity

RPA is a single-stranded DNA binding protein that physically associates with the BLM complex. RPA stimulates BLM helicase activity as well as the double Holliday junction dissolution activity of the BLM-topoisomerase IIIα complex. We investigated the effect of RPA on the ssDNA decatenase activity of topoisomerase IIIα. We found that RPA and other ssDNA binding proteins inhibit decatenation by topoisomerase IIIα. Complex formation between BLM, TopoIIIα, and RMI1 ablates inhibition of decatenation by ssDNA binding proteins. Together, these data indicate that inhibition by RPA does not involve species-specific interactions between RPA and BLM-TopoIIIα-RMI1, which contrasts with RPA modulation of double Holliday junction dissolution. We propose that topoisomerase IIIα and RPA compete to bind to single-stranded regions of catenanes. Interactions with BLM and RMI1 enhance toposiomerase IIIα activity, promoting decatenation in the presence of RPA

The Werner Syndrome Helicase/Exonuclease Processes Mobile D-Loops through Branch Migration and Degradation

RecQ DNA helicases are critical for preserving genome integrity. Of the five RecQ family members identified in humans, only the Werner syndrome protein (WRN) possesses exonuclease activity. Loss of WRN causes the progeroid disorder Werner syndrome which is marked by cancer predisposition. Cellular evidence indicates that WRN disrupts potentially deleterious intermediates in homologous recombination (HR) that arise in genomic and telomeric regions during DNA replication and repair. Precisely how the WRN biochemical activities process these structures is unknown, especially since the DNA unwinding activity is poorly processive. We generated biologically relevant mobile D-loops which mimic the initial DNA strand invasion step in HR to investigate whether WRN biochemical activities can disrupt this joint molecule. We show that WRN helicase alone can promote branch migration through an 84 base pair duplex region to completely displace the invading strand from the D-loop. However, substrate processing is altered in the presence of the WRN exonuclease activity which degrades the invading strand both prior to and after release from the D-loop. Furthermore, telomeric D-loops are more refractory to disruption by WRN, which has implications for tighter regulation of D-loop processing at telomeres. Finally, we show that WRN can recognize and initiate branch migration from both the 5′ and 3′ ends of the invading strand in the D-loops. These findings led us to propose a novel model for WRN D-loop disruption. Our biochemical results offer an explanation for the cellular studies that indicate both WRN activities function in processing HR intermediates

Comparison of Proliferation and Genomic Instability Responses to WRN Silencing in Hematopoietic HL60 and TK6 Cells

BACKGROUND: Werner syndrome (WS) results from defects in the RecQ helicase (WRN) and is characterized by premature aging and accelerated tumorigenesis. Contradictorily, WRN deficient human fibroblasts derived from WS patients show a characteristically slower cell proliferation rate, as do primary fibroblasts and human cancer cell lines with WRN depletion. Previous studies reported that WRN silencing in combination with deficiency in other genes led to significantly accelerated cellular proliferation and tumorigenesis. The aim of the present study was to examine the effects of silencing WRN in p53 deficient HL60 and p53 wild-type TK6 hematopoietic cells, in order to further the understanding of WRN-associated tumorigenesis. METHODOLOGY/PRINCIPAL FINDINGS: We found that silencing WRN accelerated the proliferation of HL60 cells and decreased the cell growth rate of TK6 cells. Loss of WRN increased DNA damage in both cell types as measured by COMET assay, but elicited different responses in each cell line. In HL60 cells, but not in TK6 cells, the loss of WRN led to significant increases in levels of phosphorylated RB and numbers of cells progressing from G1 phase to S phase as shown by cell cycle analysis. Moreover, WRN depletion in HL60 cells led to the hyper-activation of homologous recombination repair via up-regulation of RAD51 and BLM protein levels. This resulted in DNA damage disrepair, apparent by the increased frequencies of both spontaneous and chemically induced structural chromosomal aberrations and sister chromatid exchanges. CONCLUSIONS/SIGNIFICANCE: Together, our data suggest that the effects of WRN silencing on cell proliferation and genomic instability are modulated probably by other genetic factors, including p53, which might play a role in the carcinogenesis induced by WRN deficiency

The wonders of flap endonucleases: structure, function, mechanism and regulation.

Processing of Okazaki fragments to complete lagging strand DNA synthesis requires coordination among several proteins. RNA primers and DNA synthesised by DNA polymerase α are displaced by DNA polymerase δ to create bifurcated nucleic acid structures known as 5'-flaps. These 5'-flaps are removed by Flap Endonuclease 1 (FEN), a structure-specific nuclease whose divalent metal ion-dependent phosphodiesterase activity cleaves 5'-flaps with exquisite specificity. FENs are paradigms for the 5' nuclease superfamily, whose members perform a wide variety of roles in nucleic acid metabolism using a similar nuclease core domain that displays common biochemical properties and structural features. A detailed review of FEN structure is undertaken to show how DNA substrate recognition occurs and how FEN achieves cleavage at a single phosphate diester. A proposed double nucleotide unpairing trap (DoNUT) is discussed with regards to FEN and has relevance to the wider 5' nuclease superfamily. The homotrimeric proliferating cell nuclear antigen protein (PCNA) coordinates the actions of DNA polymerase, FEN and DNA ligase by facilitating the hand-off intermediates between each protein during Okazaki fragment maturation to maximise through-put and minimise consequences of intermediates being released into the wider cellular environment. FEN has numerous partner proteins that modulate and control its action during DNA replication and is also controlled by several post-translational modification events, all acting in concert to maintain precise and appropriate cleavage of Okazaki fragment intermediates during DNA replication

Acetylation Regulates WRN Catalytic Activities and Affects Base Excision DNA Repair

Background: The Werner protein (WRN), defective in the premature aging disorder Werner syndrome, participates in a number of DNA metabolic processes, and we have been interested in the possible regulation of its function in DNA repair by post-translational modifications. Acetylation mediated by histone acetyltransferases is of key interest because of its potential importance in aging, DNA repair and transcription. Methodology/Principal Findings: Here, we have investigated the p300 acetylation mediated changes on the function of WRN in base excision DNA repair (BER). We show that acetylation of WRN increases in cells treated with methyl methanesulfonate (MMS), suggesting that acetylation of WRN may play a role in response to DNA damage. This hypothesis is consistent with our findings that acetylation of WRN stimulates its catalytic activities in vitro and in vivo, and that acetylated WRN enhances pol b-mediated strand displacement DNA synthesis more than unacetylated WRN. Furthermore, we show that cellular exposure to the histone deacetylase inhibitor sodium butyrate stimulates long patch BER in wild type cells but not in WRN depleted cells, suggesting that acetylated WRN participates significantly in this process. Conclusion/Significance: Collectively, these results provide the first evidence for a specific role of p300 mediated WRN acetylation in regulating its function during BER

Nucleolin Inhibits G4 Oligonucleotide Unwinding by Werner Helicase

The Werner protein (WRNp), a member of the RecQ helicase family, is strongly associated with the nucleolus, as is nucleolin (NCL), an important nucleolar constituent protein. Both WRNp and NCL respond to the effects of DNA damaging agents. Therefore, we have investigated if these nuclear proteins interact and if this interaction has a possible functional significance in DNA damage repair.Here we report that WRNp interacts with the RNA-binding protein, NCL, based on immunoprecipitation, immunofluorescent co-localization in live and fixed cells, and direct binding of purified WRNp to nucleolin. We also map the binding region to the C-terminal domains of both proteins. Furthermore, treatment of U2OS cells with 15 µM of the Topoisomerase I inhibitor, camptothecin, causes the dissociation of the nucleolin-Werner complex in the nucleolus, followed by partial re-association in the nucleoplasm. Other DNA damaging agents, such as hydroxyurea, Mitomycin C, and aphidicolin do not have these effects. Nucleolin or its C-terminal fragment affected the helicase, but not the exonuclease activity of WRNp, by inhibiting WRN unwinding of G4 tetraplex DNA structures, as seen in activity assays and electrophoretic mobility shift assays (EMSA).These data suggest that nucleolin may regulate G4 DNA unwinding by WRNp, possibly in response to certain DNA damaging agents. We postulate that the NCL-WRNp complex may contain an inactive form of WRNp, which is released from the nucleolus upon DNA damage. Then, when required, WRNp is released from inhibition and can participate in the DNA repair processes

Meiosis genes in Daphnia pulex and the role of parthenogenesis in genome evolution

<p>Abstract</p> <p>Background</p> <p>Thousands of parthenogenetic animal species have been described and cytogenetic manifestations of this reproductive mode are well known. However, little is understood about the molecular determinants of parthenogenesis. The <it>Daphnia pulex </it>genome must contain the molecular machinery for different reproductive modes: sexual (both male and female meiosis) and parthenogenetic (which is either cyclical or obligate). This feature makes <it>D. pulex </it>an ideal model to investigate the genetic basis of parthenogenesis and its consequences for gene and genome evolution. Here we describe the inventory of meiotic genes and their expression patterns during meiotic and parthenogenetic reproduction to help address whether parthenogenesis uses existing meiotic and mitotic machinery, or whether novel processes may be involved.</p> <p>Results</p> <p>We report an inventory of 130 homologs representing over 40 genes encoding proteins with diverse roles in meiotic processes in the genome of <it>D. pulex</it>. Many genes involved in cell cycle regulation and sister chromatid cohesion are characterized by expansions in copy number. In contrast, most genes involved in DNA replication and homologous recombination are present as single copies. Notably, <it>RECQ2 </it>(which suppresses homologous recombination) is present in multiple copies while <it>DMC1 </it>is the only gene in our inventory that is absent in the <it>Daphnia </it>genome. Expression patterns for 44 gene copies were similar during meiosis <it>versus </it>parthenogenesis, although several genes displayed marked differences in expression level in germline and somatic tissues.</p> <p>Conclusion</p> <p>We propose that expansions in meiotic gene families in <it>D. pulex </it>may be associated with parthenogenesis. Taking into account our findings, we provide a mechanistic model of parthenogenesis, highlighting steps that must differ from meiosis including sister chromatid cohesion and kinetochore attachment.</p