Flap Endonuclease Disengages Dna2 Helicase/Nuclease from Okazaki Fragment Flaps

Okazaki fragments contain an initiator RNA/DNA primer that must be removed before the fragments are joined. In eukaryotes, the primer region is raised into a flap by the strand displacement activity of DNA polymerase {delta}. The Dna2 helicase/nuclease and then flap endonuclease 1 (FEN1) are proposed to act sequentially in flap removal. Dna2 and FEN1 both employ a tracking mechanism to enter the flap 5' end and move toward the base for cleavage. In the current model, Dna2 must enter first, but FEN1 makes the final cut at the flap base, raising the issue of how FEN1 passes the Dna2. To address this, nuclease-inactive Dna2 was incubated with a DNA flap substrate and found to bind with high affinity. FEN1 was then added, and surprisingly, there was little inhibition of FEN1 cleavage activity. FEN1 was later shown, by gel shift analysis, to remove the wild type Dna2 from the flap. RNA can be cleaved by FEN1 but not by Dna2. Pre-bound wild type Dna2 was shown to bind an RNA flap but not inhibit subsequent FEN1 cleavage. These results indicate that there is a novel interaction between the two proteins in which FEN1 disengages the Dna2 tracking mechanism. This interaction is consistent with the idea that the two proteins have evolved a special ability to cooperate in Okazaki fragment processing

Dna2p Helicase/Nuclease Is a Tracking Protein, Like FEN1, for Flap Cleavage during Okazaki Fragment Maturation

During cellular DNA replication the lagging strand is generated as discontinuous segments called Okazaki fragments. Each contains an initiator RNA primer that is removed prior to joining of the strands. Primer removal in eukaryotes requires displacement of the primer into a flap that is cleaved off by flap endonuclease 1 (FEN1). FEN1 employs a unique tracking mechanism that requires the recognition of the free 5' terminus and then movement to the base of the flap for cleavage. Abnormally long flaps are coated by replication protein A (RPA), inhibiting FEN1 cleavage. A second nuclease, Dna2p, is needed to cleave an RPA-coated flap producing a short RPA-free flap, favored by FEN1. Here we show that Dna2p is also a tracking protein. Annealed primers or conjugated biotin-streptavidin complex block Dna2p entry and movement. Single-stranded binding protein-coated flaps inhibit Dna2p cleavage. Like FEN1, Dna2p can track over substrates with a non-Watson Crick base, such as a biotin, or a missing base within a chain. Unlike FEN1, Dna2p shows evidence of a "threading-like" mechanism that does not support tracking over a branched substrate. We propose that the two nucleases both track, Dna2p first and then FEN1, to remove initiator RNA via long flap intermediates

Dynamic removal of replication protein A by Dna2 facilitates primer cleavage during Okazaki fragment processing in Saccharomyces cerevisiae

Eukaryotic Okazaki fragments are initiated by an RNA/DNA primer, which is removed before the fragments are joined. Polymerase d displaces the primer into a flap for processing. Dna2 nuclease/helicase and flap endonuclease 1 (FEN1) are proposed to cleave the flap. The single-stranded DNA binding protein, replication protein A (RPA), governs cleavage activity. Flap-bound RPA inhibits FEN1. This necessitates cleavage by Dna2, which is stimulated by RPA. FEN1 then cuts the remaining RPA-free flap to create a nick for ligation. Cleavage by Dna2 requires that it enter the 5'-end and track down the flap. Since Dna2 cleaves the RPA-bound flap, we investigated the mechanism by which Dna2 accesses the protein-coated flap for cleavage. Using a nuclease-defective Dna2 mutant, we showed that just binding of Dna2 dissociates the flap-bound RPA. Facile dissociation is specific to substrates with a genuine flap, and will not occur with an RPA-coated single strand. We also compared the cleavage patterns of Dna2 with and without RPA to better define RPA stimulation of Dna2. Stimulation derived from removal of DNA folding in the flap. Apparently, coordinated with its dissociation, RPA relinquishes the flap to Dna2 for tracking in a way that does not allow flap structure to reform. We also found that RPA strand melting activity promotes excessive flap elongation, but it is suppressed by Dna2-promoted RPA dissociation. Overall, results indicate that Dna2 and RPA coordinate their functions for efficient flap cleavage and preparation for FEN1

Pif1 Helicase Lengthens Some Okazaki Fragment Flaps Necessitating Dna2 Nuclease/Helicase Action in the Two-nuclease Processing Pathway

We have developed a system to reconstitute all of the proposed steps of Okazaki fragment processing using purified yeast proteins and model substrates. DNA polymerase δ was shown to extend an upstream fragment to displace a downstream fragment into a flap. In most cases, the flap was removed by flap endonuclease 1 (FEN1), in a reaction required to remove initiator RNA in vivo. The nick left after flap removal could be sealed by DNA ligase I to complete fragment joining. An alternative pathway involving FEN1 and the nuclease/helicase Dna2 has been proposed for flaps that become long enough to bind replication protein A (RPA). RPA binding can inhibit FEN1, but Dna2 can shorten RPA-bound flaps so that RPA dissociates. Recent reconstitution results indicated that Pif1 helicase, a known component of fragment processing, accelerated flap displacement, allowing the inhibitory action of RPA. In results presented here, Pif1 promoted DNA polymerase δ to displace strands that achieve a length to bind RPA, but also to be Dna2 substrates. Significantly, RPA binding to long flaps inhibited the formation of the final ligation products in the reconstituted system without Dna2. However, Dna2 reversed that inhibition to restore efficient ligation. These results suggest that the two-nuclease pathway is employed in cells to process long flap intermediates promoted by Pif1

Influence du changement climatique sur la diversité inter et intra-spécifique des plantes cultivées à Tougou au nord du Burkina Faso

L’objectif de l’étude est d’évaluer la diversité taxonomique et la variabilité inter et intra-spécifique des plantes cultivées non spontanées dans un contexte de changement climatique. Les données sont collectées à l’aide d’une enquête au près de 75 producteurs menée suivant des interviews semi-structurées. Les résultats ont révélé l’existence de 122 écotypes appartenant à 49 espèces, 39 genres et 22 familles. La diversité interspécifique représentait 73% de la diversité des plantes cultivées au plan national. La variabilité intraspécifique était faible comparée à la variabilité au plan national. Elle était plus élevée chez les céréales et les cultures potagères pluviales avec 2 à 10 écotypes par espèce contre 1 à 3 écotypes par espèce chez les cultures maraîchères. Les écotypes ayant un cycle de plus de 90 jours représentant 10,5% de l’ensemble des écotypes recensés, sont menacés de disparition à cause du changement climatique. Une stratégie de collecte, de conservation, d’amélioration et de promotion de ces ressources ainsi que la restauration des écosystèmes dégradés s’avèrent nécessaire pour le maintien de la phytodiversité cultivée.Mots clés: Burkina Faso, agro biodiversité, écotype, espèces menacées, conservatio

Sustained response to infliximab treatment in two cases of early rheumatoid arthritis that has been maintained after drug withdrawal

The authors report two cases of active seropositive rheumatoid arthritis who were treated in an early phase of the disease with infliximab plus methotrexate obtaining a clinical remission. The benefit was maintained after the discontinuation of the anti-TNF alpha inhibitor for adverse events, indicating that the early administration of the drug may be followed by a sustained remission

Issue des accouchements sur utérus cicatriciel dans un hôpital universitaire au Burkina

Certains auteurs ont tendance à privilégier la césarienne comme méthode de prise en charge d’une parturiente porteuse d’un utérus cicatriciel.D’autres auteurs préconisent un accouchement par voie basse quand des paramètres cliniques précis sont observés. Le but de cette étude estd’analyser la prise en charge et l’issue des accouchements sur utérus cicatriciel au Centre Hospitalier Universitaire Souro Sanou de Bobo-Dioulasso et de la comparer aux différentes approches recommandées. Nous avons menés une étude transversale dans le Département de Gynécologie d’Obstétrique et de Médecine de la Reproduction du Centre Hospitalier Universitaire Sanou Souro de Bobo Dioulasso du 1er août 2006 au 1er août 2007 et a concerné 252 parturientes ayant un utérus cicatriciel parmi 4256 accouchements déroulés pendant la même période. Les accouchements sur utérus cicatriciels ont représenté 5,92 % de l’ensemble des accouchements dans notre département. La moyenne d'âge des patientes était de 26,2 ans et la parité moyenne de 4,3. Une césarienne d’emblée a été pratiquée chez 44% des parturientes ayant un utérus cicatriciel et 56 % parmi elles ont fait l'objet d'une épreuve utérine. Sur l’ensemble des épreuves utérines, 61% des parturientes ont accouché par voie basse. La mortalité maternelle était nulle et La mortalité périnatale était relativement importante. Les conditions d’acceptabilité de la voie basse ont été les mêmes chez toutes les patientes et un check liste a été proposé pour une meilleure prise en charge. L'épreuve utérine en salle d’accouchement doit être la règle à chaque fois que cela est possible chez une parturiente porteuse d’utérus cicatriciel. L’établissement d’un check liste pour accouchement par voie basse sur utérus cicatriciel facilite les prises de décision.Key words: Utérus cicatriciels, épreuve utérine, accouchement par voie basse, check-lis

Biochemical analyses indicate that binding and cleavage specificities define the ordered processing of human Okazaki fragments by Dna2 and FEN1

In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap during synthesis. Using biochemical techniques, binding and cleavage assays presented here indicate that when the flap is ∼30 nt long the nuclease Dna2 can bind with high affinity to the flap and downstream double strand and begin cleavage. When the polymerase idles or dissociates the Dna2 can reorient for additional contacts with the upstream primer region, allowing the nuclease to remain stably bound as the flap is further shortened. The DNA can then equilibrate to a double flap that can bind Dna2 and flap endonuclease (FEN1) simultaneously. When Dna2 shortens the flap even more, FEN1 can displace the Dna2 and cleave at the flap base to make a nick for ligation

Importance of the confirmatory assay for the detection of the HBsAg in the epidemiological studies and in the diagnosis of the viral Hepatitis B

Several epidemiological studies have reported high prevalence of HBsAg among pregnant women in Burkina Faso. They used various algorithms, as it is also done for the routine diagnostic. Knowing this antigen carriage rate in such a population or in other clinic attendees is important for the implementation of a national immunisation programme and the monitoring of patients with hepatitis B. Often, the screening tests were not confirmed in spite of the existence of known false positive and false negative results. The aim of this study was to determine a more accurate prevalence of HBsAg, among the pregnant women in Burkina Faso. From October 2006 to January 2007, blood samples were collected from 1139 pregnant women. Each sample was analyzed for HBsAg, using two assays and according to manufacturers’ instructions vis, Hepanostika®HBsAg Uniform II B9 (Bio-Mérieux; France) and HBsAg (V2) Abbott AxSYM® system (Abbott Diagnostics). All the positive samples were tested with a confirmatory neutralization assay- Hepanostika®HBsAg Uniform II B9 Confirmatory (Bio-Merieux). The mean age of the pregnant women was 24.85years [range: 15-45years] and the age range of 20-24 (37%) and 25-29 (25.4%) years were the most represented. The overall rate of HBsAg-positive pregnant women with the two screening assays was 20.9%. The HBsAg detection rate was significantly higher with Hepanostika® UniformII B9 (16.9%) than with HBsAg (V2) AxSYM system assay (12.1%), with

An Alternative Pathway for Okazaki Fragment Processing - Resolution of Fold-Back Flaps by Pif1 Helicase

Two pathways have been proposed for eukaryotic Okazaki fragment RNA primer removal. Results presented here provide evidence for an alternative pathway. Primer extension by DNA polymerase δ (pol δ) displaces the downstream fragment into an RNA-initiated flap. Most flaps are cleaved by flap endonuclease 1 (FEN1) while short, and the remaining nicks joined in the first pathway. A small fraction escapes immediate FEN1 cleavage and is further lengthened by Pif1 helicase. Long flaps are bound by replication protein A (RPA), which inhibits FEN1. In the second pathway, Dna2 nuclease cleaves an RPA-bound flap and displaces RPA, leaving a short flap for FEN1. Pif1 flap lengthening creates a requirement for Dna2. This relationship should not have evolved unless Pif1 had an important role in fragment processing. In this study, biochemical reconstitution experiments were used to gain insight into this role. Pif1 did not promote synthesis through GC-rich sequences, which impede strand displacement. Pif1 was also unable to open fold-back flaps that are immune to cleavage by either FEN1 or Dna2 and cannot be bound by RPA. However, Pif1 working with pol δ readily unwound a full-length Okazaki fragment initiated by a fold-back flap. Additionally, a fold-back in the template slowed pol δ synthesis, so that the fragment could be removed before ligation to the lagging strand. These results suggest an alternative pathway in which Pif1 removes Okazaki fragments initiated by fold-back flaps in vivo