The signature motif of the Saccharomyces cerevisiae Pif1 DNA helicase is essential in vivo for mitochondrial and nuclear functions and in vitro for ATPase activity

Pif1 family DNA helicases are conserved from bacteria to humans and have critical and diverse functions in vivo that promote genome integrity. Pif1 family helicases share a 23 amino acid region, called the Pif1 signature motif (SM) that is unique to this family. To determine the importance of the SM, we did mutational and functional analysis of the SM from the Saccharomyces cerevisiae Pif1 (ScPif1). The mutations deleted portions of the SM, made one or multiple single amino acid changes in the SM, replaced the SM with its counterpart from a bacterial Pif1 family helicase and substituted an α-helical domain from another helicase for the part of the SM that forms an α helix. Mutants were tested for maintenance of mitochondrial DNA, inhibition of telomerase at telomeres and double strand breaks, and promotion of Okazaki fragment maturation. Although certain single amino acid changes in the SM can be tolerated, the presence and sequence of the ScPif1 SM were essential for all tested in vivo functions. Consistent with the in vivo analyses, in vitro studies showed that the presence and sequence of the ScPif1 SM were critical for ATPase activity but not substrate binding

Regulation of yeast DNA polymerase δ-mediated strand displacement synthesis by 5\u27-flaps

The strand displacement activity of DNA polymerase δ is strongly stimulated by its interaction with proliferating cell nuclear antigen (PCNA). However, inactivation of the 3′–5′ exonuclease activity is sufficient to allow the polymerase to carry out strand displacement even in the absence of PCNA. We have examined in vitro the basic biochemical properties that allow Pol δ-exo(−) to carry out strand displacement synthesis and discovered that it is regulated by the 5′-flaps in the DNA strand to be displaced. Under conditions where Pol δ carries out strand displacement synthesis, the presence of long 5′-flaps or addition in trans of ssDNA suppress this activity. This suggests the presence of a secondary DNA binding site on the enzyme that is responsible for modulation of strand displacement activity. The inhibitory effect of a long 5′-flap can be suppressed by its interaction with single-stranded DNA binding proteins. However, this relief of flap-inhibition does not simply originate from binding of Replication Protein A to the flap and sequestering it. Interaction of Pol δ with PCNA eliminates flap-mediated inhibition of strand displacement synthesis by masking the secondary DNA site on the polymerase. These data suggest that in addition to enhancing the processivity of the polymerase PCNA is an allosteric modulator of other Pol δ activities

Pif1 removes a Rap1-dependent barrier to the strand displacement activity of DNA polymerase δ

Using an in vitro reconstituted system in this work we provide direct evidence that the yeast repressor/activator protein 1 (Rap1), tightly bound to its consensus site, forms a strong non-polar barrier for the strand displacement activity of DNA polymerase δ. We propose that relief of inhibition may be mediated by the activity of an accessory helicase. To this end, we show that Pif1, a 5′–3′ helicase, not only stimulates the strand displacement activity of Pol δ but it also allows efficient replication through the block, by removing bound Rap1 in front of the polymerase. This stimulatory activity of Pif1 is not limited to the displacement of a single Rap1 molecule; Pif1 also allows Pol δ to carry out DNA synthesis across an array of bound Rap1 molecules that mimics a telomeric DNA-protein assembly. This activity of Pif1 represents a novel function of this helicase during DNA replication

Ultrasound Effectiveness of Steroid Injection for hand Psoriatic Dactylitis: Results from a Longitudinal Observational Study

Introduction: To assess clinical and ultrasound effectiveness of steroid injection (local treatment, LT) into the digital flexor tendon sheath for the treatment of psoriatic dactylitis compared to systemic treatment (ST) alone. Methods: In this observational, multicentre, prospective study, 88 cases of symptomatic hand dactylitis were evaluated clinically and sonographically by high-frequency ultrasound (US) probe in both greyscale (GS) and power Doppler (PD). The presence of flexor tenosynovitis (FT), soft tissue oedema (STO), peritendon extensor inflammation and synovitis was assessed (including DACtylitis glObal Sonographic—DACTOS—score) before treatment, at 1-month (T1) and 3-months (T3) follow-up. LT was proposed to all patients. Patients refusing LT were treated with oral NSAIDs. Patients continued the same baseline csDMARDs and/or corticosteroid therapy during the whole follow-up period. US response was defined for DACTOS score < 3 and US remission for DACTOS score = 0. Results: At T3 evaluation the ST group showed a significantly higher persistence (grade > 1) of FT and STO (p < 0.001 for all) and MCP synovitis (p = 0.001). US remission was achieved only in the LT group (at T3 31% vs. 0, p < 0.001). The percentage of patients with DACTOS < 3 was significantly greater in the LT group compared with ST group, at both T1 (49% vs. 5%, p < 0.001) and T3 evaluation (76% vs. 7%, p < 0.001). In multiple conditional logistic regression analysis, the only factor associated with US remission was LT (T3 odds ratio = 41.21, p < 0.001). Conclusions: US confirmed the effectiveness of steroid injection for dactylitis by demonstrating that it involves the resolution of extra-articular inflammation, in particular FT and STO

Rad51 Polymerization Reveals a New Chromatin Remodeling Mechanism

Rad51 protein is a well known protagonist of homologous recombination in eukaryotic cells. Rad51 polymerization on single-stranded DNA and its role in presynaptic filament formation have been extensively documented. Rad51 polymerizes also on double-stranded DNA but the significance of this filament formation remains unclear. We explored the behavior of Saccharomyces cerevisiae Rad51 on dsDNA and the influence of nucleosomes on Rad51 polymerization mechanism to investigate its putative role in chromatin accessibility to recombination machinery. We combined biochemical approaches, transmission electron microscopy (TEM) and atomic force microscopy (AFM) for analysis of the effects of the Rad51 filament on chromatinized templates. Quantitative analyses clearly demonstrated the occurrence of chromatin remodeling during nucleoprotein filament formation. During Rad51 polymerization, recombinase proteins moved all the nucleosomal arrays in front of the progressing filament. This polymerization process had a powerful remodeling effect, as Rad51 destabilized the nucleosomes along considerable stretches of DNA. Similar behavior was observed with RecA. Thus, recombinase polymerization is a powerful mechanism of chromatin remodeling. These remarkable features open up new possibilities for understanding DNA recombination and reveal new types of ATP-dependent chromatin dynamics

eHospital Safety Net ProgeHospital Safety Net Program: Defining the Transfusion Care Gap

The eHospital Safety Net Program is designed to remotely monitor gaps in adult patient care across the 21 hospitals in Northern California to reduce risk and prevent harm. A physician team identified transfusions as a care gap, however, the initial definition was broad and did not provide actionable data. This project used the PDSA model for change developed by W. Edward Deming, to test and refine the transfusion care gap definition. The eHospital team of nurses evaluated the frontline nursing documentation tool in the electronic medical record and developed a list of critical documentation items related to safe transfusion administration. A series of five PDSA cycles were conducted during February, 2015, to refine the critical items and standardize the care gap definition. As of April, all 21 hospitals were enrolled in the eHospital Safety Net Program. The preliminary results display the raw count for each critical item missing from frontline documentation. These results afford the adult care services team actionable data to assist in providing safe care. In addition, results can be used for comparison between hospitals for the purpose of identifying high performers and sharing knowledge on successful improvement strategies to benefit patients