Structural Synergy and Molecular Crosstalk between Bacterial Helicase Loaders and Replication Initiators

SummaryThe loading of oligomeric helicases onto replication origins marks an essential step in replisome assembly. In cells, dedicated AAA+ ATPases regulate loading, however, the mechanism by which these factors recruit and deposit helicases has remained unclear. To better understand this process, we determined the structure of the ATPase region of the bacterial helicase loader DnaC from Aquifex aeolicus to 2.7 Å resolution. The structure shows that DnaC is a close paralog of the bacterial replication initiator, DnaA, and unexpectedly shares an ability to form a helical assembly similar to that of ATP-bound DnaA. Complementation and ssDNA-binding assays validate the importance of homomeric DnaC interactions, while pull-down experiments show that the DnaC and DnaA AAA+ domains interact in a nucleotide-dependent manner. These findings implicate DnaC as a molecular adaptor that uses ATP-activated DnaA as a docking site for regulating the recruitment and correct spatial deposition of the DnaB helicase onto origins

An expert panel Delphi consensus statement on the use of palliative care in the management of patients with pulmonary arterial hypertension

Mortality in pulmonary arterial hypertension (PAH) remains high and referral to palliative or supportive care (P/SC) specialist services is recommended when appropriate. However, access to P/SC is frequently a challenge for patients with a noncancer diagnosis and few patients living with PAH report P/SC involvement in their care. A modified Delphi process of three questionnaires completed by a multidisciplinary panel ( = 15) was used to develop expert consensus statements regarding the use of P/SC to support patients with PAH. Panelists rated their agreement with each statement on a Likert scale. There was a strong consensus that patients should be referred to P/SC when disease symptoms become unmanageable or for end-of-life care. Services that achieved consensus were pain management techniques, end-of-life care, and psychosocial recommendations. Palliative or supportive care should be discussed with patients, preferably in-person, when disease symptoms become unmanageable, when starting treatment, when treatment-related adverse events occur or become refractory to initial intervention. Care partners and patient support groups were considered important in improving a patient\u27s overall health outcomes, treatment adherence, and perception of care. Most patients with PAH experience cognitive and/or psychosocial changes and those who receive psychosocial management have better persistence and/or compliance with their treatment. These consensus statements provide guidance to healthcare providers on the who and when of referral to palliative care services, as well as the importance of focusing on the psychosocial aspects of patient care and quality of life

Harnessing type I CRISPR–Cas systems for genome engineering in human cells

Type I CRISPR–Cas systems are the most abundant adaptive immune systems in bacteria and archaea1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation5–7. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain8–11 and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI–Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade–Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR–Cas systems can be harnessed for genome engineering applications in eukaryotic cells