Crystal Structure of T7 Gene 4 Ring Helicase Indicates a Mechanism for Sequential Hydrolysis of Nucleotides

AbstractWe have determined the crystal structure of an active, hexameric fragment of the gene 4 helicase from bacteriophage T7. The structure reveals how subunit contacts stabilize the hexamer. Deviation from expected six-fold symmetry of the hexamer indicates that the structure is of an intermediate on the catalytic pathway. The structural consequences of the asymmetry suggest a “binding change” mechanism to explain how cooperative binding and hydrolysis of nucleotides are coupled to conformational changes in the ring that most likely accompany duplex unwinding. The structure of a complex with a nonhydrolyzable ATP analog provides additional evidence for this hypothesis, with only four of the six possible nucleotide binding sites being occupied in this conformation of the hexamer. This model suggests a mechanism for DNA translocation

Structures of RecBCD in complex with phage-encoded inhibitor proteins reveal distinctive strategies for evasion of a bacterial immunity hub

Following infection of bacterial cells, bacteriophage modulate double-stranded DNA break repair pathways to protect themselves from host immunity systems and prioritise their own recombinases. Here, we present biochemical and structural analysis of two phage proteins, gp5.9 and Abc2, which target the DNA break resection complex RecBCD. These exemplify two contrasting mechanisms for control of DNA break repair in which the RecBCD complex is either inhibited or co-opted for the benefit of the invading phage. Gp5.9 completely inhibits RecBCD by preventing it from binding to DNA. The RecBCD-gp5.9 structure shows that gp5.9 acts by substrate mimicry, binding predominantly to the RecB arm domain and competing sterically for the DNA binding site. Gp5.9 adopts a parallel coiled-coil architecture that is unprecedented for a natural DNA mimic protein. In contrast, binding of Abc2 does not substantially affect the biochemical activities of isolated RecBCD. The RecBCD-Abc2 structure shows that Abc2 binds to the Chi-recognition domains of the RecC subunit in a position that might enable it to mediate the loading of phage recombinases onto its single-stranded DNA products

Insights into Chi recognition from the structure of an AddAB-type helicase–nuclease complex

Homologous recombination DNA repair requires double-strand break resection by helicase–nuclease enzymes. The crystal structure of bacterial AddAB in complex with DNA substrates shows that it employs an inactive helicase site to recognize ‘Chi' recombination hotspot sequences that regulate resection

Chi hotspots trigger a conformational change in the helicase-like domain of AddAB to activate homologous recombination

In bacteria, the repair of double-stranded DNA breaks is modulated by Chi sequences. These are recognised by helicase-nuclease complexes that process DNA ends for homologous recombination. Chi activates recombination by changing the biochemical properties of the helicase-nuclease, transforming it from a destructive exonuclease into a recombination-promoting repair enzyme. This transition is thought to be controlled by the Chi-dependent opening of a molecular latch, which enables part of the DNA substrate to evade degradation beyond Chi. Here, we show that disruption of the latch improves Chi recognition efficiency and stabilizes the interaction of AddAB with Chi, even in mutants that are impaired for Chi binding. Chi recognition elicits a structural change in AddAB that maps to a region of AddB which resembles a helicase domain, and which harbours both the Chi recognition locus and the latch. Mutation of the latch potentiates the change and moderately reduces the duration of a translocation pause at Chi. However, this mutant displays properties of Chi-modified AddAB even in the complete absence of bona fide hotspot sequences. The results are used to develop a model for AddAB regulation in which allosteric communication between Chi binding and latch opening ensures quality control during recombination hotspot recognition

Антикризисное управление

Учебно-методический комплекс (УМК) по учебной дисциплине «Ан-тикризисное управление» создан в соответствии с требованиями Положения об учебно-методическом комплексе на уровне высшего образования, утвер-жденного постановлением Министерства образования Республики Беларусь от 26.07.2011 № 167, предназначен для реализации содержания образова-тельной программы для обучающихся первой ступени высшего образования для студентов специальности 1-26 02-02 «Менеджмент». Содержание разделов УМК соответствует образовательному стандарту высшего образования данной специальности. Главная цель УМК – оказание методической помощи магистрантам в освоении и систематизации учебного материала в процессе обучения и под-готовки к аттестации по дисциплине «Антикризисное управление». УМК включает: 1. Теоретический раздел (конспект лекций, аннотированный перечень основных учебных и научно-практических изданий). 2. Практический раздел (тематика практических занятий по дисци-плине в соответствии с учебным планом и учебной программой). 3. Контроль работы обучающихся (материалы для текущей аттестации, позволяющие определить соответствие учебной деятельности обучающихся требованиям образовательного стандарта высшего образования и учебно-программной документации, в т.ч. вопросы для подготовки к зачету). 4. Вспомогательный раздел (содержание учебного материала учебной дисциплины; методические рекомендации по организации самостоятельной работы магистрантов; информационно-аналитические материалы: список ре-комендуемой литературы, перечень электронных ресурсов и их адреса; при-мерный перечень тем для написания рефератов). Работа с УМК должна включать на первом этапе ознакомление с со-держанием учебного материала учебной дисциплины, посредством которого можно получить информацию о тематике лекций и практических занятий, перечнях рассматриваемых вопросов и рекомендуемой для их изучения ли-тературы. Для подготовки к практическим занятиям необходимо использо-вать материалы, представленные в Теоретическом и Практическом разделах. В основу структуры программы легла учебная программа «Антикри-зисное управление» Белорусского государственного университета для выс-ших учебных заведений по специальности Э.01.03.00 «Экономика и управле-ния на предприятии» (регистрационный № ТД-182/баз.)

Teaching a new dog old tricks?

AbstractThe recently determined crystal structures of fragments of the human and vaccinia virus type IB topoisomerases reveal unexpected similarity with the lambda family of site-specific recombinases. The conservation of structure suggests a common mechanism, indicating that topoisomerase activity may be the consequence of uncoupling DNA strand cleavage/religation from synapsis

Distinct roles for ATP binding and hydrolysis at individual subunits of an archaeal clamp loader

Circular clamps are utilised by replicative polymerases to enhance processivity. The topological problem of loading a toroidal clamp onto DNA is overcome by ATP-dependent clamp loader complexes. Different organisms use related protein machines to load clamps, but the mechanisms by which they utilise ATP are surprisingly different. Using mutant clamp loaders that are deficient in either ATP binding or hydrolysis in different subunits, we show how the different subunits of an archaeal clamp loader use ATP binding and hydrolysis in distinct ways at different steps in the loading process. Binding of nucleotide by the large subunit and three of the four small subunits is sufficient for clamp loading. However, ATP hydrolysis by the small subunits is required for release of PCNA to allow formation of the complex between PCNA and the polymerase, while hydrolysis by the large subunit is required for catalytic clamp loading