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Es presenta el model atòmic a 4.5 Å de DnaB, la principal helicasa replicativa bacteriana, d'Aquifex aeolicus. És un anell hexamèric de 100 Å d'amplada i 80 Å d'alçada amb dues capes de simetria diferenciada, la dels dominis N-terminals en C3 i la dels C-terminals propera a C6. El diàmetre central és de 25 Å al llarg d'ambdues capes, principal diferència amb les estructures prèvies, on era 25 Å més estret a la capa N-terminal. L'estretament s'origina pel trencament d'una de les dues superfícies d'interacció entre monòmers N-terminals, cosa que augmenta la flexibilitat del subdomini implicat. Només l'ssDNA pot atravessar l'anell, quan a les estructures prèvies hi podia passar tant ssDNA com dsDNA. L'estructura aquí presentada és més propera a la conformació funcional de DnaB durant la realització de l'activitat helicasa, mentre que les anteriors correspondrien a la forma inactiva o a la conformació capaç de translocar-se sobre dsDNA. / DnaB is the main replicative helicase in bacteria. An atomic model for the DnaB from Aquifex aeolicus at a 4.5 Å resolution is presented. It´s a ring-shaped homohexamer (100 Å width and 80 Å hight) with two simmetry layers, a C3 N-terminal layer and an almost C6 C-terminal one. The diameter of the central channel is 25 Å along both layers, being the main diference with the previously solved structures, which were 25 Å smaller along the N-terminal layer. This is due to one of the previous interacting surphaces being lost in the current structure, thus enabling a higher felxibility of the subdomain involved. Only ssDNA can pass trhough the ring, while both ssDNA and dsDNA could in the previous structures. So, the present structure is closer to the functional conformation, while the previous ones would correspond to the inactive form or the conformation that is only able to translocate along dsDNA.

Bacillus anthracis is a Gram positive, spore forming, non-motile, rod shaped, soil bacterium, and is endemic worldwide. Currently, the biology of B. anthracis is poorly understood. B. anthracis is one of many biological weapons used today. A -/- mutant strain of B. anthracis that lacks the pathogenic plasmids was created by serial culture at 42°C. Key DNA replication genes were identified by homology as targets. The dnaB gene, essential for B. subtilis initiation of DNA replication, was my focus. A vector system was created by polymerase chain reaction (PCR) with the pMUTIN4 integration vector and the promoter region of dnaB to study the genetics of B. anthracis. An electro-transformation system was formulated to knock-out the -/- B. anthracis dnaB gene. We have successfully incorporated the pMUTIN4 vector into the chromosomal DNA of B. anthracis. We also have formulated an electro-transformation system and vector system for use in B. anthracis.

Accurate replication of the genome is essential for reproduction in all cells. However, even under normal conditions, the replication machinery may face a variety of impediments that can prevent it from completing its task. The mechanism by which cells overcome these hurdles is likely to vary depending upon the nature of the obstacle. Both UV irradiation and inactivation of replicative proteins in DnaB can inhibit the progression of the DNA replication machinery. However, the mechanism by which replication recovers following UV irradiation is different from the mechanism of recovery following the inactivation of the replicative proteins. Previous results show that following UVinduced damage in Escherichia coli, the replication fork is maintained and protected from extensive degradation by RecF, RecO, and RecR until replication can resume. By contrast, replication does not recover following inactivation of the replication protein DnaB, and the nascent DNA is extensively degraded irrespective of whether RecF is present. In this study, we verified DNA replication arrest by monitoring the total DNA accumulation and rate of DNA synthesis following UV-induced DNA damage and inactivation of thermosensitive replication alleles, such as dnaB266. We measured the amount of nascent DNA degradation, allowing us to determine how the newly synthesized strand of DNA is affected following replication fork arrest. Our data indicate that following inactivation of DnaB266, the replication fork is not maintained and is subject to extensive degradation. The degradation that occurs after DnaB266 inactivation is partially reduced in the absence of RecF-O-R, RecJ, and ExoI, suggesting that DNA processing by these enzymes occurs after DnaB arrest. In addition, two-dimensional agarose gel analysis revealed that unique structural intermediates accumulated following inactivation of DnaB266. These observations indicate that the recovery of replication when impeded by DNA lesions, such as those produced by UVirradiation, is maintained and processed through mechanisms that do not resemble the events occurring when replication proteins are inactivated.