Unveiled the mechanism by which helicases load onto DNA

A work just published in Molecular Cell by Dr. Ernesto Arias, principal investigator of the Cryo-EM of Macromolecular Machines group at Centro de Investigaciones Biológicas (CSIC), reveals the high-resolution cryo-EM structures of the E. Coli helicase-loader complex with and without DNA, and sheds light on the mechanism by which helicases are recruited to the origin of replication of DNA.

Duplication of genetic information is an essential process for all living organisms. DNA replication, thus, has to occur in a timely and faithful manner to prevent the generation of mutations and chromosomal aberrations that may be lethal for the cell. During this process, hexameric, ring-shaped molecular motors called helicases unwind the parental duplex and lead the progression of the molecular machinery responsible for duplicating the DNA. One of the key steps that controls the onset of DNA replication is the recruitment, or loading, of the helicase at the origin of replication. In cells, where the DNA molecules do not contain free ends, helicase loading does not occur spontaneously, but requires the help of dedicated factors. In E. coli, a model organism, the replicative helicase and loader are known as DnaB and DnaC, respectively. The DnaB/DnaC system has been used as a textbook model for understanding DNA replication initiation, however, after decades of research; the molecular bases of this process remain unknown. This work, result of an international collaboration, offers a high-resolution view of the E. coli DnaB/DnaC complex in a pre- and post-loading state, and answers long-standing questions in the field.

The high-resolution cryo-EM experiments performed at the CIB have allowed to obtain sub-4 Å-resolution structures, essential for understanding fundamental steps of the process at atomic level. In the absence of DNA, six DnaC protomers latch onto a DnaB hexamer using an extended N-terminal domain to break the helicase ring and allow DNA passage into the central channel of the motor protein. Upon binding DNA, DnaC hydrolyzes ATP, allowing DnaB to re-seal around the nucleic acid and isomerize into a topologically closed state. Interestingly, nucleic acid engagement remodels the helicase that adopts now a translocation-competent configuration, which is also primed to recruit other key factors of the DNA replication machinery. Collectively, this work provides the first high-resolution view of how replicative helicase loading occurs in bacteria and explains how this mechanism both parallels and diverges from homologous hexameric helicases and other essential cellular systems.

Moreover, since DNA replication is crucial for cellular proliferation, from a biomedical perspective the results obtained by Arias et al. provide important structural and functional information that might help to design new drugs for controlling bacterial infections.

Reference: Physical basis for the loading of a bacterial replicative helicase onto DNA. Ernesto Arias-Palomo, Neha Puri, Valerie L. O'Shea Murray, Qianyun Yan and James M. Berger. Molecular Cell (2019) https://doi.org/10.1016/j.molcel.2019.01.023

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