Contacts and Confirmations that Regulate Hexameric Helicases at the Replication Fork

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Duplication of the genome requires efficient, rapid, and coordinated activities of many enzymes that interact within a larger replisome complex. The hexameric helicase is at the leading front of the replisome and is a nexus of control for both the initiation and elongation phases of DNA replication. These proteins are comprised of a N‐terminal DNA binding domain and a C‐terminal motor domain that assemble into a toroidal hexameric structure. Two main superfamilies of hexameric helicases exist: the RecA family, which contain helicases from phage and bacteria, and the AAA+ family, which contain helicases from archaea and eukaryotes. Interestingly, the measured translocation polarity of 5′‐3′ or 3′‐5′, respectively, dictates the orientation of these helicases at the replication fork.Using site specific DNA cleavage experiments and presteady‐state FRET, we have unequivocally shown the then N‐terminal domain of the archaeal MCM helicase leads the way for unwinding (N‐first). The MCM hexamer itself can bind model DNA substrates in multiple conformations; however, the correct ‘N‐first’ conformation is dictated by 1) the presence of an excluded strand, 2) the ssDNA polarity (3′‐5′), and 3) the C‐terminal winged‐helix (WH). This orientation is opposite to that of the bacterial DnaB helicase which proceeds C‐first in the 5′‐3′ direction.To determine the cellular impact of the excluded strand interaction in the steric exclusion and unwinding model (SEW), we created and tested several CRISPR/Cas9 genetic mutations of E. coli dnaB that correlate with mutations on the exterior surface. These DnaB mutations alter the conformation of the hexamer creating a spectrum of constricted states that all result in faster DNA unwinding. Genetic dnaB mutations lead to decoupling within the replisome, buildup of ssDNA, activation of the SOS response, and loss of genomic integrity. Therefore, the SEW interaction and the central channel diameter is adaptive and dynamic to properly maintain the DNA unwinding within the confines of the replisome to regulate speed of DNA replication and prevent chromosomal instability.Support or Funding InformationNational Science Foundation Division of Molecular and Cellular Biosciences (NSF1613534)