Unveiling the orientation, conformations, and contacts of replicative hexameric DNA helicases that govern DNA replication dynamics.
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Perera, Himasha M., 1990-
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Replication of chromosomal DNA is an intricate process carefully orchestrated in all living organisms by a multiprotein molecular machine known as the replisome. The replicative DNA helicase motor protein is at the forefront of the replisome performing a vital process in DNA replication by unwinding duplex DNA at the replication fork. As a stable component that serves as the platform for the assembly of other replisome proteins, significant efforts have been made over the past several decades to characterize functional properties of this enzyme using model organisms. This collection of work describes our contributions to unveil the orientation, conformations, and contacts of replicative hexameric DNA helicases from two different model organisms, archaeal Saccharolobus solfataricus (Sso) and bacterial Escherichia coli (E. coli). DNA replication is controlled by helicase loading at replication origins, preferentially encircling one DNA strand prior to unwinding in a defined orientation. Using archaeal SsoMCM helicase we discovered that the active unwinding orientation of SsoMCM helicase places its N-terminal domain at the DNA replication fork. This work also explores how an interdisciplinary combination of biochemical, chemical, structural biology, and kinetic methods can provide greater insight into more thoroughly understanding active unwinding orientation of helicases in other organisms. DNA replication requires tight coordination between DNA unwinding and synthesis in the replisome. In E. coli replisome, this coordination is maintained by the interaction between E. coli DnaB helicase and Polymerase III holoenzyme (HE) that performs unwinding and synthesis respectively. Using site specific mutations in the DnaB helicase, we discovered specific helicase contacts that accelerates unwinding and decouple the replisome leading to impaired DNA synthesis. These results set the platform for future investigations of specific molecular contacts and kinetic control within the DnaB helicase that leads to replisome decoupling. Altogether, this work contributes to the fascination of understanding the role of replicative DNA helicases in DNA replication in detail which will undoubtedly be valuable in designing targeted therapeutic approaches for various diseases including cancer.