In vitro biochemical reconstitution and electron microscopy of early pre-DNA replication complex intermediates using human proteins

Abstract

Cell division is essential for cellular life, from single-celled organisms to higher-order metazoans. To support cellular and organism function – including proliferation, genomic information must be duplicated precisely once under highly regulated conditions to ensure genetic integrity. Dysregulation in this process can have detrimental consequences, such as carcinogenesis and developmental disorders. Across the tree of life, DNA replication initiates from discrete sites (known as origins). These are recognised by specific protein initiators that collaborate with co-factor proteins to load the machinery used for DNA replication. The core of this machinery is the replicative helicase, essential for unwinding duplex DNA to allow for semiconservative bidirectional replication. This research set out to examine the mechanistic aspects behind origin recognition and helicase loading using human proteins. Full-length H. sapiens proteins were purified and tested using a newly established in vitro method to assess helicase loading. Using a combination of biochemical and structural approaches, intermediates during the helicase loading process were studied in greater detail. Regulation of DNA replication is highly dependent on intrinsic ATP hydrolysis of these proteins, although the precise mechanisms behind this are not well-defined. Using hydrolysis mutants, we were able to study how coordinated ATP hydrolysis contributes to helicase loading, as well as identifying and trapping short-lived intermediates during this process. Taken together, the results and methodologies in this thesis provide fundamental insight into how human replication machinery is assembled on a molecular level and how individual characteristics of these proteins contribute to faithful and efficient replication of the genome.