The structural basis of replicative helicase loading onto DNA

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The DNA in our cells and that of other lifeforms gives the precise instructions on how life is shaped and works. To grow, renew and reproduce, biological cells must first duplicate their DNA, so that each daughter cell can receive the full genetic make-up from the mother cell. Only upon duplication, cell division occurs. DNA duplication is a carefully choreographed process, which is carried out by the "workers of the cell", large protein machineries, co-operatively assembled along many checkpoints to ensure faithful copying. Since the molecule DNA itself is shaped as a double helix, composed of two wound-up strands, it has to be unwound to grant access to the genetic information for the copy machinery. Unwinding is carried out by a tightly integrated complex of proteins, forming the enzyme called DNA helicase, but its activity can be occasionally misregulated, causing cellular stress or ageing, resulting in human disease or growth defects in plants. In the normal cell, the ring-shaped, pre-mature helicase is first being wrapped around DNA before its activation. We have recently uncovered detailed insights into the interactions of the pre-mature helicase with the protein that helps loading it around DNA and identified a stretch in a protein called Cdt1 that might act as a clamp to open and close the ring. The opening process is particular important later in the unwinding process and therefore has to be explored to extend our understanding of the normal versus an abnormal process. To study the helicase during the process of loading around DNA in vitro, we created several helicase variants that "freeze" the helicase loading process at specific points and enable us to study the outcome in detail using high-resolution cryo-electron microscopy and sophisticated computational methods. The mechanistic insight into accurate helicase loading onto DNA gained from the presented study will generate an overview of several essential early steps towards eventual unwinding of the DNA and DNA duplication. While this is particularly important for a basic understanding of how our cell works, it has important implications for human diseases such as cancer but also will more immediately support research into inhibitors of infectious diseases.