Structural insights into DNA lagging strand replication and translesion synthesis

DNA replication requires the coordination of many different enzymes at the primer terminus. The eukaryotic sliding clamp, proliferating cell nuclear antigen (PCNA), encircles DNA at primer-template junctions, providing a platform for DNA polymerases and other enzymes to bind and increasing their processivity and activity on DNA. Many DNA processing enzymes contain a conserved PIP-box motif, which binds a hydrophobic pocket on PCNA.

This thesis focuses on two processes: Okazaki fragment maturation and translesion synthesis (TLS). In the former, the lagging strand polymerase (Pol) δ synthesises each Okazaki fragment upstream of the last, displacing a portion of the downstream fragment. Flap endonuclease (FEN1) cleaves the resulting flap, and a ligase enzyme seals the nick. In TLS, Pol δ stalls opposite a DNA lesion. It must then be replaced by a TLS polymerase, which can bypass the lesion, before resuming synthesis downstream.

Both processes involve sequential action of enzymes. As PCNA is a homotrimer with three PIP-box binding pockets, it may recruit more than one of these enzymes simultaneously for a fast and efficient changeover. This simultaneous binding, the “toolbelt” model, has been demonstrated experimentally in archaea and bacteria. Evidence of PCNA toolbelts in eukaryotes has proven more elusive, and this thesis set out to explore the notion further.

Using single-particle cryo-electron microscopy, the work presented here has uncovered structural data of different polymerases forming holoenzymes with PCNA and primer-template DNA. This work shows that Pol δ binds PCNA at the side, leaving room for a second binding partner – and indeed we show structural evidence that FEN1 can bind PCNA simultaneously, forming a toolbelt. TLS Pol κ also binds towards one side of PCNA, inviting the hypothesis of a TLS toolbelt.

Together, this work provides an insight into how the interactions of enzymes with PCNA facilitates enzyme switching at the primer terminus.