Molecular mechanisms at the crossroads between DNA replication and chromosome cohesion establishment
Structural Biology
Final Report Abstract
DNA replication is fundamental to the propagation of cellular life. The basic process of separating a DNA duplex and copying the two strands must be well coordinated and tightly regulated. Any disturbances, termed replication stress, can be highly detrimental to cells, and must be dealt with and signalled to the whole cell to prevent cancer formation. On top of this, the replication machinery, termed the ‘replisome’, also has to transfer epigenetic marks and organisational elements from the front of the replication fork to the two new strands. One particularly enigmatic process is cohesion establishment, where cohesin molecules encircle the new DNA duplexes to ensure that they are transferred to the correct daughter cell during mitosis. The importance of this for human health is underlined by the severe developmental disorders termed cohesinopathies which occur when there are defects in cohesion. Furthermore, during cancer, cells experience increased replication stress, and many replisome proteins become essential, making them excellent drug targets to selectively kill cancer cells. To further our understanding of cohesion establishment and replication stress we determined how two replisome components function at a molecular level: the Ctf18-RFC alternative clamp loader, and the Tof1-Csm3 fork protection complex. Ctf18-RFC loads the sliding clamp PCNA, which is needed to DNA polymerases to synthesise DNA processively, and also as an interaction hub to regulate processes at the DNA replication fork. However, it is unclear when and where Ctf18-RFC loads PCNA. Using a combination of integrative structural biology and yeast genetics we showed that Ctf18-RFC forms a constitutive complex with the leading strand polymerase DNA Pol ε, positioning Ctf18-RFC as the leading strand clamp loader. The flexibility of this interaction enables the polymerase machine to cycle through the DNA synthesis reaction without interference. Even slightly disrupting the interaction prevented activation of the DNA replication checkpoint. We proposed that Ctf18-RFC is positioned to sense stalling by DNA Pol ε. Our work also showed that Ctf18-RFC works together with two other cohesion establishment factors, Mrc1 and Chl1, to maintain genome stability during replication. Our work paves the way for future work to study how the PCNA loading activity of Ctf18-RFC works in replication checkpoint activation and cohesion establishment. The Tof1-Csm3 complex protects stalled replication forks while also enabling cohesion establishment and checkpoint responses. To understand the molecular basis of its diverse functions we solved the crystal structure of this complex. This revealed that it forms an extended alpha solenoid protein, suggesting a role as a scaffolding protein at the fork. To further investigate this, we characterised and mapped its interactions with the checkpoint mediator Mrc1 and with DNA. This information will now help to dissect the role of these interactions in vivo.
Publications
- Crystal Structure and interactions of the Tof1-Csm3 (Timeless-Tipin) Fork Protection Complex. 2020 Nucleic Acids Research, 48 (12), 6996-7004
Grabarczyk DB
(See online at https://doi.org/10.1093/nar/gkaa456) - Ctf18-RFC and DNA Pol ε form a stable leading strand polymerase/clamp loader complex required for normal and perturbed DNA replication. 2020 Nucleic Acids Research, 48 (14), 8128-45
Stokes K, Winczura A, Song B, De Piccoli G and Grabarczyk DB
(See online at https://doi.org/10.1093/nar/gkaa541)
