Structural and biochemical study of replication fork protection complex in S-phase checkpoint activation

Abstract

Faithful transmission of genetic information from parent cells to daughter cells is crucial for the survival of all eukaryotic organisms. To achieve high fidelity of inheritance, cells need to resist constant insults on genome from both environmental and endogenous sources which may lead to loss of genome integrity. Eukaryotic cells have evolved the surveillance mechanisms termed checkpoints for DNA damage detection and response. The S-phase checkpoint is activated by DNA damages during the replication process. Although the cascade of the S-phase checkpoint activation has been revealed to a great extent, the molecular basis of its intricate protein interaction network is not well understood. My studies focus on the interaction network of the replication fork protection complex (FPC) including Tipin and Timeless, which preserves the replication fork structure and facilitates the sufficient phosphorylation of Chk1 in the S-phase checkpoint activation. In Chapter 2, X-ray crystallography and biochemical binding assays were employed to define the complete RPA32 recognition mode shared by multiple RPA32-interacting proteins, which represents the molecular mechanism of Tipin recruitment by RPA32. Tipin has been considered as a constitutive binding partner of Timeless. In Chapter 3, my study focused on Timeless and has for the first time identified the physical interaction between Timeless and PARP-1. The crystal structures were determined for the Timeless PARP-1-binding domain in free form and in complex with PARP-1 catalytic domain which is the first reported PARP-1 crystal structure in complex with any of its interacting proteins. In vitro PARP-1 auto-modification assay indicates that the association of Timeless does not interfere with PARP-1 enzymatic activity. Interestingly, Timeless specifically interacts with PARP-1, but not PARP-2 or PARP-3. The PARP-1 and Timeless interaction raises the possibility that PARP-1 functions together with Timeless in the S-phase checkpoint pathway, while the preliminary data also supports that Timeless functions in homologous recombination for DSB repair which has not been demonstrated before.

The second part of my work presented in Chapter 4 focuses on human UHRF1. UHRF1 has been extensively characterized for its function in histone methylation and DNA methylation maintenance, while the molecular mechanism of histone H3 recognition by UHRF1 remains elusive. The crystal structure of UHRF1 PHD finger in complex with histone H3 N-terminal tail is presented, demonstrating that PHD finger of UHRF1 recognizes the N-terminus of histone H3 containing unmodified R2 and K4. Structural analysis together with biochemical assays indicated that UHRF1 PHD finger and the adjacent dTudor domain act as one functional unit to specifically recognize the H3K9me3 mark. Based on these observations, a novel model is proposed demonstrating that histone methylation and DNA methylation reinforce each other through UHRF1, which is supported by experimental evidence from other groups in later studies.