Wade, BO; (2017) Structural and Biochemical Studies on Cohesion Establishment. Doctoral thesis , UCL (University College London).

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Abstract

Sister-chromatid cohesion is a vital cellular process which involves the topological entrapment of sister-chromatids inside the ring of a protein complex called cohesin (Smc1, Smc3, Scc3 and Scc1). The early loss of cohesion can lead to chromosome mis-segregation, which in humans can cause the development of cancers. As a result, cells have evolved complex regulatory mechanisms to control cohesion throughout the cell cycle. Broadly, the cohesion cycle can be split into four stages loading, anti-establishment, establishment and release. The establishment of sisterchromatid cohesion occurs during S-phase with the passage of the replication fork, which is due to the acetylation of Smc3 at K112 and K113 through the action of the acetyltransferase called Eco1. Apart from this acetyltransferase, a number of other non-essential establishment factors have been identified, which include Chl1, Ctf4 and RFCCtf18. RFCCtf18 is a replication factor C (RFC) like complex formed of the seven subunits Ctf18, Rfc2-5, Dcc1 and Ctf8. Apart from sister-chromatid cohesion RFCCtf18 has also been shown to function in intra-S phase checkpoints. To further improve our understanding on the establishment of sister-chromatid cohesion the structures of the heterotrimer Ctf18C-Dcc1-Ctf8 was solved along with the acetyltransferase domain (ACT) of X. laevis Eco2 (xEco2) bound to two substrate peptides. The structure of Ctf18C-Dcc1-Ctf8 revealed that Dcc1 contained three tandem winged helix (WH) domains which could bind to both ssDNA and dsDNA. These WH domains were involved in recruiting RFCCtf18 to stalled replication forks. The structure of xEco2 ACT domain indicated that it had different binding mechanisms for both K112 and K113 substrates. To further investigate this, a mass spectrometry assay was employed to examine the relative rates of acetylation of K112 and K113. It was discovered that K112 acetylation occurs at a faster rate than the acetylation of K113.

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