Spagnolo, Laura

Boettcher, Bettina

Cannone, Giuseppe

Darwin Trust of Edinburgh

2016-10-28T13:42:27Z

2016-10-28T13:42:27Z

2015-06-29

Previous studies suggest a degree of homology between eukaryotic replication, transcription and translation proteins and archaeal ones. Hence, Archaea are considered a simplified model for understanding the complex molecular machinery involved in eukaryotic DNA metabolism. DNA replication in eukaryotic cells is widely studied. In recent years, DNA replication studies expanded on the archaeal DNA replication machinery. P. abyssi was the first archaeon whose genome was fully sequenced. Genome sequencing and comparative genomics have highlighted an MCM-like protein in P. abyssi. In this study, I report the biochemical and structural characterisation of PabMCM. PabMCM is explored as model for understanding more complex eukaryotic MCM proteins and unravelling the biochemical mechanism by which MCM proteins release their helicase activity. The crenarchaeon Sulfolobus solfataricus possesses a simplified toolset for DNA replication compared to Eukaryotes. In particular, S. solfataricus has a subset of the eukaryotic Okazaki fragment maturation factors, among which there are a heterotrimeric DNA sliding clamp, (the proliferating cell nuclear antigen, PCNA), the DNA polymerase B1 (PolB1), the flap endonuclease (Fen1) and the ATP-dependent DNA ligase I (LigI). PCNA functions as a scaffold with each subunit having a specific binding affinity for each of the factors involved in Okazaki fragment maturation. Here, the 3D reconstruction of PCNA in complex with the Okazaki fragment maturation proteins PolB1, LigI and Fen1 is reported.

http://hdl.handle.net/1842/17070

en

The University of Edinburgh

Zhang, J., Rouillon, C., Kerou, M., Reeks, J., Brugger, K., Graham, S., Reimann, J., Cannone, G., Liu, H., Albers, S.V. et al. (2012) Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity. Mol. Cell 45, 303–313

Rouillon, C., Zhou, M., Zhang, J., Politis, A., Beilsten, V., Cannone, G., Graham, S., Robinson, C., Spagnolo, L. and White, M. (2013) Structure of the CRISPR surveillance complex CSM reveals key similarities with Cascade. Mol. Cell 52, 124–134

eukaryotic DNA metabolism

eukaryotic MCM proteins

PCNA

Okazaki fragment maturation

Structural investigation of the archaeal replicative machinery by electron microscopy and digital image processing

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy