Logo Logo
Hilfe
Kontakt
Switch language to English
The role of ORC6 in the human cell
The role of ORC6 in the human cell
Orc6 is a crucial component of the replication initiation machinery in eukaryotes. Its study helps us to better understand one of the most basic cell functions: DNA replication. The first step in replication initiation is the assembly and DNA binding of the origin recognition complex (ORC). Although members of ORC are highly conserved and well studied, Orc6 evolved faster than the other members of the complex, has a different structure and is less well understood than Orc1-5. The low number of studies on Orc6 in human cells and the low predictability of results obtained from model systems necessitates an in depth analysis of HsOrc6. The aim of this study is to better understand the role of Orc6 in the human cell by exploring the mechanism of Orc6-Orc1-5 interaction and Orc6-DNA binding, studying the function of Orc6 in live cells, and mapping its interaction network to find new pathways and functions of the protein. The study concludes that Orc6 interacts via its C terminus with Orc1-5 in vitro. It might also have a second pre-RC interaction domain, probably close to the nuclear localization signal. Orc6 is not required for Orc1-5-DNA binding, but is able to enhance the process in vitro. It is also able to bind DNA in the absence of Orc1-5 with high affinity. In vivo experiments present evidence on the ability of Orc6 to recruit the replication machinery to itself on DNA. Although this ability is less efficient than that of EBNA1, a viral factor, it is sufficiently strong to support autonomous replication of plasmids in human cell lines for at least four weeks. Truncated variants of the protein containing at least one of the predicted Orc1-5 interaction sites are sufficient to allow replication. This finding can be used to design autonomous-replicating plasmids for use in gene therapy. Mapping the interaction network of Orc6 leads to the conclusion that HsOrc6 is only weakly attached to pre-RC factors, and is likely to have an important role in mitosis and possibly be involved in other pathways.
Orc6, protein, DNA, molecular biology, replication, ORC, complex, cell, human, proteomics, biochemistry
Ravasz, Mate
2014
Englisch
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Ravasz, Mate (2014): The role of ORC6 in the human cell. Dissertation, LMU München: Fakultät für Biologie
[thumbnail of Ravasz_Mate.pdf]
Vorschau
PDF
Ravasz_Mate.pdf

3MB

Abstract

Orc6 is a crucial component of the replication initiation machinery in eukaryotes. Its study helps us to better understand one of the most basic cell functions: DNA replication. The first step in replication initiation is the assembly and DNA binding of the origin recognition complex (ORC). Although members of ORC are highly conserved and well studied, Orc6 evolved faster than the other members of the complex, has a different structure and is less well understood than Orc1-5. The low number of studies on Orc6 in human cells and the low predictability of results obtained from model systems necessitates an in depth analysis of HsOrc6. The aim of this study is to better understand the role of Orc6 in the human cell by exploring the mechanism of Orc6-Orc1-5 interaction and Orc6-DNA binding, studying the function of Orc6 in live cells, and mapping its interaction network to find new pathways and functions of the protein. The study concludes that Orc6 interacts via its C terminus with Orc1-5 in vitro. It might also have a second pre-RC interaction domain, probably close to the nuclear localization signal. Orc6 is not required for Orc1-5-DNA binding, but is able to enhance the process in vitro. It is also able to bind DNA in the absence of Orc1-5 with high affinity. In vivo experiments present evidence on the ability of Orc6 to recruit the replication machinery to itself on DNA. Although this ability is less efficient than that of EBNA1, a viral factor, it is sufficiently strong to support autonomous replication of plasmids in human cell lines for at least four weeks. Truncated variants of the protein containing at least one of the predicted Orc1-5 interaction sites are sufficient to allow replication. This finding can be used to design autonomous-replicating plasmids for use in gene therapy. Mapping the interaction network of Orc6 leads to the conclusion that HsOrc6 is only weakly attached to pre-RC factors, and is likely to have an important role in mitosis and possibly be involved in other pathways.