Final Report Summary - DNA REPLICATION (In vitro reconstitution of the replication machinery on a chromatin template)
Enormous progress has been made in the yeast Saccharomyces cerevisiae to understand the regulation of the temporal control of DNA replication. However, the important interplay between chromatin and DNA replication still remains poorly understood. In particular, major questions include how the replication machinery overcomes the nucleosome-barrier to ensure 1) initiation and 2) elongation of the replicative helicase/polymerase after origin firing. These questions underlie the main goals of the project, which aimed to establish an in vitro system to connect DNA replication and chromatin.
Previous research already connected chromatin and DNA replication by showing that local chromatin environment and chromatin modifications impact the activation of replication origins. Interestingly, origins of replication are nucleosome free and the position of the adjacent nucleosome is important for origin function in yeast. This is dependent on the ORC complex and it is believed that alterations of ORC-dependent nucleosome configuration at a yeast origin of replication compromises origin function by disrupting pre-RC formation. Additionally, ATP dependent and independent chromatin remodelers are known to interact with the replication machinery and influence DNA replication. The underlying molecular mechanisms, however, remain elusive.
The ultimate goal of this proposal was to shed light on the important question of how replication origins are chosen and how DNA replication functions in a chromatin context.
During the two years of the fellowship, I have successfully implemented the reconstitution of helicase loading and replication initiation on a chromatin template.
The main results I achieved are:
1) Chromatin is beneficial for origin-specific ORC binding and MCM loading by excluding non-specific ORC binding.
2) ORC is important for the precise deposition of nucleosomes around an origin
3) A minimal replisome cannot evict nucleosomes in front of the replication fork.
4) One histone chaperone is sufficient for replicating chromatin in vitro
Having an in vitro system to replicate chromatin is an invaluable resource to study the effects of e.g. chromatin remodellers and histone modifications on chromatin replication. As mis-regulation of DNA replication is implicated in cancer, my newly established in vitro system will help study DNA replication on chromatin in detail and will have great impact on cancer research.
Previous research already connected chromatin and DNA replication by showing that local chromatin environment and chromatin modifications impact the activation of replication origins. Interestingly, origins of replication are nucleosome free and the position of the adjacent nucleosome is important for origin function in yeast. This is dependent on the ORC complex and it is believed that alterations of ORC-dependent nucleosome configuration at a yeast origin of replication compromises origin function by disrupting pre-RC formation. Additionally, ATP dependent and independent chromatin remodelers are known to interact with the replication machinery and influence DNA replication. The underlying molecular mechanisms, however, remain elusive.
The ultimate goal of this proposal was to shed light on the important question of how replication origins are chosen and how DNA replication functions in a chromatin context.
During the two years of the fellowship, I have successfully implemented the reconstitution of helicase loading and replication initiation on a chromatin template.
The main results I achieved are:
1) Chromatin is beneficial for origin-specific ORC binding and MCM loading by excluding non-specific ORC binding.
2) ORC is important for the precise deposition of nucleosomes around an origin
3) A minimal replisome cannot evict nucleosomes in front of the replication fork.
4) One histone chaperone is sufficient for replicating chromatin in vitro
Having an in vitro system to replicate chromatin is an invaluable resource to study the effects of e.g. chromatin remodellers and histone modifications on chromatin replication. As mis-regulation of DNA replication is implicated in cancer, my newly established in vitro system will help study DNA replication on chromatin in detail and will have great impact on cancer research.