Dissertation
The Timeless protein coordinates multiple pathways at the dna replication fork to preserve genomic integrity
Doctor of Philosophy (Ph.D.), Drexel University
Apr 2011
DOI:
https://doi.org/10.17918/00009847
Abstract
The DNA replication machinery is responsible for the high fidelity duplication of the human genome, containing over three billion nucleotides. Dysfunction of the replication processes lead to spontaneous, heritable genetic errors such as mutations, rearrangements, and breakage of genomic DNA. In humans, these can cause diseases or developmental abnormalities, such as cancer or birth defects. DNA replication is the process of DNA polymerase enzymes copying template DNA unwound by replicative helicases. Accessory proteins ensure that replication proceeds in a highly efficient and accurate manner in the eukaryotic cell. Timeless and Tipin proteins comprise the mammalian Fork Protection Complex (FPC). The FPC has been implicated in replication fork stability, DNA replication checkpoint activation, and proper sister chromatid pairing, mostly in fungal and nematode systems, although the mechanisms of action are still unclear. Human genome size and regulation add additional complexity to DNA transactions in mammalian cells; therefore it is important to characterize the mammalian FPC and determine its function in human DNA replication. We demonstrate that Timeless-Tipin complex in human cells preserves genome integrity during DNA replication. We establish localization of human FPC proteins specifically to the DNA replication fork on chromatin during S-phase. We show that Timeless-Tipin act to prevent DNA damage accumulation by allowing proper recovery from genotoxic insults. We also demonstrate that proper FPC function is important for the regulated pairing of sister chromatids during S-phase. We have linked FPC to cohesion establishment through interaction with cohesin proteins. We have uncovered a physical and functional interaction between Timeless and ChlR1, a helicase known to promote sister chromatid cohesion. Additionally, we have elucidated a role for Timeless in replication of telomeres. The replication of telomeres is substantially delayed after Timeless knockdown. In the absence of Timeless, telomeres shorten and accumulate DNA damage. We have shown that Timeless regulates telomere replication through interaction with telomere repeat binding proteins. Taken together, these data are consistent with a genome-wide role for Timeless in replication fork stabilization and DNA replication fork progression at difficult templates throughout the genome. Thus, we have established Timeless as an important coordinator of multiple processes with DNA replication.
Metrics
34 File views/ downloads
19 Record Views
Details
- Title
- The Timeless protein coordinates multiple pathways at the dna replication fork to preserve genomic integrity
- Creators
- Adam Robert Leman
- Contributors
- Eishi Noguchi (Advisor) - Drexel University, Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xiii, 221 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- College of Medicine; Drexel University
- Other Identifier
- 991021888958104721