Complete and faithful replication of the genome is essential during every cell division cycle in order to maintain genome integrity and prevent disease. Replication forks frequently encounter obstacles such as DNA lesions, difficult to replicate sequences, and protein complexes bound to DNA (such as transcription machinery) that can impede their progress and the completion of DNA synthesis. A large number of proteins and protein complexes are involved in facilitating accurate and complete DNA replication and restoring proper chromatin architecture behind the replication fork. Defects in many of these processes lead to a failure to overcome these obstacles causing genome instability and disease. While most of the essential components of the eukaryotic replication machinery are known, many proteins that have roles in influencing the fidelity and efficiency of DNA replication remain to be identified and characterized. Obtaining a complete inventory of all the proteins that travel with the replication fork, including those involved in DNA replication, chromatin maturation, and the replication stress response, is required to fully understand how genome stability is maintained and what processes present at replication forks contribute to it.
Aim 1 of this proposal will utilize iPOND (isolation of proteins on nascent DNA) to provide a comprehensive inventory of the replication fork proteome, and Aim 2 will employ biochemistry and cell biology to functionally characterize one of the novel replication fork associated proteins. The catalog of proteins associated with replication forks generated from these experiments will be a valuable resource for identifying functions of understudied proteins and has the potential to increase our understanding of human diseases.
Defects in processes that facilitate the complete and faithful replication of the genome lead to genome instability and disease. While most of the essential components of the eukaryotic replication machinery are known, many proteins that have roles in influencing the fidelity and efficiency of DNA replication remain to be identified and characterized. This proposal will provide a comprehensive inventory of the replication fork proteome and functionally characterize the identified proteins to provide critical insights into the replication fork associated processes that maintain genome stability and prevent disease.