Accurate and complete replication of the genome is essential for both proliferation and genomic stability. Genomic DNA is constantly under assault from both exogenous and endogenous agents. The resulting DNA lesions can cause stalling of the replication fork and activation of the replication stress response (RSR). This activates a group of proteins that protect and restart the replication fork so that genomic integrity is maintained. Incorrect repair can lead to cancer and aging, and defects in the RSR predispose individuals to develop cancer. Our overall goal is to define the molecular mechanisms that protect the genome during replication stress. We have discovered that DNA Helicase B (HELB) protects stalled replication forks from aberrant degradation by nucleases and promotes replication restart. This is consistent with data showing reduced recovery from replication stress with HELB knockdown; however, the mechanism by which HELB promotes recovery from replication stress is unknown. In addition, we have found that HELB enhances replication through G-quadruplex (G4) structures, suggesting that HELB may enable the replication fork to progress through difficult to replicate sites. Since replication stalling can result in epigenetic instability, HELB may be involved in epigenetic maintenance in addition to genomic maintenance. HELB has also been implicated in initiation of DNA replication and regulation of end resection in homologous recombination (HR). Due to its varied roles in multiple processes, regulation of HELB activity is likely required. Indeed, we have evidence that HELB activity is regulated by post- translational modifications (PTMs). We hypothesize that HELB maintains genomic and epigenetic integrity by protecting nascent DNA from degradation, aiding in replication restart, and assisting in replication through difficult sites. We will test this hypothesis through the following Specific Aims: (1) Ascertain the role of HELB in protection and restart of stalled DNA replication forks, (2) Determine the function of HELB in replication through difficult to replicate regions of the genome, and (3) Delineate the role of HELB PTMs and protein-protein interactions (PPIs) in the replication stress response.

P20GM121293 Sub-Project 6782

2022-07-20

2027-06-30