Abstract
DNA replication requires a plethora of proteins to maintain its accuracy during replicative stress. In order to fully understand how DNA replication is sustained at proper pace, it is important to study the mechanisms that are guarding DNA replication during normal and perturbed conditions. Using the iPOND (isolation of proteins on nascent DNA) based screen we uncovered a new protein, GNL3 (aka nucleostemin), to be associated with replisome. GNL3 is overexpressed in several cancers and is involved in maintaining genomic integrity in stem and cancer cells, however its precise role(s) is unclear.One of the key mechanisms that protects the genomic stability during replication stress is the proper regulation of origin firing. In this project we show that GNL3 limits replicative stress by limiting replication origins firing. We proved that GNL3 is in in proximity of nascent DNA using different approaches and that its depletion reduces forks speed but increases forks density and replication origin firing. Conversely, overexpression of GNL3 leads to a decrease in origin firing. When subjected to exogenous replicative stress, cells impaired for GNL3 exhibits an increased MRN-dependent resection and RPA phosphorylation. Interestingly , we found that inhibition of origin firing using CDC7 inhibitor decreased resection in absence of GNL3 but not in absence of BRCA1, suggesting that GNL3 protects the integrity of stalled forks indirectly by regulating origin firing efficiency. In addition, using various approaches (BioID, PLA, coIP), we established that ORC2 and GNL3 interact together in the nucleolus. We propose that GNL3 level is crucial to determine the correct distribution of ORC2 on chromatin to regulate origins licensing. Our data present insights into a new role of GNL3 in the regulation of origin firing that protects genomic stability.