Regulatory mechanisms of mammalian replication origins

Resumen

In mammalian cells, DNA replication starts from thousands of replication origins whose activation is tightly regulated in the cell division cycle. In this study, we have aimed at better understanding the regulation of origin selection and activation in mouse embryonic stem cells and human cancer cells. In the first part of this dissertation, we set out to investigate the dynamics of origin activation in response to stress conditions that slow down or stall replication forks. This situation promotes the activation of otherwise ‘dormant’ origins, which provide a backup mechanism to complete replication and prevent genomic instability. Using the SNS-Seq technique based on deep-sequencing of short nascent DNA strands, we have mapped the genomic positions of origins in control growth conditions and two experimental settings that trigger the activation of extra origins: (a) exposure to DNA polymerase inhibitor aphidicolin; (b) overexpression of CDC6, a limiting factor for origin licensing and activation in primary murine cells. Using SNS-Seq data we also determined the efficiency of activation of each individual origin in the cell population. Constitutive origins, in contrast to stress-responsive ones, show a strong preference towards open, transcriptionally active chromatin and display higher efficiency. Our results strongly suggest that the main response to stress is mediated by modulating the activity of pre-existing origins rather than the activation of new ones. We have also carried out an unprecedented integration of linear origin maps into 3D chromatin networks that reveals how origins tend to group together in clusters that likely correspond to DNA replication factories. Origin connections are found within the same topologically associated domain (TAD), but also between origins located in different TADs. We report for the first time that the connectivity of an origin is directly proportional to its efficiency of activation. In the second part, we aimed at dissecting a novel mechanism that regulates CDC6 protein stability. Downregulation of CDC7 kinase, known to activate the MCM helicase at replication origins, caused a drop in cellular CDC6 levels. CDC6 was phosphorylated by CDC7 in vitro and became destabilized in vivo when all possible CDC7-dependent phosphorylation sites were mutated. We report a previously unknown role of CDC7 in the regulation of CDC6 stability that is mediated by a combination of direct and indirect mechanisms.