Factors Affecting the Diversity of DNA Replication Licensing Control in Eukaryotes

Abstract

Replication of eukaryotic genomes is limited to once per cell cycle, by a two-step mechanism. DNA replication origins are first "licensed" during G1 phase by loading of an inactive DNA helicase (Mcm2-7) into pre-replicative complexes (pre-RCs). Initiation then occurs during S phase, triggered by cyclin-dependent kinases (CDKs), which promote recruitment of proteins required for helicase activation and replisome assembly. CDKs and the anaphase promoting complex/cyclosome (APC/C) restrict licensing to G1 phase by directly and indirectly regulating pre-RC components, including ORC, Cdc6, Cdt1, and Mcm2-7. Despite the fundamental importance of licensing regulation, the mechanisms by which pre-RC components are regulated differ widely across Eukarya. Here we show that even within the genus Saccharomyces, Cdc6 is regulated differently in different species. We propose that two factors contribute to the rapid evolution of licensing regulation. The first is redundancy: eliminating any single pre-RC-regulatory mechanism has very little affect on viability. The second is interchangeability: we show that regulatory mechanisms can be swapped between pre-RC components without compromising the block to re-replication. These experiments provide a framework for understanding the diversity of licensing regulation in eukaryotes and provide new tools for manipulating the chromosome-replication cycle.