Abstract
To ensure genomic integrity, the genome must be duplicated exactly once per cell cycle. Disruption of replication licensing mechanisms may lead to re-replication and genomic instability. Cdt1, also known as Double-parked (Dup) in Drosophila, is a key regulator of the assembly of the pre-replicative complex (pre-RC) and its activity is strictly limited to G1 by multiple mechanisms including Cul4-Ddb1 mediated proteolysis and inhibition by geminin. We assayed the genomic consequences of disregulating the replication licensing mechanisms by RNAi depletion of geminin. We found that not all origins of replication were sensitive to geminin depletion and that heterochromatic sequences were preferentially re-replicated in the absence of licensing mechanisms. The preferential re-activation of heterochromatic origins of replication was unexpected because these are typically the last sequences to be duplicated in a normal cell cycle. We found that the re-replication of heterochromatin was regulated not at the level of pre-RC activation, but rather by the formation of the pre-RC. Unlike the global assembly of the pre-RC that occurs throughout the genome in G1, in the absence of geminin, limited pre-RC assembly was restricted to the heterochromatin by elevated cyclin A-CDK activity. These results suggest that there are chromatin and cell cycle specific controls that regulate the re-assembly of the pre-RC outside of G1.
Highlights
The precise duplication of the genome is a fundamental requirement to maintain genomic integrity
Catastrophic consequences may occur if the cell fails to either completely copy the genome or if it duplicates some regions of the genome more than once in a cell cycle
The cell must coordinate thousands of DNA replication start sites to ensure that the entire genome is copied and that no replication origin is activated more than once in a cell cycle. The cell accomplishes this coordination by confining the selection and activation of replication origins to discrete phases of the cell cycle
Summary
The precise duplication of the genome is a fundamental requirement to maintain genomic integrity. Failure to properly license replication origins will result in either re-initation of DNA replication or underreplication, and may contribute to genomic instability. A critical feature of this licensing system is that the assembly of the prereplicative complex (pre-RC) at origins of replication is separated from replication initiation by strictly limiting each process to distinct phases of the cell cycle [1,2]. In G1, when CDK levels are low, Cdc and Cdt function to load the replicative helicase complex (MCMs) at ORC binding sites to form the pre-RC. The pre-RC is subsequently activated in S phase by cyclin and Dbf dependent kinase (CDK and DDK) activities which results in the loading of DNA polymerases and the initiation of bi-directional DNA replication
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