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Abstract
The role of Mcm7 ubiquitylation during termination of eukaryotic DNA replication
Sara Priego Moreno, Nicholas Campion, Rachael Hollins and Agnieszka Gambus*
School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT
To ensure duplication of the whole genome DNA replication initiates from thousands of origins of replication. The pre-loaded inactive helicase (Mcm2-7 double hexameric complexes) becomes activated during replication initiation by loading of additional components: Cdc45 and GINS in a carefully regulated process. The progressing replication fork moves through the chromatin until it encounters fork from the neighbouring origin. During the termination of replication forks the replisomes disassemble by an unknown mechanism and topisomerase II resolves the daughter DNA molecules. If not resolved properly, the terminating forks are at high risk of stalling and fork reversal, leading to DNA damage and genomic instability.
Using the Xenopus laevis egg extract system, we have shown that blocking polyubiquitylation results in the prolonged association of the active helicase with replicating chromatin. This is not accompanied by defect in nascent DNA synthesis, S-phase checkpoint activation nor excessive origin firing. Instead, we propose that without effective polyubiquitylation it is the disassembly of the active helicase at the termination of replication forks that is defective.
The Mcm7 subunit of the Mcm2-7 complex is the only component of the active helicase that we find polyubiquitylated during S-phase. Mcm7 ubiquitylation coincides with the presence of replication forks on chromatin and is blocked when forks cannot terminate. Both the disassembly phenotype and Mcm7 ubiquitylation are driven by lysine 48-linked ubiquitin chains, but not proteasomal degradation. Instead we propose an alternative model of replisome removal from chromatin.
Altogether, our data provides first insight into the mechanism of replisome disassembly during termination of eukaryotic DNA replication.
Sara Priego Moreno, Nicholas Campion, Rachael Hollins and Agnieszka Gambus*
School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT
To ensure duplication of the whole genome DNA replication initiates from thousands of origins of replication. The pre-loaded inactive helicase (Mcm2-7 double hexameric complexes) becomes activated during replication initiation by loading of additional components: Cdc45 and GINS in a carefully regulated process. The progressing replication fork moves through the chromatin until it encounters fork from the neighbouring origin. During the termination of replication forks the replisomes disassemble by an unknown mechanism and topisomerase II resolves the daughter DNA molecules. If not resolved properly, the terminating forks are at high risk of stalling and fork reversal, leading to DNA damage and genomic instability.
Using the Xenopus laevis egg extract system, we have shown that blocking polyubiquitylation results in the prolonged association of the active helicase with replicating chromatin. This is not accompanied by defect in nascent DNA synthesis, S-phase checkpoint activation nor excessive origin firing. Instead, we propose that without effective polyubiquitylation it is the disassembly of the active helicase at the termination of replication forks that is defective.
The Mcm7 subunit of the Mcm2-7 complex is the only component of the active helicase that we find polyubiquitylated during S-phase. Mcm7 ubiquitylation coincides with the presence of replication forks on chromatin and is blocked when forks cannot terminate. Both the disassembly phenotype and Mcm7 ubiquitylation are driven by lysine 48-linked ubiquitin chains, but not proteasomal degradation. Instead we propose an alternative model of replisome removal from chromatin.
Altogether, our data provides first insight into the mechanism of replisome disassembly during termination of eukaryotic DNA replication.
| Original language | English |
|---|---|
| Publication status | Published - 10 Sept 2013 |
| Event | Eukaryotic DNA replication and genome maintenance, Cold Spring Harbor Laboratories Meeting - Cold Spring Harbor, New York , United States Duration: 9 Sept 2013 → 13 Sept 2013 |
Conference
| Conference | Eukaryotic DNA replication and genome maintenance, Cold Spring Harbor Laboratories Meeting |
|---|---|
| Country/Territory | United States |
| City | Cold Spring Harbor, New York |
| Period | 9/09/13 → 13/09/13 |
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- 3 Finished
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WT ISSF 12/13 - Identification of Proteins Regulated by BRCA1/BARD1 Ubiquitin Ligase
1/03/13 → 30/06/13
Project: Research
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Prizes
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MRC Career Development Fellowship
Gambus, Aga (Recipient), 1 Mar 2013
Prize: Fellowship awarded competitively