Building a double hexamer of DNA helicase at eukaryotic replication origins

Background: Understanding DNA replication initiation is essential to understand the mis-regulation of replication seen in cancer and other human disorders. DNA replication initiates from DNA replication origins. In eukaryotes, replication is dependent on cell cycle kinases which function during S phase. Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) act to phosphorylate the DNA helicase (composed of mini chromosome maintenance proteins: Mcm2-7) and firing factors to activate replication origins. It has recently been found that Rif1 can oppose DDK phosphorylation. Rif1 can recruit protein phosphatase 1 (PP1) to dephosphorylate MCM and restricts origin firing. In this study, we investigate a potential role for another phosphatase, protein phosphatase 2A (PP2A), in regulating DNA replication initiation. The PP2A regulatory subunit Rts1 was previously identified in a large-scale genomic screen to have a genetic interaction with ORC2 (a DNA replication licensing factor). Deletion of RTS1 synthetically rescued the temperature-sensitive (ts-) phenotype of ORC2 mutants. Methods: We deleted RTS1 in multiple ts-replication factor Saccharomyces cerevisiae strains, including ORC2. Dilution series assays were carried out to compare qualitatively the growth of double mutant ∆rts1 ts-replication factor strains relative to the respective single mutant strains. Results: No synthetic rescue of temperature-sensitivity was observed. Instead we found an additive phenotype, indicating gene products function in separate biological processes. These findings are in agreement with a recent genomic screen which found that RTS1 deletion in several ts-replication factor strains led to increased temperature-sensitivity. Conclusions: We find no evidence that Rts1 is involved in the dephosphorylation of DNA replication initiation factors.

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RNF4 Regulates the BLM Helicase in Recovery From Replication Fork Collapse

Frontiers in Genetics ◽

10.3389/fgene.2021.753535 ◽

2021 ◽

Vol 12 ◽

Author(s):

Nathan Ellis ◽

Jianmei Zhu ◽

Mary K Yagle ◽

Wei-Chih Yang ◽

Jing Huang ◽

...

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Replication Stress ◽

Dna Helicase ◽

Replication Forks ◽

Replication Origins ◽

Dna Double Strand Breaks ◽

Response Factors ◽

Fork Stalling ◽

In Cells

Sumoylation is an important enhancer of responses to DNA replication stress and the SUMO-targeted ubiquitin E3 ligase RNF4 regulates these responses by ubiquitylation of sumoylated DNA damage response factors. The specific targets and functional consequences of RNF4 regulation in response to replication stress, however, have not been fully characterized. Here we demonstrated that RNF4 is required for the restart of DNA replication following prolonged hydroxyurea (HU)-induced replication stress. Contrary to its role in repair of γ-irradiation-induced DNA double-strand breaks (DSBs), our analysis revealed that RNF4 does not significantly impact recognition or repair of replication stress-associated DSBs. Rather, using DNA fiber assays, we found that the firing of new DNA replication origins, which is required for replication restart following prolonged stress, was inhibited in cells depleted of RNF4. We also provided evidence that RNF4 recognizes and ubiquitylates sumoylated Bloom syndrome DNA helicase BLM and thereby promotes its proteosome-mediated turnover at damaged DNA replication forks. Consistent with it being a functionally important RNF4 substrate, co-depletion of BLM rescued defects in the firing of new replication origins observed in cells depleted of RNF4 alone. We concluded that RNF4 acts to remove sumoylated BLM from collapsed DNA replication forks, which is required to facilitate normal resumption of DNA synthesis after prolonged replication fork stalling and collapse.

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A conserved MCM single-stranded DNA binding element is essential for replication initiation

eLife ◽

10.7554/elife.01993 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 60

Author(s):

Clifford A Froelich ◽

Sukhyun Kang ◽

Leslie B Epling ◽

Stephen P Bell ◽

Eric J Enemark

Keyword(s):

Pyrococcus Furiosus ◽

Dna Helicase ◽

Replication Initiation ◽

Binding Motif ◽

Replication Origins ◽

Dna Interactions ◽

Single Stranded Dna ◽

Ssdna Binding ◽

Helicase Loading ◽

Mcm Helicase

The ring-shaped MCM helicase is essential to all phases of DNA replication. The complex loads at replication origins as an inactive double-hexamer encircling duplex DNA. Helicase activation converts this species to two active single hexamers that encircle single-stranded DNA (ssDNA). The molecular details of MCM DNA interactions during these events are unknown. We determined the crystal structure of the Pyrococcus furiosus MCM N-terminal domain hexamer bound to ssDNA and define a conserved MCM-ssDNA binding motif (MSSB). Intriguingly, ssDNA binds the MCM ring interior perpendicular to the central channel with defined polarity. In eukaryotes, the MSSB is conserved in several Mcm2-7 subunits, and MSSB mutant combinations in S. cerevisiae Mcm2-7 are not viable. Mutant Mcm2-7 complexes assemble and are recruited to replication origins, but are defective in helicase loading and activation. Our findings identify an important MCM-ssDNA interaction and suggest it functions during helicase activation to select the strand for translocation.

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Faculty Opinions recommendation of Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.714597887.790202811 ◽

2012 ◽

Author(s):

Nick Rhind

Keyword(s):

Dna Helicase ◽

Replication Origins

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Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53

10.1101/2020.05.04.077628 ◽

2020 ◽

Author(s):

Syafiq Abd Wahab ◽

Dirk Remus

Keyword(s):

Kinase Activity ◽

Dna Helicase ◽

Replication Origins ◽

Genome Maintenance ◽

Activation Reaction ◽

Origin Activation ◽

Multiple Protein ◽

Rad53 Kinase ◽

Hexameric Form ◽

Antagonistic Control

ABSTRACTEukaryotic replication origins are licensed by the loading of the replicative DNA helicase, Mcm2-7, in inactive double hexameric form around DNA. Subsequent origin activation is under control of multiple protein kinases that either promote or inhibit origin activation, which is important for genome maintenance. Using the reconstituted budding yeast DNA replication system, we find that the flexible N-terminal tail of Mcm2 promotes the stable recruitment of Dbf4-dependent kinase (DDK) to Mcm2-7 double hexamers, which in turn promotes DDK phosphorylation of Mcm4 and -6 and subsequent origin activation. Conversely, we demonstrate that the checkpoint kinase, Rad53, inhibits DDK binding to Mcm2-7 double hexamers. Unexpectedly, this function is not dependent on Rad53 kinase activity, but requires Rad53 activation by trans-autophosphorylation, suggesting steric inhibition of DDK by activated Rad53. These findings identify critical determinants of the origin activation reaction and uncover a novel mechanism for checkpoint-dependent origin inhibition.

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Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53

eLife ◽

10.7554/elife.58571 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Syafiq Abd Wahab ◽

Dirk Remus

Keyword(s):

Kinase Activity ◽

Dna Helicase ◽

Replication Origins ◽

Genome Maintenance ◽

Activation Reaction ◽

Origin Activation ◽

Multiple Protein ◽

Rad53 Kinase ◽

Hexameric Form ◽

Antagonistic Control

Eukaryotic replication origins are licensed by the loading of the replicative DNA helicase, Mcm2-7, in inactive double hexameric form around DNA. Subsequent origin activation is under control of multiple protein kinases that either promote or inhibit origin activation, which is important for genome maintenance. Using the reconstituted budding yeast DNA replication system, we find that the flexible N-terminal extension (NTE) of Mcm2 promotes the stable recruitment of Dbf4-dependent kinase (DDK) to Mcm2-7 double hexamers, which in turn promotes DDK phosphorylation of Mcm4 and −6 and subsequent origin activation. Conversely, we demonstrate that the checkpoint kinase, Rad53, inhibits DDK binding to Mcm2-7 double hexamers. Unexpectedly, this function is not dependent on Rad53 kinase activity, suggesting steric inhibition of DDK by activated Rad53. These findings identify critical determinants of the origin activation reaction and uncover a novel mechanism for checkpoint-dependent origin inhibition.

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