Replication origin-flanking roadblocks reveal origin-licensing dynamics and altered sequence dependence

The Journal of Biological Chemistry
Megan D WarnerStephen P Bell

Abstract

In eukaryotes, DNA replication initiates from multiple origins of replication for timely genome duplication. These sites are selected by origin licensing, during which the core enzyme of the eukaryotic DNA replicative helicase, the Mcm2-7 (minichromosome maintenance) complex, is loaded at each origin. This origin licensing requires loading two Mcm2-7 helicases around origin DNA in a head-to-head orientation. Current models suggest that the origin-recognition complex (ORC) and cell-division cycle 6 (Cdc6) proteins recognize and encircle origin DNA and assemble an Mcm2-7 double-hexamer around adjacent double-stranded DNA. To test this model and assess the location of Mcm2-7 initial loading, we placed DNA-protein roadblocks at defined positions adjacent to the essential ORC-binding site within Saccharomyces cerevisiae origin DNA. Roadblocks were made either by covalent cross-linking of the HpaII methyltransferase to DNA or through binding of a transcription activator-like effector (TALE) protein. Contrary to the sites of Mcm2-7 recruitment being precisely defined, only single roadblocks that inhibited ORC-DNA binding showed helicase loading defects. We observed inhibition of helicase loading without inhibition of ORC-DNA binding o...Continue Reading

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Citations

Dec 18, 2018·ELife·Tito CandelliDomenico Libri
Mar 27, 2019·PloS One·Ramon Y Rios-MoralesStephen P Bell
Nov 22, 2019·Nature·Thomas C R MillerAlessandro Costa
Oct 7, 2020·Molecular Cancer Research : MCR·Jeffrey C MartinJoyce E Ohm
Mar 28, 2021·Nature Communications·Humberto SánchezNynke H Dekker
May 22, 2021·Technology in Cancer Research & Treatment·Kai CuiYa-Lan Dong
Dec 10, 2021·ELife·Shalini GuptaStephen P Bell

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