DNA polymerase ε relies on a unique domain for efficient replisome assembly and strand synthesis

DNA polymerase epsilon (Pol ε) is required for genome duplication and tumor suppression. It supports both replisome assembly and leading strand synthesis; however, the underlying mechanisms remain to be elucidated. Here we report that a conserved domain within the Pol ε catalytic core influences both of these replication steps in budding yeast. Modeling cancer-associated mutations in this domain reveals its unexpected effect on incorporating Pol ε into the four-member pre-loading complex during replisome assembly. In addition, genetic and biochemical data suggest that the examined domain supports Pol ε catalytic activity and symmetric movement of replication forks. Contrary to previously characterized Pol ε cancer variants, the examined mutants cause genome hyper-rearrangement rather than hyper-mutation. Our work thus suggests a role of the Pol ε catalytic core in replisome formation, a reliance of Pol ε strand synthesis on a unique domain, and a potential tumor-suppressive effect of Pol ε in curbing genome re-arrangements.


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For GCR assay and mutation rate assay, sample sizes were chosen based previous studies. Data exclusions We did not exclude data.

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anti-Myc: The 9E10 monoclonal antibody reacts with human c-myc, a 62 kDa transcription factor that plays a role in cell cycle progression, apoptosis and cellular transformation. c-Myc is commonly added to proteins of interest using recombinant DNA technology. anti-HA: Anti-HA High Affinity is a monoclonal antibody to the HA-peptide (clone 3F10 The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
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Yeast cells were spun down from culture, and fixed in ice old 70% ethanol. Cell were washed with and resuspended in NaCtirate buffer. RNase and Proteinase K were added sequentially to digest RNA and protein. Sytox green was used to stain the cells.

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