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| Status |
Public on Sep 02, 2022 |
| Title |
Balanced act of a leading strand DNA polymerase specific domain and its exonuclease domain promotes genome-wide replication fork symmetry |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Other
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| Summary |
During genome replication, the leading strand DNA polymerase conducts continuous synthesis over long stretches of DNA within each replicon. Processive DNA synthesis supports symmetric progression of sister replication forks and efficient genome duplication. To address the mechanisms underlying leading strand polymerase-mediated synthesis, we examine one of its conserved domains, referred to as POPS, in the budding yeast Pol2 enzyme. We provide evidence that POPS supports replication fork symmetry and efficient genome replication via balancing the function of the Pol2 exonuclease domain. We found that the defective growth, slow S phase progression, and impaired genome synthesis associated with a POPS mutation were rescued by abolishing the Pol2 exonuclease activity. The suppressive effects further extended to the increased DNA re-arrangements in the POPS mutant and its negative genetic interactions with mutants of other genome maintenance factors. Significantly, our single molecule replicon-seq data demonstrate that the POPS mutant exhibited genome-wide replication fork asymmetry, and this defect was improved by eliminating the Pol2 exonuclease activity, thus providing a basis for its rescuing of a range of POPS mutant phenotypes. Collectively, these data suggest a model in which balanced activity between a unique Pol2 catalytic domain, and its exonuclease domain facilitates replication fork symmetry and genome maintenance.
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| Overall design |
MCM4 was fused to Miccrococcal nuclease ( MNase) to generate DNA double strand break at the site of replisomes. DNA ends are repaired and MinION compatible DNA adaptors are ligated. Full length molecules are sequenced. Because cells have been released from a G1 arrest in presence of BrdU, we can select for replicon reads (reads that contain BrdU) informatically using DNAscent.
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| Contributor(s) |
Meng X, Claussin C, Regan-Mochrie G, Whitehouse L, Zhao X |
| Citation missing |
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| Submission date |
Aug 26, 2022 |
| Last update date |
Sep 02, 2022 |
| Contact name |
clemence claussin |
| E-mail(s) |
claussin.clemence@gmail.com
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| Organization name |
Memorial sloan kettering cancer center
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| Department |
molecular biology
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| Lab |
whitehouse iestyn
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| Street address |
1275 York Avenue
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| City |
new york |
| State/province |
New York |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platforms (1) |
| GPL25739 |
MinION (Saccharomyces cerevisiae) |
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| Samples (8)
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| Relations |
| BioProject |
PRJNA874015 |
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