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Diminished origin-licensing capacity specifically sensitizes tumor cells to replication stress.

ABSTRACT: Previous studies have shown that dormant licensed replication origins can be exploited to enhance recovery from replication stress. Since tumor cells express high levels of origin-licensing proteins, we examined whether depletion of such factors might specifically sensitize tumor versus nontumor cells. Consistent with previous findings, we observed that three tumor-derived cell lines overexpress ORC1, a licensing component, compared with four nontumor cell lines and that a greater level of ORC1 was required to maintain viability in the tumor cells. We determined siRNA-mediated knockdown conditions for each line that maximally reduced ORC1 but did not impact upon viability, which we considered would optimally deplete dormant origins. ORC1 depletion hypersensitized the tumor-derived cells to hydroxyurea and H202 but did not affect the sensitivity of the nontumor lines. Similar results were observed following depletion of ORC6 or CDC6. Furthermore, codepletion of p53 and ORC1 modestly impaired viability of 1BR3hTERT nontumor fibroblasts and more dramatically caused hypersensitivity to hydroxyurea. Finally, overexpression of the c-Myc oncogene combined with ORC1 depletion in nontumor BJhTERT cells diminished viability. Collectively, these findings suggest that tumor cells may have a reliance on origin-licensing capacity, suggesting that licensing factors could represent a target for drug-based cancer therapy.

SUBMITTER: Zimmerman KM

PROVIDER: S-EPMC3797919 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Diminished origin-licensing capacity specifically sensitizes tumor cells to replication stress.

Zimmerman Kristin M KM Jones Rebecca M RM Petermann Eva E Jeggo Penelope A PA

Molecular cancer research : MCR 20130130 4

Previous studies have shown that dormant licensed replication origins can be exploited to enhance recovery from replication stress. Since tumor cells express high levels of origin-licensing proteins, we examined whether depletion of such factors might specifically sensitize tumor versus nontumor cells. Consistent with previous findings, we observed that three tumor-derived cell lines overexpress ORC1, a licensing component, compared with four nontumor cell lines and that a greater level of ORC1 ...[more]

PMID: 23364533

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Project description:During origin licensing, the eukaryotic replicative helicase Mcm2-7 forms head-to-head double hexamers to prime origins for bidirectional replication. Recent single-molecule and structural studies revealed that one molecule of the helicase loader ORC can sequentially load two Mcm2-7 hexamers to ensure proper head-to-head helicase alignment. To perform this task, ORC must release from its initial high-affinity DNA binding site and "flip" to bind a weaker, inverted DNA site. However, the mechanism of this binding-site switch remains unclear. In this study, we used single-molecule Förster resonance energy transfer (sm-FRET) to study the changing interactions between DNA and ORC or Mcm2-7. We found that the loss of DNA bending that occurs during DNA deposition into the Mcm2-7 central channel increases the rate of ORC dissociation from DNA. Further studies revealed temporally-controlled DNA sliding of helicase-loading intermediates, and that the first sliding complex includes ORC, Mcm2-7, and Cdt1. We demonstrate that sequential events of DNA unbending, Cdc6 release, and sliding lead to a stepwise decrease in ORC stability on DNA, facilitating ORC dissociation from its strong binding site during site switching. In addition, the controlled sliding we observed provides insight into how ORC accesses secondary DNA binding sites at different locations relative to the initial binding site. Our study highlights the importance of dynamic protein-DNA interactions in the loading of two oppositely-oriented Mcm2-7 helicases to ensure bidirectional DNA replication.Significance statementBidirectional DNA replication, in which two replication forks travel in opposite directions from each origin of replication, is required for complete genome duplication. To prepare for this event, two copies of the Mcm2-7 replicative helicase are loaded at each origin in opposite orientations. Using single-molecule assays, we studied the sequence of changing protein-DNA interactions involved in this process. These stepwise changes gradually reduce the DNA-binding strength of ORC, the primary DNA binding protein involved in this event. This reduced affinity promotes ORC dissociation and rebinding in the opposite orientation on the DNA, facilitating the sequential assembly of two Mcm2-7 molecules in opposite orientations. Our findings identify a coordinated series of events that drive proper DNA replication initiation.

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Diminished origin-licensing capacity specifically sensitizes tumor cells to replication stress.

Publications

Diminished origin-licensing capacity specifically sensitizes tumor cells to replication stress.

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