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Acidic phospholipids with unsaturated fatty acids inhibit the binding of origin recognition complex to origin DNA
Jong-Ryul Lee1, Hitomi Takenaka, Naoko Takahashi
1Faculty of Pharmaceutical Sciences, Okayama University, 700-8530, Japan.
Abstract:
Origin Recognition Complex (ORC) is a candidate initiator of chromosomal DNA replication in eukaryotes. We recently reported that cardiolipin inhibits the interaction of Origin Recognition Complex ORC with origin DNA, as is the case of DnaA, the initiator of chromosomal DNA replication in prokaryotes. We report here that another acidic phospholipid, phosphatidylglycerol (PG), also inhibits the interaction. Synthetic PG with only unsaturated fatty acids inhibits ORC-binding to origin DNA more strongly than PG with only saturated fatty acids. On the other hand, phosphatidylcholine (neutral phospholipid) does not affect the ORC-origin interaction, regardless of the presence of saturated or unsaturated fatty acids. These results suggest that an acidic moiety and unsaturated fatty acids are important factors for the inhibitory effect of phospholipids on ORC binding to origin DNA, as is the case for DnaA. The inhibitory effect of cardiolipin on ORC binding to origin DNA was more apparent at 30 degrees C than at 4 degrees C. Furthermore, chlorpromazine restored the ORC-origin interaction in the presence of cardiolipin. Since the presence of unsaturated fatty acids, low incubation temperatures, and the addition of chlorpromazine all decrease membrane fluidity, these results suggest that membrane fluidity is important for the inhibitory effect of acidic phospholipids on ORC-binding to origin DNA, as is the case for DnaA.
Insights
Acidic phospholipids, like cardiolipin and phosphatidylglycerol, inhibit eukaryotic Origin Recognition Complex (ORC) binding to DNA. Membrane fluidity and unsaturated fatty acids are key factors in this inhibition.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The Origin Recognition Complex (ORC) is crucial for initiating eukaryotic chromosomal DNA replication.
- Previous studies indicated cardiolipin inhibits ORC-DNA interactions, similar to DnaA in prokaryotes.
Purpose of the Study:
- To investigate the effect of other acidic phospholipids, specifically phosphatidylglycerol (PG), on ORC-origin DNA binding.
- To determine the role of fatty acid saturation and membrane fluidity in phospholipid-mediated inhibition of ORC binding.
Main Methods:
- In vitro assays measuring the binding of ORC to origin DNA in the presence of various phospholipids.
- Experiments varying fatty acid saturation (saturated vs. unsaturated) in phospholipids.
- Temperature-dependent binding studies and experiments with chlorpromazine to assess membrane fluidity effects.
Main Results:
- Phosphatidylglycerol (PG), another acidic phospholipid, was found to inhibit ORC-origin DNA interaction.
- Unsaturated fatty acids in PG enhanced the inhibitory effect compared to saturated fatty acids.
- Neutral phospholipids (phosphatidylcholine) did not inhibit ORC binding.
- Inhibitory effects were more pronounced at higher temperatures, suggesting a role for membrane fluidity.
- Chlorpromazine, which decreases membrane fluidity, restored ORC-origin interaction in the presence of cardiolipin.
Conclusions:
- Both acidic nature and unsaturated fatty acids are important for phospholipid inhibition of ORC-origin DNA binding.
- Membrane fluidity plays a significant role in the inhibitory mechanism, mirroring observations with prokaryotic DnaA.
- These findings provide insights into the regulation of DNA replication initiation by membrane properties.