Dataset Information

Mitochondrial origin-binding protein UMSBP mediates DNA replication and segregation in trypanosomes.

ABSTRACT: Kinetoplast DNA (kDNA) is the remarkable mitochondrial genome of trypanosomatids. Its major components are several thousands of topologically linked DNA minicircles, whose replication origins are bound by the universal minicircle sequence-binding protein (UMSBP). The cellular function of UMSBP has been studied in Trypanosoma brucei by using RNAi analysis. Silencing of the trypanosomal UMSBP genes resulted in remarkable effects on the trypanosome cell cycle. It significantly inhibited the initiation of minicircle replication, blocked nuclear DNA division, and impaired the segregation of the kDNA network and the flagellar basal body, resulting in growth arrest. These observations, revealing the function of UMSBP in kDNA replication initiation and segregation as well as in mitochondrial and nuclear division, imply a potential role for UMSBP in linking kDNA replication and segregation to the nuclear S-phase control during the trypanosome cell cycle.

SUBMITTER: Milman N

PROVIDER: S-EPMC2148276 | biostudies-literature | 2007 Dec

REPOSITORIES: biostudies-literature

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Mitochondrial origin-binding protein UMSBP mediates DNA replication and segregation in trypanosomes.

Milman Neta N Motyka Shawn A SA Englund Paul T PT Robinson Derrick D Shlomai Joseph J

Proceedings of the National Academy of Sciences of the United States of America 20071128 49

Kinetoplast DNA (kDNA) is the remarkable mitochondrial genome of trypanosomatids. Its major components are several thousands of topologically linked DNA minicircles, whose replication origins are bound by the universal minicircle sequence-binding protein (UMSBP). The cellular function of UMSBP has been studied in Trypanosoma brucei by using RNAi analysis. Silencing of the trypanosomal UMSBP genes resulted in remarkable effects on the trypanosome cell cycle. It significantly inhibited the initiat ...[more]

PMID: 18048338

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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.

Dataset Information

Mitochondrial origin-binding protein UMSBP mediates DNA replication and segregation in trypanosomes.

Publications

Mitochondrial origin-binding protein UMSBP mediates DNA replication and segregation in trypanosomes.

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