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Plastome mutator-induced alterations arise in Oenothera chloroplast DNA through template slippage.

ABSTRACT: The plastome mutator of Oenothera hookeri strain Johansen causes deletions and duplications at target sites defined by direct repeats in the plastid genome. Previous studies characterized the mutations long after they had occurred and could not discriminate between the possibilities that the plastome mutator acted through unequal homologous recombination or template slippage. From the known hotspots, the rRNA spacer in the large inverted repeat was chosen for this study because it contains both direct and indirect repeats. Identical deletions were recovered from independently derived plants; the altered regions were always flanked by direct repeats. The regions in which the deletions occurred have the potential to form secondary structures that would stabilize the intervening sequence. Of the two affected regions, the one with the stronger potential secondary structure was altered more frequently. Because no duplication products or inversions were recovered, it is proposed that the plastome mutator acts through template slippage rather than through a recombination mechanism.

SUBMITTER: Stoike LL

PROVIDER: S-EPMC1460143 | biostudies-other | 1998 May

REPOSITORIES: biostudies-other

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Plastome mutator-induced alterations arise in Oenothera chloroplast DNA through template slippage.

Stoike L L LL Sears B B BB

Genetics 19980501 1

The plastome mutator of Oenothera hookeri strain Johansen causes deletions and duplications at target sites defined by direct repeats in the plastid genome. Previous studies characterized the mutations long after they had occurred and could not discriminate between the possibilities that the plastome mutator acted through unequal homologous recombination or template slippage. From the known hotspots, the rRNA spacer in the large inverted repeat was chosen for this study because it contains both ...[more]

PMID: 9584108

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Project description:Helicases are vital enzymes that carry out strand separation of duplex nucleic acids during replication, repair and recombination. Bacteriophage T7 gene product 4 is a model hexameric helicase that has been observed to use dTTP, but not ATP, to unwind double-stranded (ds)DNA as it translocates from 5' to 3' along single-stranded (ss)DNA. Whether and how different subunits of the helicase coordinate their chemo-mechanical activities and DNA binding during translocation is still under debate. Here we address this question using a single-molecule approach to monitor helicase unwinding. We found that T7 helicase does in fact unwind dsDNA in the presence of ATP and that the unwinding rate is even faster than that with dTTP. However, unwinding traces showed a remarkable sawtooth pattern where processive unwinding was repeatedly interrupted by sudden slippage events, ultimately preventing unwinding over a substantial distance. This behaviour was not observed with dTTP alone and was greatly reduced when ATP solution was supplemented with a small amount of dTTP. These findings presented an opportunity to use nucleotide mixtures to investigate helicase subunit coordination. We found that T7 helicase binds and hydrolyses ATP and dTTP by competitive kinetics such that the unwinding rate is dictated simply by their respective maximum rates V(max), Michaelis constants K(M) and concentrations. In contrast, processivity does not follow a simple competitive behaviour and shows a cooperative dependence on nucleotide concentrations. This does not agree with an uncoordinated mechanism where each subunit functions independently, but supports a model where nearly all subunits coordinate their chemo-mechanical activities and DNA binding. Our data indicate that only one subunit at a time can accept a nucleotide while other subunits are nucleotide-ligated and thus they interact with the DNA to ensure processivity. Such subunit coordination may be general to many ring-shaped helicases and reveals a potential mechanism for regulation of DNA unwinding during replication.

Dataset Information

Plastome mutator-induced alterations arise in Oenothera chloroplast DNA through template slippage.

Publications

Plastome mutator-induced alterations arise in Oenothera chloroplast DNA through template slippage.

Similar Datasets

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