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DNA processing during entry in transformation of Streptococcus pneumoniae
1Unité de Microbiologie et de Génétique Moléculaires du CNRS-UPR9007, Université Paul Sabatier, Toulouse, France.
The Journal of Biological Chemistry
|March 15, 1993
Summary
Streptococcus pneumoniae uses a DNA uptake model where one strand enters the cell while the other is degraded. This study confirms DNA entry and degradation occur at similar rates and opposite polarities, requiring an intact DNA backbone.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The established model for DNA processing during Streptococcus pneumoniae transformation involves double-strand DNA cleavage, followed by linear uptake of one strand from a 3'-end and simultaneous degradation of the other.
- Key predictions of this model include opposing polarities for DNA degradation and entry, and degradation rates at least matching entry rates.
Purpose of the Study:
- To experimentally validate the polarity and rate predictions of the current DNA processing model during Streptococcus pneumoniae transformation.
- To investigate the mechanism of DNA strand processing, entry, and degradation in Streptococcus pneumoniae.
Main Methods:
- Utilized in vitro constructed, single-strand labeled donor DNA molecules for transformation experiments.
- Employed uniformly labeled, 3'-end labeled, and 5'-end labeled DNA to track strand processing and degradation.
- Performed kinetic analysis of DNA entry and degradation rates at 31°C.
- Tested the effect of single-strand interruptions on DNA uptake.
Main Results:
- Uniformly labeled DNA uptake resulted in acid-soluble label recovery in the medium, indicating degradation.
- 3'-end labeled DNA was largely susceptible to degradation, while 5'-end labeled DNA was mostly resistant.
- DNA entry and degradation occurred at similar rates (approx. 100 nucleotides/s at 31°C) and with opposite polarities (entry 3'→5', degradation 5'→3').
- A single-strand interruption significantly inhibited the uptake of downstream DNA sequences, confirming a model prediction.
Conclusions:
- The findings support the model of DNA processing during Streptococcus pneumoniae transformation, with concomitant entry and degradation occurring at similar rates and opposite polarities.
- An intact sugar-phosphate backbone is necessary for the continuous uptake machinery.
- DNA entry proceeds in a 3' to 5' direction, while degradation occurs 5' to 3'.