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A new technique to check DNA repair mechanism

A new technique to check DNA repair mechanism

In an article entitled, “Single-molecule DNA repair in live bacteria,” published in April 29, 2013, issue of PNAS (www.pnas.org/cgi/doi:10.1073/pnas.1301804110), the lead author Stephan Uphoff with four other associates from three research groups have reported a technique to observe DNA repair in living bacteria. Damage caused by mutagens to the nucleotide bases of DNA is reversed by a process called base excision repair, executed by a suite of enzymes. Two such enzymes (DNA polymerase I, which replaces missing nucleotides, and DNA ligase, which ligates DNA strands to help close nicks) perform the final steps of the repair process in a range of organisms from bacteria to humans, but the enzymes have not been directly observed in action. Stephan Uphoff and colleagues used a technique called photo activated localization microscopy to visualize the activity of single molecules of both enzymes in live Escherichia coli bacteria. Interpreting tracks of single enzyme molecules bound to DNA as evidence of DNA synthesis and ligation, the authors found that the DNA repair sites were spread throughout the cell. In addition, they reported that individual repair events lasted 2.1 seconds for the polymerase, and 2.5 seconds for the ligase; within minutes of experimentally induced DNA damage, the activities of both enzymes increased five-fold over baseline. Only 2.7% of approximately 400 copies of polymerase and 3.8% of around 200 copies of ligase are active in replication and repair at any given time in a bacterial cell with undamaged DNA. It was also revealed that when the cell was subjected to DNA damage, both enzymes spent more than 80% of their lifespan searching for toxic intermediates to repair, thus minimizing the lifetimes of gaps and nicks. Furthermore, direct measurements of DNA repair rates in living cells might lead to quantitative models that can help researchers predict how organisms might respond to DNA damage. [Summarized by Samsad Razzaque, Research Associate at Plant Biotechnology Lab, DU]

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