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After introducing a nick in the system, the negative supercoil gradually unwinds (c) until it reaches its final, circular, plasmid state (d). [2] Nicked DNA can be the result of DNA damage or purposeful, regulated biomolecular reactions carried out in the cell. During processing, DNA can be nicked by physical shearing, over-drying, or enzymes.
Nick translation [1] (or head translation), developed in 1977 by Peter Rigby and Paul Berg, is a tagging technique in molecular biology in which DNA polymerase I is used to replace some of the nucleotides of a DNA sequence with their labeled analogues, creating a tagged DNA sequence which can be used as a probe in fluorescent in situ hybridization (FISH) or blotting techniques.
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria ; however, plasmids are sometimes present in archaea and eukaryotic organisms .
Genetic and biochemical studies have shown that the helicase is essential for plasmid rolling-circle replication and repair of DNA damage caused by exposure to ultraviolet radiation. It catalyzes the unwinding of double-stranded plasmid DNA that has been nicked at the replication origin by the replication initiation protein.
Other genes that code for relaxosome components include TraH, which stabilizes the relaxosome's structural formation, TraI, which encodes for the relaxase protein, TraJ, which recruits the complex to the oriT site, TraK, which increases the 'nicked' state of the target plasmid, and TraY, which imparts single-stranded DNA character on the oriT site.
DNA Pol I has a 5′ to 3′ exonuclease activity in addition to its polymerase activity, and uses its exonuclease activity to degrade the RNA primers ahead of it as it extends the DNA strand behind it, in a process called nick translation. Pol I is much less processive than Pol III because its primary function in DNA replication is to create ...
If a DNA segment under twist strain is closed into a circle by joining its two ends, and then allowed to move freely, it takes on different shape, such as a figure-eight. This shape is referred to as a supercoil. (The noun form "supercoil" is often used when describing DNA topology.) The DNA of most organisms is usually negatively supercoiled.
The other strand of the plasmid, the strand that was not nicked by the relaxase, is a template for further synthesis by DNA polymerase. [ 17 ] Once the relaxase reaches the upstream section of the oriT again where there is an inverted repeat , the process is terminated by reuniting the ends of the plasmid and releasing a single-stranded plasmid ...