Now that we know that replication is semiconservative. It was time to identify the enzyme that catalysed the process. It was possible to make predictions about this enzyme. First, it should require a template, that is one of the two strands of a double helix, from which it would synthesize a complementary strand using nucleotide precursors.
Here's Arthur Kornberg experiment, in which a DNA-dependent DNA polymerase was first detected. DNA was extracted from E. coli cultures and then heated to denature the double helical DNA.
The single-stranded DNA was then mixed with four deoxynucleotide triphosphates and added to an E. coli lysate that was prepared simply by bursting open the cells from a different culture.
One of the deoxynucleotides in this case, was radioactive, so that any DNA that was synthesized, even if only a small amount could be detected as large radioactive molecules.
After incubating this mixture for a while, the DNA was extracted and separated by size. This is a cartoon of a separation that might have been done using a size separating resin shown in a column in here, and material coming out of the bottom of the column in the size order shown.
The different size fractions, that is these test tubes of DNA, were collected and analysed to see which were radioactive, or if any were radioactive. And sure enough, large radioactive molecules had been made.
This DNA was extracted and then hydrolysed back down to nucleotide monomers. And when the base composition of this extracted DNA was determined, it showed that the newly made DNA had the same proportion of A, G, C and T as the parental E. coli DNA.
Kornberg concluded that the bacterial lysate contained a polymerase with the expected properties. Kornberg further analysed his polymerase and showed that the enzyme was about a thousand amino acids long, and it was present at about 400 copies per cell. He also showed that his bacterial lysates, or his purified enzymes could catalyse DNA synthesis using DNA from different species as a template. But there was a problem.
Mutants of E. coli were found that grew slowly, but still had normal levels of this polymerase. And still other mutants that had unusually low levels of this polymerase were found that grew at normal rates.
In the graph, the increase in the number of cells is plotted against time. The control, or wild-type curve has appeared first. The growth curve for the slow growing mutant, with normal levels of polymerase, appears next. And the curve for the polymerase- deficient mutant that nevertheless grew at normal rates, appeared last.
The conclusion had to be, another DNA polymerase was responsible for most of the replication of DNA in the bacterium. Arthur Kornberg's son Thomas, was the one who worked out the characteristics of two faster-acting DNA polymerases than the one his father had discovered.
We now know, after many years of looking around, that this DNA polymerase in E. coli is also involved in repair mechanisms when DNA is incorrectly replicated. And DNA polymerase 3 (the one with the asterisk) turns out to be the main polymerase of replication in E. coli.
Biochemists soon determined most of the catalytic mechanism of DNA polymerase. And perhaps not surprisingly, most of these activities turn out to be the same for all DNA polymerases, not only the ones in E. coli. But the ones subsequently isolated from many different eukaryotic, as well as prokaryotic cells.
So all DNA polymerases require a DNA template. They cannot start catalysis of new strand synthesis without a template. It also turns out that all DNA polymerases require a, primer, that is, a short pre-existing strand on which to add new nucleotides.
All known DNA polymerases cannot begin a new DNA strand from a single nucleotide. And of course, all DNA polymerases build new DNA from deoxynucleotide triphosphates precursors.
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Covered
|
Discovery Of DNA Polymerase
 |
| Arthur Kornberg |
Here's Arthur Kornberg experiment, in which a DNA-dependent DNA polymerase was first detected. DNA was extracted from E. coli cultures and then heated to denature the double helical DNA.
The single-stranded DNA was then mixed with four deoxynucleotide triphosphates and added to an E. coli lysate that was prepared simply by bursting open the cells from a different culture.
One of the deoxynucleotides in this case, was radioactive, so that any DNA that was synthesized, even if only a small amount could be detected as large radioactive molecules.
After incubating this mixture for a while, the DNA was extracted and separated by size. This is a cartoon of a separation that might have been done using a size separating resin shown in a column in here, and material coming out of the bottom of the column in the size order shown.
The different size fractions, that is these test tubes of DNA, were collected and analysed to see which were radioactive, or if any were radioactive. And sure enough, large radioactive molecules had been made.
This DNA was extracted and then hydrolysed back down to nucleotide monomers. And when the base composition of this extracted DNA was determined, it showed that the newly made DNA had the same proportion of A, G, C and T as the parental E. coli DNA.
Kornberg concluded that the bacterial lysate contained a polymerase with the expected properties. Kornberg further analysed his polymerase and showed that the enzyme was about a thousand amino acids long, and it was present at about 400 copies per cell. He also showed that his bacterial lysates, or his purified enzymes could catalyse DNA synthesis using DNA from different species as a template. But there was a problem.
Mutants of E. coli were found that grew slowly, but still had normal levels of this polymerase. And still other mutants that had unusually low levels of this polymerase were found that grew at normal rates.
In the graph, the increase in the number of cells is plotted against time. The control, or wild-type curve has appeared first. The growth curve for the slow growing mutant, with normal levels of polymerase, appears next. And the curve for the polymerase- deficient mutant that nevertheless grew at normal rates, appeared last.
Results Of The Experiment
The conclusion had to be, another DNA polymerase was responsible for most of the replication of DNA in the bacterium. Arthur Kornberg's son Thomas, was the one who worked out the characteristics of two faster-acting DNA polymerases than the one his father had discovered.
The 3 E. coli enzymes are compared in this slide. They're called Pol 1, Pol 2 and Pol 3 for short.
- Pol 1 (father Kornberg's polymerase) was the slow acting one, at 400 copies per cell, and as you'll see, it has a function in DNA replication related to a repair feature.
- DNA polymerase 2 was much faster acting. There were however a tenth the number of copies of this enzyme per cell.
Conclusion
We now know, after many years of looking around, that this DNA polymerase in E. coli is also involved in repair mechanisms when DNA is incorrectly replicated. And DNA polymerase 3 (the one with the asterisk) turns out to be the main polymerase of replication in E. coli.
Biochemists soon determined most of the catalytic mechanism of DNA polymerase. And perhaps not surprisingly, most of these activities turn out to be the same for all DNA polymerases, not only the ones in E. coli. But the ones subsequently isolated from many different eukaryotic, as well as prokaryotic cells.
So all DNA polymerases require a DNA template. They cannot start catalysis of new strand synthesis without a template. It also turns out that all DNA polymerases require a, primer, that is, a short pre-existing strand on which to add new nucleotides.
All known DNA polymerases cannot begin a new DNA strand from a single nucleotide. And of course, all DNA polymerases build new DNA from deoxynucleotide triphosphates precursors.
Read Also:
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