Robert J. Brooker - Genetica
Esperimento di genetica 11.2
DNA Replication Can Be Studied In Vitro
Much of our understanding of bacterial DNA replication has come
from thousands of experiments in which DNA replication has been
studied in vitro. This approach was pioneered by Arthur Kornberg
in the 1950s, who received a Nobel Prize for his efforts in 1959.
Figure EG11.2.1 describes Kornberg’s approach to monitor
DNA replication in vitro. In this experiment, an extract of proteins
from E. coli was used. Although we will not consider the procedures for purifying replication proteins, an alternative approach is to
purify individual proteins from the extract and study their functions
individually. In either case, the proteins would be mixed with template DNA and radiolabeled nucleotides. Kornberg correctly hypothesized that deoxyribonucleoside triphosphates (dNTPs) are the
precursors for DNA synthesis. Also, he knew that deoxyribonucleoside triphosphates are soluble in an acidic solution, whereas long
strands of DNA will precipitate out of solution at an acidic pH. This
precipitation event provides a method to separate nucleotides—in
this case, deoxyribonucleoside triphosphates—from strands of
DNA. Therefore, after the proteins, template DNA, and nucleotides
were incubated for a sufficient time to allow the synthesis of new
strands, step 3 of this procedure involved the addition of perchloric
acid. This step precipitated strands of DNA, which were then separated from the radiolabeled nucleotides via centrifugation. Newly
made strands of DNA, which were radiolabeled, sediment to the
pellet, while radiolabeled nucleotides that had not been incorporated
into new strands remained in the supernatant.
THE HYPOTHESIS
DNA synthesis can occur in vitro if all the necessary components
are present.
TESTING THE HYPOTHESIS — FIGURE 11.2.1 In vitro synthesis of DNA strands.
Starting material: An extract of proteins from E. coli.
© 2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia
Robert J. Brooker - Genetica
THE DATA
Conditions
Complete system
Control (template DNA omitted)
Amount of Radiolabeled DNA*
3,300
0
*Calculated in picomoles of 32P-labeled DNA.
INTERPRETING THE DATA
As shown in the data of Figure EG11.2.1, when the E. coli proteins
were mixed with nonlabeled template DNA and radiolabeled deoxyribonucleoside triphosphates, an acid-precipitable, radiolabeled
product was formed. This product was newly synthesized DNA
strands. As a control, if nonradiolabeled template DNA was omitted
from the assay, no radiolabeled DNA was made. This is the expected result, because the template DNA is necessary to make new
daughter strands. Taken together, these results indicate that this
technique can be used to measure the synthesis of DNA in vitro.
The in vitro approach has provided the foundation to study the
replication process at the molecular level. A common experimental
strategy is to purify proteins from cell extracts and to determine
their roles in the replication process. In other words, purified proteins, such as those described in Table 11.1, can be mixed with nucleotides, template DNA, and other substances in a test tube to determine if the synthesis of new DNA strands occurs. This approach
still continues, particularly as we try to understand the added complexities of eukaryotic DNA replication.
© 2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia