A Positron Named Priscilla Scientific Discovery at the Frontier (1994) / Chapter Skim
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4 Doubling Up: How the Genetic Code Replicates Itself
4 Doubling Up: How the Genetic Code Replicates Itself
Pages 98-123
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From page 98... ...
Although it had been known for hundreds of years that plants and animals had some sort of genetic material that carried information from generation to generation, what it was and how it worked remained unknown until 1953, when the American lames Watson and the Englishman Francis Crick made their announcement from Cambridge, England. Their description of DNA as a twisted, ladder-like structure with rungs of complementary pairs of simple chemical bases offered a basic architecture for the genetic code.
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From page 99... ...
- could now be tackled. Moreover, broad problems-such as the way DNA duplication fits into the total cell cycle, the ongoing process of cell growth and division could be addressed (see Box beginning on p.
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From page 100... ...
phase, which lies between the previous nuclear division and the start of DNA synthesis. Once cells reach a critical size in G1, a decision is made whether to commit fit is possible for cells to exit from the cell cycle and still be very much alive.
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From page 101... ...
The complete cell cycle in a mammalian cell needs about 16 hours, and the S phase can take almost half ~at. The time devoted to DNA synthesis is even more striking In E
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From page 102... ...
Pulling It All Together Although much is known about what happens in the individual stages of the cell cycle, one of the biggest challenges facing cell biology is to learn
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From page 103... ...
For example, stopping DNA duplication keeps cells from entering mitosis. Although early studies of the cell cycle focused on external extracellular signals, such as hormones and other growth factors, in recent years many clues have emerged about regulatory signals that come from within the cell.
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From page 104... ...
These so-called replication bubbles were identified in the chromosomes of animal, plant, and bacterial viruses as well as those of more advanced organisms, such as yeast and some mammals. Other researchers fed microbial and animal cells labeled isotopes at the start of DNA synthesis, with normal isotopes offered later in the same round of DNA synthesis.
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From page 105... ...
, proposed in 1963 by Francois Jacob and Sydney Brenner, which suggested that DNA replication is controlled by a biochemical regulator, known as the initiator, which triggered the start of DNA duplication by achng on a specific collection of DNA bases, called the replicator, or replication origin. But this work did not offer definitive proof of the existence of the postulated replicator and its helpmate, the initiator.
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From page 106... ...
Now that some details are known about the various proteins needed to guide DNA replication, cell growth, and division in normal cells, efforts can be made to design drugs that correct some of the biochemical imbalances in cancer cells. By adding or deleting key proteins, such anticancer drugs may inject a level of management and structure that is missing in cancer cells.
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From page 107... ...
Another complication that might affect chemotherapeutic strategies is the finding that inhibiting RNA or protein synthesis protects cells from the cell-killing action of the topoisomerase poison, Amsacrine. So now there is a search for factors that could inhibit, or enhance, the impact of topoisomerase poisons.
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From page 108... ...
Flanking this essential core were other DNA sequences in the virus genome that stimulated DNA replication. A key issue, of course, was what the initiator protein actually did to trigger DNA duplication.
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From page 109... ...
Was the system used by viruses, FIGURE 4.3 Initiation of SV40 DNA replication. {Courtesy of Bruce Stillman, Cold Spring Harbor Laboratory.)
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From page 110... ...
Such conservation among species hints that these nucleotides are important for biological function and may well interact with initiator proteins that are also conserved among the species. A key challenge, then, was to demonstrate that this general strategy for starting duplication of DNA having a protein identify a specific origin sequence, among millions of others on the double helix, and then having it uncoil the DNA also worked for the more advanced organisms known as eukaryotes, organisms that have a nucleus and have the genome divided among multiple chromosomes.
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From page 111... ...
Then 3' 5' As, ~,~/ A_ 5 ~primase DRAM\ ~ hel~'case ~_ 3' PChIA RPA FIGURE 4.4 The multiprotein complex of the replisome keeps DNA synthesis progressing smoothly. In eukaryotes it includes two polymerases, alpha and delta; primase; helicase (T antigen in the case of SV40 virus DNA replications; replication protein A (a single-stranded b in cling prote inJ; replica tic n factor c; and the proliferating cell nuclear antigen.
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From page 112... ...
{Courtesy of Bruce Stillman, CoIcl Spring Harbor Laboratory.J 112 ~ · · · a___ ~__ l tarry ._ | Late 1 you initiate from all of them and you'll replicate the genome." Like a parallel-processing supercomputer, and very unlike a simple virus, eukaryotic cells have hundreds, thousands, or even tens of thousands of replication processes going on simultaneously (see Figure 4.5~. Indeed, it is now known that in the particular experimental yeast favored by researchers, called Saccharomyces cerevisiae, at least 400 replication origins exist in its 16 chromosomes.
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From page 113... ...
With this information, a "footprint" of the initiator protein binding to a particular set of DNA sequences was pieced together, and, using special biochemical techniques, the protein was extracted and purified from the complex. This protein complex, it was found, bound to all of the yeast's replication origins.
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From page 114... ...
In fact, loachim Li of the University of California, San Francisco, and Bruce Alberts, formerly at that institution and now president of the National Academy of Sciences, have noted that, "By following the fate of the Saccharomyces cerevisiae initiator protein during the replication reaction and throughout the cell cycle, we can expect to learn how the cell prepares itself for a new round of initiation and how it prevents those preparations from occurring prematurely." Another possibility is to use information about the yeast origin recognition protein to fill gaps in our understanding of the cell cycle (see Figure 4.6~. For example, much attention has been given to the study of the various biochemical events of the G1 phase immediately prior to the synthesis of DNA.
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From page 115... ...
. be possible to compare the predicted amino acid sequences of the ORC polypeptides to those of known proteins; any similarities that turn up will suggest roles for the individual polypeptides in the initiation process." And, add Li and Alberts, "The discovery of homologues to the ORC, and analysis of their DNA binding sites, could provide a shortcut to defining replication origins in [other]
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From page 116... ...
Although the polypeptide components of the machinery may differ in detail from organism to organism, enough is known about them to conclude that some key polypeptides fill certain roles in DNA replication in all organisms. The proteins described here go into action after the initiator proteins described earlier.
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From page 117... ...
The single-stranded DNA binding protein speedily dissociates as a different enzyme, DNA polymerase, approaches. The Polymerases The DNA polymerases were first recognized for their ability to add deoxynucleotides into DNA.
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From page 118... ...
Freeman and Company.J 118 tori strands Movement of growing fork RNA oligonucleotides (primer) copied from DNA DNA polymerase elongates RNA primers with new DNA DNA polymerase removes 5' RNA at end of neighboring fragment and fills gap DNA ligase · ~ Jolns adjacent fragments Lagging-strand synthesis Old DNA Lagging strand , Leading strand RNA primer ,-_ New Okazaki fragment DNA Ligation
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From page 119... ...
It is believed that the giant asymmetric polymerase III clamps on to the growing DNA chain throughout the polymerization, instead of dissociating and binding randomly to other growing chains. Exonucleases, Polymerases, and [igases That Piece Together the Leading and Lagging Strands As mentioned earlier, two different types of DNA strands are made at the replication fork: the leading strand builds in the direction of the replication fork, and the other, the lagging strand, builds in the opposite direction.
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From page 120... ...
The concerted action of these various accessory proteins keeps synthesis of the leading and lagging strands clean flowing. The Topoisomerases Adjust Swivel to DNA The protein apparatus of the replisome appears to prevent tangles at the outset of synthesis, but another set of proteins, the topoisomerases, probably help resolve tangles that are created later in synthesis and exist in newly replicated DNA: they seem essential to the successful conclusion of replication and the separation of finished chromosomal copies.
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From page 121... ...
coli, for example, is that one segment of the DNA polymerase III holoenzyme, the ~ subunit, proofreads newly synthesized DNA by recognizing the distortions produced by an incorrectly paired base. This is probably done by some physical means where the enzyme detects a malformation in the three-dimensional structure of the DNA.
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From page 122... ...
Both these lines of study may reveal more molecular details about DNA repair In advanced organisms, including humans. But they could also offer an explanation of how uncorrected damage to DNA leads to the uncontrolled proliferation of DNA and cell growth characteristic of - cancer.
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From page 123... ...
1991. What controls the cell cycle.
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