Exam IV Flashcards
- In order for DNA was to be accepted as the genetic material, scientists needed to show DNA
o Was present in the cell nucleus and in condensed chromosomes
o Doubled during S phase of the cell cycle
o Was twice as abundant in the diploid cells as in the haploid cells of a given organism
o Showed the same patterns of transmission as the genetic information it was supposed to car
- DNA was first isolated in 1868 by
Fredrich Miescher.
Miescher isolated cell nuclei rom white blood cells in
a fibrous substance came out of solution. He called it nuclein and found it contained the elements C, H, O, N, P.
- People stained cells and confirmed two predictions of DNA
o Virtually all nondivding somatic cells of a particular organism have the same amount of nuclear DNA
o Similar experiments show that after meiosis, gametes have half the amount of nuclear DNA as somatic cells
- Chromosomes in eukaryotic cells contain DNA, but they also contain
proteins that are bound to DNA. Therefore, it was difficult for scientists to rule out that genetic information might be carrier on proteins.
- Many viruses, including bacteriophage, are composed of DNA and only one
or a few kinds of proteins. When a bacteriophage infects a bacterium, it takes about 20 minutes for the virus to hijack the bacterium’s metabolic capabilities and turn the bacterium into a virus factory. Minutes later, the bacterium is dead and hundreds of viruses are released
- The transition from bacterium to virus producer is
a change in the genetic program of the bacterial cell.
- Careful chemical analyses and observations by electron microscope showed that only
the viral DNA is injected into the cell during infection. This was further evidence that DNA and not protein was the genetic material
- Scientists rely on experiments to provide
proof of a cause and effect relationship.
- In order to confirm that DNA was the genetic material, biologists used
model organisms such as bacteria in transformation experiments. They found that the addition of DNA from one strain of bacterium could genetically transform another strain of bacterium
o Bacterium strain A + strain B DNA -> bacterium strain B
- The transformation of mammalian cells carrying genetic mutations provided another model system for showing that
that DNA is the genetic material. For example, certain cells were found to lack the gene for thymidine kinase, an enzyme that catalyzes the first step in a pathway that converts thymidine into dTTP. Such cells cannot grow in a medium that contains thymidine as the only source for dTTP synthesis. However when the cells were incubated with DNA containing the gene for thymidine kinase, some of the cells became transformed with the TK gene and were able two grow.
- For successful genetic transformation,
DNA must pass through the cell membrane into the cytoplasm and get incorporated into a host cell chromosome.
- In early transformation experiments, a major stumbling block was the first step,
because DNA is negatively charged and so are the surfaces of cell membranes. Since like charges repel, DNA does not tend to bind to cell membranes.
- Bios found they could circumvent this by
by incubating the DNA and cells in a solution.
- Transgenic
new genetically transformed organism
- The most crucial evidence for the structure of DNA was obtained using
XRAY crystallography
- Some chemical substances, when they are isolated and purified, can be made to form crystals.
The positions of atoms in a crystalized substance can be inferred from the diffraction pattern of X rays passing through the substance. The structure of DNA would not have been characterized without the crystallography prepared in the early 1950s by the English chemist Rosalind Franklin.
- Franklin’s work, in turn, depended on the success of the English biophysicist
Maurice Wilkins, who prepared samples containing very uniformly oriented DNA fibers. These fibers and the crystallographs Franklin prepared from them suggested a spiral or helical molecule
- DNA is a polymer of
nucleotides
- Each of these nucleotides consist of
a molecule of the sugar deoxyribose, a phosphate group, and a nitrogen base. The only differences between the four nucleotides of DNA are their nitrogenous bases: the purines adenine, guanine, and the pyrimidines cytosine and thymine
- Erwin Chargaff found the rule
that the amount of adenine equaled the amount of thymine and the amount of guanine equaled the amount of cytosine. The DNA would be known as Chargaff’s rule
- Watson and Crick used the chemical model to solve the structure. Watson and crick attempted to combine all that had been learned so far about DNA structure into a single coherent model.
Franklin’s crystallography results convinced them that the DNA molecule must be helical-it must have a spiral shape like a spring. Density measurements and previous model building results suggested that there are two polynucleotide chains in the molecule. Modeling studies also showed that the strands run in opposite directions, that is, they are anti-parallel.
- Watson and crick suggested that:
o The nucleotide bases are on the interior of the two strands, with a sugar phosphate backbone on the outside
o To satisfy Chargaff’s rule, a purine on one strand is always paired with a pyrimidine on the opposite strand. These base pairs have the same width down the double helix, a uniformity shown by x ray diffraction
- Four features summarize the structure of DNA
o It is a double stranded helix of uniform diameter
o It is right handed. Hold your right hand with the thumb pointing up. Imagine the curve of the helix following the direction of your fingers as it winds upward, and you have the idea
o It is antiparallel. Each strand is built in the 5-3 direction, but the two strands run in opposite directions to one another. So at one end of the double stranded molecule there is a 3OH exposed on the deoxyribose sugar of one strand and a 5 phosphate on the other
o The outer edges of the nitrogenous bases are exposed in the major and minor grooves. These grooves exist because the helices formed by the backbones of the two DNA strands are not evenly spaced relative to one another. The exposed outer edges of the base pairs are accessible for additional hydrogen bonding. Notice that the arrangements of unpaired atoms and groups differ in the AT base pairs compared with the GC base pairs. Thus the surfaces of the AT and GC base pairs are chemically distant. Allow other molecules, such a proteins, to recognize specific base pair sequences and bind to them. The atoms and groups in the major groove are more accessible, and tend to bind other molecules more frequently, than those in the minor groove. This binding of proteins to specific base pair sequences is the key to protein DNA interactions, which are necessary for the replication and expression of the genetic information in DNA
- The genetic material performs four important functions, and the DNA structure proposed by Watson and Crick was elegantly suited to three of them
o Storage of genetic information. With its millions of nucleotides, the base sequence of a DNA molecule can encode and store an enormous amount of information. Variations in DNA sequences can account for differences among species and individuals
o Precise replication during the cell division cycle. Replication could be accomplished by complementary based pairing, A with T and G with C.
o Susceptibility to mutations – the structure of DNA suggested an obvious mechanism for mutations: they might be simple changes in the linear sequence of base pairs.
o Expression of the coded information as phenotypes – the way this function is accomplished is not obvious in the structure of DNA. However, the nucleotide sequence of DNA is copied into RNA. The linear sequence of nucleotides in RNA is translated into a linear sequence of amino acids- a protein. The folded forms of proteins determine many of the phenotypes of an organism
- Semiconservative replication
each strand of the parental DNA acts as a template for a new strand which is added by base pairing
Conservative mode of replication would show
the parental DNA intact with both strands labeled, and the new DNA with both strands unlabeled. This does not occur. Instead, the resulting DNA molecules are always hybrids providing experimental evidence to support the semiconservative model of replication.
- DNA replication involves a number of different enzymes and other proteins. It takes place in two general steps
o The DNA double helix is unwound to separate the two template strands and make them available for new base pairing
o As new nucleotides form complementary base pairs with template DNA, they are covalently linked together by phosphodiester bonds, forming a polymer whose base sequence is complementary to the bases in the template strand. The template DNA is read in the 3-5 direction
- During DNA synthesis, nucleotides are added to
the 3” end of the growing new strand-the end at which the DNA strand has a free hydroxyl group on the 3’ carbon of its terminal deoxyribose,
- A free nucleotide can have one of three
phosphate groups attached to its pentose sugar, The raw materials for DNA synthesis are the four nucleotides deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), and deoxyguanosine triphosphate (dGTP) or deoxyribonucleotides.
- These nucleotides can carry
three phosphate groups.
- During DNA synthesis, the two outer phosphate groups
groups are released in an exothermic reaction. This provides energy for the formation of a phosphodiester bond between the third phosphate group of the incoming nucleotide and the 3’ position of the sugar at the end of the DNA chain
- DNA replication begins with
the binding of a large protein complex to a specific site on the DNA molecule.
- This dna protein complex contains several different proteins
among them the enzyme DNA polymerase, which catalyzes the addition of nucleotides as the new DNA chain grows.
- All chromosomes have at least one region called
the origin of replication, to which the pre-replication complex binds.
- Binding occurs when
when proteins in the complex recognize specific DNA sequences within the ori
- Once the pre-replication complex binds to it,
the DNA unwinds and replication proceeds in both directions around the circle, forming two replication forks.
Ecoli
- The single circular chromosome of the bacterium Escherichia coli has 4x10^6 base pair4s of DNA. The 245 bp ori sequence is at a fixed location on the chromosome.
- Once the pre-replication complex binds to it, the DNA unwinds and replication proceeds in both directions around the circle, forming two replication forks.
- The replication rate in E. coli is approximately 1— bp per second, so it takes about 40 minutes to fully replication the chromosome.
- Rapidly dividing e coli cells divide every 20 minutes. In these cells, new rounds of replication begin at the ori of each new chromosome before the first chromosome has fully replicated, In this way the cell can divide in less time than the time needed to finish replicating the original chromosome
- Eukaryotic chromosomes are much longer than those of prokaryotes-up to a billion bp- and are
linear, not circular. If replication occurred from a single ori, it would take weeks to fully replicate a chromosome. So eukaryotic chromosomes have multiple origins of replication, scattered at intervals of 10,000 – 40,000
- DNA polymerase elongates a polynucleotide to
a preexisting strand. However it cannot begin this process without a short starter strand, called a primer
- In most organisms this primer is a
a short single strand of rna, but in some organisms its dna
- The primer is complementary to
the DNA template and is synthesized one nucleotide at a time by an enzyme called a primase. The DNA polymerase then adds nucleotides to the 3’ end of the primer and continues until replication of that section of DNA has been completed.
- Then RNA primer is
degraded, DNA is added in its place and resulting DNA fragments are connected by the action of other enzymes. When dna replication is complete each new strand consists of DNA only
- DNA polymerase are much larger than their
substrate, the dNTPs, and the template DNA, which is very thin.
- Molecular models of the enzyme substrate template
template complex from bacteria show that the enzyme is shaped like an open hand with a palm, a thumb, and fingers.
- Within the palm is the
the active site of the enzyme, which brings together each dNTP substrate and the template.
- The finger regions have precise shapes that can
recognize the different shapes of the four nucleotide bases. They bind to the bases by hydrogen bonding and rotate inwards
- Most cells contain more than one kind of DNA polymerase, but
only one of them is responsible for chromosomal DNA replication.
- Most cells contain more than one kind of DNA polymerase, but only one of them is responsible for chromosomal DNA replication.
- The others are involved in primer removal and DNA repair.
- Most cells contain more than one kind of DNA polymerase, but only one of them is responsible for chromosomal DNA replication.
- The others are involved in primer removal and DNA repair.
- A single replication fork opens in
one direction.
- One newly synthesized strand – the leading strand-is oriented so that it can
grow continuously at its 3 end as the fork opens up.
- The other new strand-the lagging strand- must be synthesized differently
because it grows in the direction away from the replication fork
- Synthesis of the lagging strand requires the synthesis of relatively
relatively short, discontinuous stretches of sequence (100 – 200 nucleotides in eukaryotes; 1,000-2,000 nucleotides in prokaryotes)
- These discontinuous stretches are synthesized just as
the leading strand is, by the addition of new nucleotides one at a time to the 3 end, but the new strand grows away from the replication fork
- These stretches of new DNA are called
Okazaki fragments (Reiji)
- To summarize, while the leading strand grows continuously forward, the lagging strand grows in shorter backward stretches with gaps between them
- To summarize, while the leading strand grows continuously forward, the lagging strand grows in shorter backward stretches with gaps between them
- A single primer is needed to initiate synthesis of the leading strand, but each Okazki fragments requires
requires its own primer to be synthesizes by the primase. DNA polymerase then synthesizes an Okazki fragment by adding nucleotides to one primer until it reaches the primer of the previous fragment.
- At this point, a different DNA polymerase removes the old primer and replaces it with
DNA. Left behind is tiny nick-the final phosphodiester linkage between the adjacent Okazaki fragments is missing
- The enzyme DNA ligase catalyzes the
formation of the diester bond linking the fragments and making the lagging strand whole.
- DNA replication may appear complex, but it occurs with astonishing speed and accuracy. There’s less than
1 base in a million error.
- DNA replication would not proceed as rapidly as it does if DNA polymerase went through such a cycle for
each nucleotide. Instead polymerase is processive-that is, it catalyzes many sequential polymerization reactions each time it binds to a DNA molecule
o Substrate bind to enzyme -> many products are formed -> enzyme is released -> cycle repeats
- When the terminal RNA primer is removed from the replicating end of a linear eukaryotic chromosome,
no DNA can be synthesized to replace it because there is no 3” end to extend.
- So the new chromosome has a bit of single stranded DNA at the end. This situation activates a mechanism for
for cutting off the single stranded region, along with some of the intact double stranded DNA. Thus the chromosome becomes slightly shorter with each cell division
- Another problem with chromosome ends is that they must be
protected from being joined to other chromosomes by the DNA repair system. When DNA is damaged by external and internal agents, it is repaired by a combination of DNA polymerase and DNA ligase activities. This system might mistakenly recognize chromosome ends as breaks and join two chromosomes together. This would create havoc with genomic integrity.
- To prevent chromosomes from joining,
many eukaryotes have strings of repetitive sequences at the ends of their chromosomes called telomeres.
