CHP 14
DNA polymerase III
catalyze the addition of nucleotides to the growing complimentary strands; Contains 10 different polypeptide chains; Dimer w/ 2 similar multi-subunit complexes (multi-functional); Different proteins play key role within each complex; Single large catalytic a subunit - catalyzes 5'-3' nucleotide addition; e subunit - 3'-5' proofreading activity; B2 dimer that clamps the pol III around the DNA double helix; pol III threads the DNA through the enzyme complex; 1000 nucleotides per second; DNA pol III can add nucleotides only to the 3' end of a DNA strand (end w/ the free 3'-OH group)
Meselson-Stahl Experiment
clearly established semiconservative DNA replication; Bacteria grown in a medium containing 15N (heavy) which becomes incorporated into bacterial DNA after many generations; Bacteria transferred to 14N (light) medium and collected at various time points; DNA isolated and centrifuged in CsCl salt solution; DNA separated according to it weight; Results: 15N-labeled DNA - all heavy; One generation after placing in 14N medium - hybrid of heavy/light; Two generations in 14N medium - 2 classes 1) hybrid of heavy/light and 2) light/light
alternative theories for DNA replication
conservative replication, dispersive replication
Chargaff's Rules
different species had equal amounts of purines (A+G) and pyrimidines (T + C), but different ratios of A+T to G+C
single-strand binding protein definition
during replication, protein that binds to the single-stranded DNA; this helps in keeping the two strands of DNA apart so that they may serve as templates
lagging strand definition
during replication, the strand that is replicated in short fragments and away from the replication fork
DNA pol III in prokaryotes
enzyme required for DNA synthesis
ligase definition
enzyme that catalyzes the formation of a phosphodiester linkage between the 3' OH and 5' phosphate ends of the DNA
primase definition
enzyme that synthesizes the RNA primer; the primer is needed for DNA pol to start synthesis of a new DNA strand
relieving the torque generated by unwinding
for replication to proceed at 1000 nucleotides per second, the parental DNA must rotate 100 revolutions per second - topoisomerases or gyrases cleave a strand of the helix, allow it to swivel around the intact strand and then reseal it
What kind of chemical bond is found between paired bases of the DNA double helix?
hydrogen
semiconservative DNA replication
if unzip the DNA strands, fill in the complimentary nucleotides for form 2 daughter strands w/ the same sequence; one stand of the original duplex is now part of a new duplex
What is the role of DNA ligase in the elongation of the lagging strand during DNA replication?
join Okazaki fragments together
You briefly expose bacteria undergoing DNA replication to radioactively labeled nucleotides. When you centrifuge the DNA isolated from the bacteria, the DNA separates into two classes. One class of labeled DNA includes very large molecules (thousands or even millions of nucleotides long), and the other includes short stretches of DNA (several hundred to a few thousand nucleotides in length). These two classes of DNA probably represent ...
leading strands and Okazaki fragments.
What covalently connects segments of DNA?
ligase
building a primer
new DNA cannot be synthesized on exposed parental templates until a primer is constructed (DNA pol III requires 3' primer to extend from); Primer is a short stretch of RNA built by RNA polymerase (primase); RNA b/c starting point many times contains errors and RNA marks this as a temporary sequence that is replaced by DNA later in replication
Where does DNA polymerase obtain the energy to add nucleotides?
nucleoside triphosphates ATP, GTP, TTP and CTP, high-energy molecules that can serve both as the source of DNA nucleotides and the source of energy to drive the polymerization; when the bond between the phosphates is "broken," the energy released is used to form the phosphodiester bond between the incoming nucleotide and the growing chain
A space probe returns with a culture of a microorganism found on a distant planet. Analysis shows that it is a carbon-based life-form that has DNA. You grow the cells in 15N medium for several generations and then transfer them to 14N medium. What pattern would you expect if the DNA was replicated in a conservative manner?
one high and one low line
In the late 1950s, Meselson and Stahl grew bacteria in a medium containing "heavy" nitrogen (15N) and then transferred them to a medium containing 14N. What results would be expected after one round of DNA replication in the presence of 14N?
one midway line
5 interlocking steps of the replication process
opening the DNA double helix, building a primer, assembling complimentary strands, removing the primer, joining the Okazaki fragments
dispersive replication
parental duplex DNA is randomly dispersed throughout the new copy of duplex DNA (mixture of parental and new DNA)
conservative replication
parental duplex stays intact and generate DNA copies consisting of entirely new molecules
DNA pol II in prokaryotes
primarily required for repair
What synthesizes short segments of RNA?
primase
transformation definition
process in which external DNA is taken up by a cell
Chargaff's Rules
proportion of A always equals T and proportion of G always equals C; always equal proportion of purines (G and A) and pyrimidines (T and C)
sliding clamp definition
ring-shaped protein that holds the DNA pol on the DNA strand
primer definition
short stretch of nucleotides that is required to initiate replication; in the case of replication, the primer has RNA nucleotides
What helps to hold the DNA strands apart while they are being replicated?
single-strand binding proteins
opening the DNA double helix
stable double stranded DNA helix must be opened and strands separated from each other: initiating replication; unwinding the duplex; stabilizing the single strands; relieving the torque generated by unwinding
leading strand definition
strand that is synthesized continuously in the 5'-3' direction, which is synthesized in the direction of the replication fork
semi-conservative replication method
suggests that each of the two parental DNA strands acts as a template for new DNA to be synthesized; after replication, each double-stranded DNA includes one parental or "old" strand and one "new" strand
lagging strand
synthesized discontinuously as a series of short segments (Okazaki Fragments) that are later joined together; 100 to 200 nucleotides long in eukaryotes; 1000 to 2000 nucleotides long in prokaryotes; Okazaki fragments synthesized 5' to 3' away from replication fork
The leading and the lagging strands differ in that ...
the leading strand is synthesized in the same direction as the movement of the replication fork, and the lagging strand is synthesized in the opposite direction.
What determines the nucleotide sequence of the newly synthesized strand during DNA replication?
the nucleotide sequence of the template strand
conservative replication method
the parental DNA remains together, and the newly formed daughter strands are together
What is the function of DNA polymerase III?
to add nucleotides to the end of a growing DNA strand
stabilizing the single strands
unwound DNA is stabilized by single-stranded binding proteins to keep them from rewinding
unwinding the duplex
"unwinding" enzymes bind to and move along one strand moving other aside as they move - Helicase
Friedrich Miescher
(1860s) isolated phosphate-rich chemicals from white blood cells (leukocytes); named these chemicals (which would eventually be known as DNA) nuclein because they were isolated from the nuclei of the cells
Friedrich Miescher
(1869) discovered an insoluble substance inside human cells and fish sperm; proportion of N and P in the substance atypical of all other cells; called substance "nuclein" because from nucleus; since nuclein was acidic in nature - renamed nucleic acid; nothing done for 50 years
Frederick Griffith
(1928) reported the first demonstration of bacterial transformation; conducted his experiments with Streptococcus pneumoniae, a bacterium that causes pneumonia, and worked with two strains of this bacterium called rough (R) and smooth (S); (the two cell types were called "rough" and "smooth" after the appearance of their colonies grown on a nutrient agar plate); the R strain is non-pathogenic (does not cause disease), the S strain is pathogenic (disease-causing), and has a capsule outside its cell wall, which allows the cell to escape the immune responses of the host mouse; when Griffith injected the living S strain into mice, they died from pneumonia; when Griffith injected the live R strain into mice, they survived; when he injected mice with the heat-killed S strain, they also survived; this experiment showed that the capsule alone was not the cause of death; in a third set of experiments, a mixture of live R strain and heat-killed S strain were injected into mice, and—to his surprise—the mice died; upon isolating the live bacteria from the dead mouse, only the S strain of bacteria was recovered; when this isolated S strain was injected into fresh mice, the mice died; concluded that something had passed from the heat-killed S strain into the live R strain and transformed it into the pathogenic S strain; called this the transforming principle
Oswald Avery, Colin MacLeod, and Maclyn McCarty
(1944) isolated the S strain from the dead mice and isolated the proteins and nucleic acids (RNA and DNA) as these were possible candidates for the molecule of heredity; used enzymes that specifically degraded each component and then used each mixture separately to transform the R strain; found that when DNA was degraded, the resulting mixture was no longer able to transform the bacteria, whereas all of the other combinations were able to transform the bacteria; led them to conclude that DNA was the transforming principle
In an analysis of the nucleotide composition of DNA,what will be found?
A + C = G + T
What best describes the addition of nucleotides to a growing DNA chain?
A nucleoside triphosphate is added to the 3' end of the DNA, releasing a molecule of pyrophosphate.
What investigator(s) was/were responsible for the following discovery? Phage with labeled proteins or DNA was allowed to infect bacteria. It was shown that the DNA, but not the protein, entered the bacterial cells, and was therefore concluded to be the genetic material.
Alfred Hershey and Martha Chase
For a science fair project, two students decided to repeat the Hershey and Chase experiment, with modifications. They decided to label the nitrogen of the DNA, rather than the phosphate. They reasoned that each nucleotide has only one phosphate and two to five nitrogens. Thus, labeling the nitrogens would provide a stronger signal than labeling the phosphates. Why won't this experiment work?
Amino acids (and thus proteins) also have nitrogen atoms; thus, the radioactivity would not distinguish between DNA and proteins.
primase
An RNA polymerase that synthesizes an RNA primer (sequence of 10 RNA nucleotides complimentary to the parent DNA template); DNA pol III recognizes the added primer and is able to add nucleotides complimentary to the parental strand; eventually the RNA primer sequence is removed and DNA nucleotides replace them
How do eukaryotes fit their DNA inside the nucleus?
At the most basic level, DNA is wrapped around proteins known as histones to form structures called nucleosomes. The histones are evolutionarily conserved proteins that are rich in basic amino acids and form an octamer composed of two molecules of each of four different histones. The DNA is wrapped tightly around the histone core. This nucleosome is linked to the next one with the help of a linker DNA. This is also known as the "beads on a string" structure. With the help of a fifth histone, a string of nucleosomes is further compacted into a 30-nm fiber, which is the diameter of the structure. Metaphase chromosomes are even further condensed by association with scaffolding proteins. At the metaphase stage, the chromosomes are at their most compact, approximately 700 nm in width
In trying to determine whether DNA or protein is the genetic material, Hershey and Chase made use of the fact that...
DNA contains phosphorus, but protein does not.
Okazaki fragment definition
DNA fragment that is synthesized in short stretches on the lagging strand
Suppose you are provided with an actively dividing culture of E. coli bacteria to which radioactive thymine has been added. What would happen if a cell replicates once in the presence of this radioactive base?
DNA in both daughter cells would be radioactive.
Chargaff's Analysis
DNA is not a simple repeating polymer; nucleotide composition of DNA varies; 4 nucleotides NOT present in equal proportions
Avery-MacLeod-McCarty experiment
DNA is the fundamental transforming principle; characterized the "transforming principle"' prepared mixture of dead Streptococcus and live Streptococcus that Griffith used; transforming resembled DNA in many ways: digesting the DNA (Dnase) eliminated transforming activity, digesting the protein did not reduce transforming activity; Avery's results not widely accepted - many still believed that protein was the repository of hereditary information
removing the primer
DNA pol I now removes the RNA primer and fills in the gap b/n Okazaki fragments with DNA
in prokaryotes, three main types of polymerases are known
DNA pol I, DNA pol II, and DNA pol III
assembling complimentary strands
DNA pol III bind to the replication fork; leading strand complexes w/ one half of the polymerase dimer, the lagging strand loops around and complexes w/ other half of dimer catalyzing formation of complimentary sequences on each of the two strands (leading and lagging) at the same time
What removes the RNA nucleotides from the primer and adds equivalent DNA nucleotides to the 3' end of Okazaki fragments?
DNA polymerase I
Which enzyme catalyzes the elongation of a DNA strand in the 5' → 3' direction?
DNA polymerase III
A new DNA strand elongates only in the 5' to 3' direction because ...
DNA polymerase can only add nucleotides to the free 3' end.
replication origin (origin of replication)
DNA replication begins at multiple sites in eukaryotes, but where there resides a specific nucleotide sequence
After mixing a heat-killed, phosphorescent strain of bacteria with a living non-phosphorescent strain, you discover that some of the living cells are now phosphorescent. What observations would provide the best evidence that the ability to fluoresce is a heritable trait?
Descendants of the living cells are also phosphorescent.
What investigator(s) was/were responsible for the following discovery? In DNA from any species, the amount of adenine equals the amount of thymine, and the amount of guanine equals the amount of cytosine.
Erwin Chargaff
Eukaryotic DNA Replication
Euk. DNA packaged into nucleosomes within chromosomes; Each individual zone replicates as a discrete section called a replication unit or replicon; 10,000 to 1 million bases; Each replicon has its own origin of replication; Multiple replicons may be undergoing replication at any given time; Regulation of replication to ensure that only one copy of the DNA is produced
What statement is true of chromatin?
Heterochromatin is highly condensed, whereas euchromatin is less compact.
What statement describes the eukaryotic chromosome?
It consists of a single linear molecule of double-stranded DNA.
In his transformation experiments, what did Griffith observe?
Mixing a heat-killed pathogenic strain of bacteria with a living nonpathogenic strain can convert some of the living cells into the pathogenic form.
In E. coli, there is a mutation in a gene called dnaB that alters the helicase that normally acts at the origin. What would you expect as a result of this mutation?
No replication fork will be formed.
Replication in prokaryotes differs from replication in eukaryotes because ...
Prokaryotic chromosomes have a single origin of replication, whereas eukaryotic chromosomes have many.
Why does the DNA double helix have a uniform diameter?
Purines pair with pyrimidines.
DNA Replication
The 2 DNA strands are assembled in different ways. Replication proceeds only in the 5' to 3' direction (DNA pol III only adds nucleotides to 3' end of DNA). 2 strands are antiparallel (oriented in opposite directions along the parent templates at each replication fork. New strands must be copied by different mechanisms. One that copies toward the replication fork - leading strand; built by simply adding nucleotides continuously in the 3' direction as parental DNA unwinds. One the copies away from the replication fork - lagging strand
What is meant by the description "antiparallel" regarding the strands that make up DNA?
The 5' to 3' direction of one strand runs counter to the 5' to 3' direction of the other strand.
phosphodiester bonds
The nucleotides combine with each other to produce phosphodiester bonds. The phosphate residue attached to the 5' carbon of the sugar of one nucleotide forms a second ester linkage with the hydroxyl group of the 3' carbon of the sugar of the next nucleotide, thereby forming a 5'-3' phosphodiester bond. In a polynucleotide, one end of the chain has a free 5' phosphate, and the other end has a free 3'-OH. These are called the 5' and 3' ends of the chain
nucleotide structure
The phosphate, which makes DNA and RNA acidic, is connected to the 5' carbon of the sugar by the formation of an ester linkage between phosphoric acid and the 5'-OH group (an ester is an acid + an alcohol). In DNA nucleotides, the 3' carbon of the sugar deoxyribose is attached to a hydroxyl (OH) group. In RNA nucleotides, the 2' carbon of the sugar ribose also contains a hydroxyl group. The base is attached to the 1' carbon of the sugar
When T2 phages infect bacteria and make more viruses in the presence of radioactive sulfur, what is the result?
The viral proteins will be radioactive.
enzyme DNA polymerase
adds nucleotides one-by-one to the growing DNA chain that is complementary to the template strand
joining the Okazaki fragments
after filling in gaps where RNA primer was w/ DNA, DNA ligase joins the fragments in the lagging strand
DNA pol I in prokaryotes
an important accessory enzyme in DNA replication, and along with DNA pol II, is primarily required for repair
What does transformation involve in bacteria?
assimilation of external DNA into a cell
initiating replication
binding of initiator proteins to origin of replication to open helix
dispersive replication method
both copies of DNA have double-stranded segments of parental DNA and newly synthesized DNA interspersed
nucleotides
building blocks of DNA; the important components of the nucleotide are a nitrogenous (nitrogen-bearing) base, a 5-carbon sugar (pentose), and a phosphate group
Martha Chase and Alfred Hershey
(1952) experiment provided confirmatory evidence that DNA was indeed the genetic material and not proteins; selected radioactive elements that would specifically distinguish the protein from the DNA in infected cells. They labeled one batch of phage with radioactive sulfur, 35S, to label the protein coat. Another batch of phage were labeled with radioactive phosphorus, 32P. Because phosphorous is found in DNA, but not protein, the DNA and not the protein would be tagged with radioactive phosphorus. Likewise, sulfur is absent from DNA, but present in several amino acids such as methionine and cysteine. Each batch of phage was allowed to infect the cells separately. After infection, the phage bacterial suspension was put in a blender, which caused the phage coat to detach from the host cell. Cells exposed long enough for infection to occur were then examined to see which of the two radioactive molecules had entered the cell. The phage and bacterial suspension was spun down in a centrifuge. The heavier bacterial cells settled down and formed a pellet, whereas the lighter phage particles stayed in the supernatant. In the tube that contained phage labeled with 35S, the supernatant contained the radioactively labeled phage, whereas no radioactivity was detected in the pellet. In the tube that contained the phage labeled with 32P, the radioactivity was detected in the pellet that contained the heavier bacterial cells, and no radioactivity was detected in the supernatant. Hershey and Chase concluded that it was the phage DNA that was injected into the cell and carried information to produce more phage particles, thus providing evidence that DNA was the genetic material and not proteins
gel electrophoresis
.. a technique used to separate DNA fragments of different sizes. Usually the gel is made of a chemical called agarose (a polysaccharide polymer extracted from seaweed that is high in galactose residues). Agarose powder is added to a buffer and heated. After cooling, the gel solution is poured into a casting tray. Once the gel has solidified, the DNA is loaded on the gel and electric current is applied. The DNA has a net negative charge and moves from the negative electrode toward the positive electrode. The electric current is applied for sufficient time to let the DNA separate according to size; the smallest fragments will be farthest from the well (where the DNA was loaded), and the heavier molecular weight fragments will be closest to the well. Once the DNA is separated, the gel is stained with a DNA-specific dye for viewing it
Meselson and Stahl
... grew E. coli for several generations in a medium containing a "heavy" isotope of nitrogen (15N), which gets incorporated into nitrogenous bases, and eventually into the DNA. The E. coli culture was then placed into medium containing 14N and allowed to grow for several generations. After each of the first few generations, the cells were harvested and the DNA was isolated, then centrifuged at high speeds in an ultracentrifuge. During the centrifugation, the DNA was loaded into a gradient (typically a solution of salt such as cesium chloride or sucrose) and spun at high speeds of 50,000 to 60,000 rpm. Under these circumstances, the DNA will form a band according to its buoyant density: the density within the gradient at which it floats. DNA grown in 15N will form a band at a higher density position (i.e., farther down the centrifuge tube) than that grown in 14N. Meselson and Stahl noted that after one generation of growth in 14N after they had been shifted from 15N, the single band observed was intermediate in position in between DNA of cells grown exclusively in 15N and 14N. This suggested either a semi-conservative or dispersive mode of replication. The DNA harvested from cells grown for two generations in 14N formed two bands: one DNA band was at the intermediate position between 15N and 14N, and the other corresponded to the band of 14N DNA. These results could only be explained if DNA replicates in a semi-conservative manner. And for this reason, therefore, the other two models were ruled out
DNA polymerase has two important restrictions
... it is able to add nucleotides only in the 5' to 3' direction (a new DNA strand can be only extended in this direction). It also requires a free 3'-OH group to which it can add nucleotides by forming a phosphodiester bond between the 3'-OH end and the 5' phosphate of the next nucleotide. This essentially means that it cannot add nucleotides if a free 3'-OH group is not available
Watson and Crick proposal of DNA structure
... proposed that DNA is made up of two strands that are twisted around each other to form a right-handed helix. Base pairing takes place between a purine and pyrimidine on opposite strands. ... The base pairs are stabilized by hydrogen bonds: adenine and thymine form two hydrogen bonds and cytosine and guanine form three hydrogen bonds. The two strands are anti-parallel in nature; that is, the 3' end of one strand faces the 5' end of the other strand. The sugar and phosphate of the nucleotides form the backbone of the structure, whereas the nitrogenous bases are stacked inside, like the rungs of a ladder. Each base pair is separated from the next base pair by a distance of 0.34 nm, and each turn of the helix measures 3.4 nm. Therefore, 10 base pairs are present per turn of the helix. The diameter of the DNA double-helix is 2 nm, and it is uniform throughout. Only the pairing between a purine and pyrimidine and the antiparallel orientation of the two DNA strands can explain the uniform diameter. The twisting of the two strands around each other results in the formation of uniformly spaced major and minor grooves
summary of the process of DNA replication in prokaryotes (10)
1. DNA unwinds at the origin of replication. 2. Helicase opens up the DNA-forming replication forks; these are extended bidirectionally. 3. Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA. 4. Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling. 5. Primase synthesizes RNA primers complementary to the DNA strand. 6. DNA polymerase III starts adding nucleotides to the 3'-OH end of the primer. 7. Elongation of both the lagging and the leading strand continues. 8. RNA primers are removed by exonuclease activity. 9. Gaps are filled by DNA pol I by adding dNTPs. 10. The gap between the two DNA fragments is sealed by DNA ligase, which helps in the formation of phosphodiester bonds.
Cytosine makes up 38% of the nucleotides in a sample of DNA from an organism. Approximately what percentage of the nucleotides in this sample will be thymine?
12%
Phoebus Levene
1920s, determined basic structure of nucleic acid (3 components): phosphate group (PO4), ribose sugar, nitrogen containing base; purine - adenine (A) and guanine (G); pyrimidine - thymine (T) and cytosine (C); RNA has uracil instead of T; each base is called a nucleotide
Hershey-Chase Experiments
1952 Hershey & Chase experimented w/ bacteriophage (viruses that infect bacteria): bacteriophage attach to bacteria, inject DNA or RNA into bacteria DNA incorporated into bacterial DNA; many new viruses in bacteria produced; bacteria eventually lyse (burst releasing new bacteria); To determine whether hereditary information is DNA or protein was the goal of the experiments: virus grown in presence of 32P (phosphorus) - incorporates only into DNA, Virus grown in presence of 35S (sulfur) - incorporates only into protein; "radiolabeled" virus allowed to infect bacteria; bacteria agitated to remove virus from the bacterial surface; 35S was found only in the virus (not inside the bacteria); 32P was not found in the virus, but now inside the bacteria; hereditary information was DNA not protein
Watson & Crick - Model of Double Helix
1953 learning from Franklin built structure of 2 strands pointed inward forming base pairs- Double Helix; 2 antiparallel strands (one running 5' to 3' and the second running 3' to 5'); also explained Chargaff's rules since there was base pairing (hydrogen bonding) b/n G-C and A-T
direction of the lagging strand
3' to 5'
At a specific area of a chromosome, the sequence of nucleotides below is present where the chain opens to form a replication fork: 3' C C T A G G C T G C A A T C C 5' An RNA primer is formed starting at the underlined T (T) of the template. What is the primer sequence?
5' A C G U U A G G 3'
direction of the leading strand
5' to 3'
Rosalind Franklin
British chemist, performed X-ray diffraction analysis on DNA; beam of X-rays directed at DNA fibers and obtained a helix or corkscrew shape
Chemical Nature of Nucleic Acids
C atoms of the ribose sugar numbered 1-5; PO4 is attached to the 5' carbon atom; nitrogenous base (G, A, T, or C) attached to the 1' carbon atom; free 3' -OH attached to the 3' carbon atom; 5' phosphate and 3' hydroxyl groups allow DNA as well as RNA to form long chains; linkage b/n phosphate group of one nucleotide and hydroxyl of another nucleotide form a covalent bond - phosphodiester bond (phosphate group linked to 2 sugars by a pair of ester bonds, results in a free 3' hydroxyl to extend chain; ALWAYS extend chain in 3' hydroxyl direction
origins of replication
There are specific nucleotide sequences where replication begins. ... The origin of replication is recognized by certain proteins that bind to this site. An enzyme called helicase unwinds the DNA by breaking the hydrogen bonds between the nitrogenous base pairs. ATP hydrolysis is required for this process. As the DNA opens up, Y-shaped structures called replication forks are formed. Two replication forks are formed at the origin of replication and these get extended bi-directionally as replication proceeds. Single-strand binding proteins coat the single strands of DNA near the replication fork to prevent the single-stranded DNA from winding back into a double helix
DNA ligase
When pol III reaches the 5' end of the lagging strand, DNA ligase joins the fragment to the lagging strand
