Chapter 14: DNA Structure and Replication
What is the replication rate in E. coli and other bacteria?
1000 nucleotides/second.
Pyrimidines
Built from a single carbon-nitrogen ring.
What isotope distribution would you expect to see if the phages used to infect E. coli were labeled with 14C, the radioactive isotope of carbon?
Carbon is found in both protein and DNA. Therefore, the radioisotope would be found in phage coats on phage infected cells, within phage-infected cells, and in progeny phages. The success of the Hershey and Chase experiment relied on using radioisotopes that were specific for DNA and protein which is why the isotopes of phosphorus and sulfur were used. The carbon radioisotope would have not been useful in the experiment to answer the question asked.
What explains DNA replication?
Complementary base pairing between the two strands.
What is the importance of complementary base pairing to DNA replication?
Complementary base pairing ensures that the new DNA double helix is a faithful copy of the parental DNA double helix. For whatever base is exposed on the template strand, the DNA polymerase inserts the nucleotide with the complementary base.
What must DNA do as hereditary material?
Faithfully store and transmit genetic information for the entire life cycle of an organism.
Complementary base pairing
Feature of DNA in which the specific purine-pyrimidine base pairs A-T (adenine-thymine) and G-C (guanine-cytosine) occur to bridge the two sugar-phosphate backbones.
Lagging Strand
The new DNA strand synthesized discontinuously during replication in the direction opposite to that of DNA unwinding.
Leading strand
The new DNA strand synthesized during replication in the direction of DNA unwinding.
The most fundamental structure of the eukaryotic chromosome
The nucleosome in which DNA wraps around an eight-protein histone complex called the nucleosome core particle.
Watson and Crick's Model for DNA Replication.
The original DNA molecule is shown in gray. A new polynucleotide chain (red) is assembled on each original chain as the two chains unwind. The template and complementary copy chains remain wound together when replication is complete, producing molecules that are half old and half new. The model is known as the semiconservative model for DNA replication.
DNA polymerase III
The principal replication polymerase in E. coli that synthesizes the majority of the new DNA.
DNA Replication
The process by which a DNA molecule is copied—replicated—to produce two identical copies.
Okazaki fragments
The short lengths of lagging strand DNA produced by discontinuous replication.
DNA polymerase I
In E. coli, the replication enzyme that replaces the RNA primer at the start of a new DNA segment with DNA.
When is telomerase expressed in humans?
In the early stages of embryo formation, in the male germ line, during development and differentiation of lymphocytes (white blood cells), and in stem cells.
X-ray Diffraction Analysis of DNA.
(A) The X-ray diffraction method to study DNA. (B) The diffraction pattern Rosalind Franklin obtained. The X-shaped pattern of spots (dashed lines) was correctly interpreted by Franklin to indicate that DNA has a helical structure similar to a spiral staircase.
Addition of a Telomere Repeat to the 3' End of a Eukaryotic Linear Chromosome by Telomerase
1. End of a chromosome showing the primer used for new DNA synthesis still in place. 2. Chromosome end after primer removal. 3. Telomerase binds to the single stranded 3' end of the chromosome by complementary base pairing between the RNA of telomerase and the telomere repeat. 4. Telomerase synthesizes new telomere DNA using telomerase RNA as the new template. 5. The longer strand is replicated by primase and DNA polymerase, and then the primer is removed, leaving a new 5' end to the bottom strand of the chromosome.
Imagine that 35S labeled both protein and DNA, whereas 32P labeled only DNA. How would Hershey and Chase's results have been different?
35S-labeled phages in this scenario will have labeled protein coats and labeled DNA. When these phages infect bacteria, radioactivity enters the cell and is found in the progeny phages. In addition, radioactivity is found in the phage material removed by the blender. 32P-labeled phages in this scenario are like the phages in Hershey and Chase's experiment; they have labeled DNA but unlabeled protein. When these phages infect bacteria, radioactivity enters the cell and is found in the progeny phages. No radioactivity is found in the phage material removed by the blender.
What direction do DNA polymerases assemble nucleotide chains?
5'-->3'
Deoxyribose
A five-carbon sugar that is a component of DNA nucleotides
Proofreading
A mechanism for correcting base pair mismatch errors made by DNA polymerase during replication.
Polymerase shape
A partially-closed human right hand in which the template DNA lies over the "palm" in a groove formed by the "fingers" and "thumb"
Phage life cycle
A phage attaches to the surface of a bacterium and infects it. Phages such as T2 quickly stop the infected cell from producing its own molecules and instead use the cell's resources for making progeny phages. After about 100 to 200 phages are assembled inside the bacterial cell, a viral enzyme breaks down the cell wall, killing the cell and releasing the new phages. The whole life cycle takes approximately 30 minutes.
Sliding clamp
A protein that encircles the DNA and binds to the DNA polymerase to tether the enzyme to the template, thereby making replication more efficient. With the clamp, many thousands of polymerizations occur before the enzyme detaches, resulting overall in a high rate of DNA synthesis.
Primer
A short nucleotide chain made of RNA that is laid down as the first series of nucleotides in a new DNA strand, or made of DNA for use in the polymerase chain reaction (PCR).
Origin of replication (ori)
A specific region at which DNA replication commences. Bacterial chromosomes have single origins of replication (ori) whereas eukaryotic chromosomes have multiple origins.
Dispersive replication model
A variant of the conservative model, neither parental strand is conserved and both chains of each replicated molecule contain old and new segments.
Bacteriophages
A virus that infects bacteria. Also referred to as a phage.
What is the replication rate in eukaryotes?
About 50-100 per second.
What else do telomeres do?
Act as caps on the ends of chromosomes, preventing the staggered ends from being recognized by the cellular machinery that detects broken DNA in need of repair.
Which bases in DNA are purines? Which are pyrimidines?
Adenine and guanine are purines. Thymine and cytosine are pyrimidines.
Telomerase
An enzyme that adds telomere repeats to chromosome ends.
DNA helicase
An enzyme that catalyzes the unwinding of DNA template strands.
Topoisomerase
An enzyme that relieves the overtwisting and strain of DNA ahead of the replication fork.
Virus
An infectious agent that contains either DNA or RNA surrounded by a protein coat. Can reproduce only in a host cell, using the cell's resources to produce more virus particles.
DNA Double Helix Diagram
Arrows and labeling of the ends show that the two polynucleotide chains of the double helix are antiparallel—they have opposite polarity in that they run in opposite directions. In the space-filling model at the top, the spaces occupied by atoms are indicated by spheres. There are 10 base pairs per turn of the helix; only 8 base pairs are visible because the other 2 are obscured where the backbones pass over each other.
What bonds form between complementary base pairs? Between a base and the deoxyribose sugar?
Complementary base pairs are held together by hydrogen bonds. Each base is attached to the deoxyribose sugar by a covalent bond.
Four nitrogenous bases
adenine (A), guanine (G), thymine (T), or cytosine (C).
Deoxyribonucleotide consists of
deoxyribose sugar, phosphate group, nitrogenous base
Guanine
A purine that base-pairs with cytosine in nucleic acids.
Adenine
A purine that base-pairs with either thymine in DNA or uracil in RNA.
Thymine
A pyrimidine that base-pairs with adenine.
Cytosine
A pyrimidine that base-pairs with guanine or nucleic acids.
What has a singular replication origin?
Bacteria chromosomes.
DNA polymerase III and DNA polymerase I are used in DNA replication in E. coli. What are their roles?
DNA polymerase III is the main DNA polymerase for replication in E. coli. This enzyme extends each primer that is synthesized on the lagging strand template, and synthesizes the leading strand. DNA polymerase I is used in lagging strand DNA synthesis. This enzyme replaces DNA polymerase when a new DNA fragment reaches the 5' end of the Okazaki fragment that was made previously. With its 5'-->3' exonuclease activity, DNA polymerase I removes the RNA primer of that Okazaki fragment, and with its 5'-->3' polymerizing activity, it replaces that primer with DNA nucleotides. In this way the Okazaki fragment is converted from an RNA-DNA hybrid into a DNA fragment.
Why is a primer needed for DNA replication? How is the primer made?
DNA polymerases cannot initiate a DNA strand; they can add DNA nucleotides only to the 39 end of an existing strand. The primer serves to provide a short stretch of nucleic acid that can be extended by DNA polymerase. The primer consists of RNA, rather than DNA, and is made by primase.
What has multiple replication origins?
Eukaryotic chromosomes.
The main features of the Double helix model are:
DNA is double stranded, consisting of two polynucleotide chains wound around each other in a right-handed double helix, like a double spiral ladder. The diameter of the double helix is 2 nm. The sugar-phosphate backbones are on the outsides of the double helix, with the base pairs on the inside. The two strands of a double helix are antiparallel (have opposite polarity), meaning that they run in opposite directions: the 3' end of one strand is opposite the 5' end of the other strand. This antiparallel arrangement is highly significant for the process of replication. Connecting the two sugar-phosphate backbones are base pairs. The base pairs lie in flat planes almost perpendicular to the long axis of the DNA molecule. 10 base pairs are stacked in a full turn of DNA. DNA has major and minor grooves
Centrifugation
DNA molecules with different densities were distinguished by a special type
Single ori
DNA replication begins from a single origin in the DNA circle, forming two forks that travel around the circle in opposite directions. Eventually, the forks meet at the opposite side from the origin to complete replication. The replicated chromosomes are distributed actively to the two halves of the bacterial cell. Subsequent binary fission of the cell produces two daughter bacterial cells, each with a copy of the chromosome.
For the semiconservative replication model, what proportion of 15N-15N, 15N-14N and 14N-14N molecules would you expect after four and five replications in 14N?
First consider the data in the Figure. After one generation in 14N, all the DNA is 15N-14N. After two generations, 1/2 the DNA is 15N-14N and 1/2 is 14N-14N. Therefore, by understanding how the semiconservative replication mechanism works, after four replications you would expect 1/8 of the DNA to be 15N-14N, and 7/8 to be 14N-14N. After five replications you would expect 1/16 of the DNA to be 15N-14N and 15/16 to be 14N-14N.
DNA Ligase
In DNA replication, an enzyme that seals nicks left after RNA primers are replaced with DNA.
Dyskeratosis congenita (DC)
Skin hyperpigmentation, white patches in the mouth, and deformity of the nails. The severity of the disorder varies among individuals. In severe cases, mortality can result from bone marrow failure, progressive loss of lung function, or cancer. The mutations in DC patients are in genes for telomerase or telomere protein components.
What explains how cancer cells can continue to divide?
Somatic cells are capable of only a certain number of mitotic divisions before they stop dividing and die. However, in most cancers, telomerase is upregulated, resulting in the preservation of chromosome length during the rapid divisions characteristic of cancer.
Sugar-phosphate backbone
Structure in a polynucleotide chain that is formed when deoxyribose sugars (in DNA) or ribose sugars (in RNA) are linked by phosphate groups in an alternating sugar-phosphate-sugar-phosphate pattern.
Replication of Antiparallel Template Strands at a Replication Fork
Synthesis of the new DNA strand on the top template strand is continuous. Synthesis on the new DNA strand on the bottom template strand is discontinuous—short lengths of DNA are made, which are then joined into a continuous chain. The overall effect is synthesis of both strands in the direction of replication fork movement.
Why are telomeres important?
Telomeres are buffers against the progressive loss of the ends of chromosomes by repeated rounds of replication. Only when the many copies of the telomere repeats have been lost are genes exposed. When those genes are lost by continued chromosome shortening and/or when chromosomes break down in the absence of telomeres, the cell is severely damaged. Telomeres also act as caps on the ends of chromosomes. The capping prevents the staggered ends from being recognized by the cellular machinery that detects broken DNA in need of repair.
Where does telomerase bind?
The DNA template strand by complementary base pairing between the telomerase RNA and the DNA.
Lagging strand template
The DNA template strand for the lagging strand.
Leading strand template
The DNA template strand for the leading strand.
How are base pairs stabilized?
The base pairs, which fit together like pieces of a jigsaw puzzle, are stabilized by hydrogen bonds—two between A and T and three between G and C.
DNA Polymerase Structure. (diagram)
The enzyme viewed from the side resembles a human right hand. The polymerization reaction site lies on the palm. When the incoming nucleotide is added, the thumb and fingers close over the site to facilitate the reaction. (B) How DNA polymerase is shown in subsequent figures of DNA replication. The figure also shows a sliding clamp anchoring the DNA polymerase to the template strand.
Chargaff's Rules
The findings that, in DNA, the amount of purines equals the amount of pyrimidines and, more specifically, that the amount of adenine equals the amount of thymine, and the amount of guanine equals the amount of cytosine.
Replisome
The key proteins and enzymes for replication assembled into a DNA replication complex.
DNA
The large, double-stranded, helical molecule that is the genetic material of all living organisms.
Semiconservative replication
The process of DNA replication in which the two parental strands separate and each serves as a template for the synthesis of new progeny double-stranded DNA molecules.
The percentage of A in a double-stranded DNA molecule is 20. What is the percentage of C in that DNA molecule?
The question focuses on the complementary basepairing rules: A = T and G = C. If A = 20% , then T = 20%, giving 40% of the DNA as A-T base pairs. Therefore, 60% of the base pairs in this DNA molecule are G-C, and the percentage of C is 30%.
Replication fork
The region of DNA synthesis where the parental strands separate and two new daughter strands elongate.
Replisomes also carry out DNA replication in eukaryotic organisms.
The replisomes are attached to specific locations on the nuclear matrix, a network of protein fibers within the nucleus. Multiple replisomes occur in a number of assemblies called replication factories. Each replisome remains stationary and, as in the E. coli replisome, the DNA to be replicated moves through it, entering as a double-stranded molecule and leaving as two identical double-stranded DNA molecules.
Is DNA or protein the genetic material?
The significant amount of 32P, the isotope used to label DNA, found within phage-infected cells and in progeny phages, indicated that DNA is the genetic material. The significant amount of 35S, the radioisotope used to label proteins, found in phage coats after infection, with little found in the infected cell or in progeny phages, showed that protein is not the genetic material.
Deoxyribonucleoside triphosphates
The substrates for the polymerization reaction catalyzed by DNA polymerases.
The Four Deoxyribonucleotide Subunits of DNA, Linked into a Polynucleotide Chain
The sugar-phosphate backbone of the chain is highlighted in gray. The connection between adjacent deoxyribose sugars is a phosphodiester bond. The polynucleotide chain has polarity; at one end, the 5' end, a phosphate group is bound to the 5' carbon of a deoxyribose sugar, whereas at the other end, the 3' end, a hydroxyl group is bound to the 3' carbon of a deoxyribose sugar.
Replication bubble
The two Y-shaped replication forks joined together at the tops of the Ys after DNA is unwound at an origin of replication.
In sum, the key molecular events of DNA replication are as follows:
The two strands of the DNA molecule unwind for replication to occur. DNA polymerase adds nucleotides to an existing chain using the parental template strand to determine which nucleotide to use. The overall direction of new synthesis is in the 5'-->3' direction, which is a direction antiparallel to that of the template strand. Nucleotides join a new chain being synthesized according to the A-T and G-C complementary base-pairing rules.
Conservative replication model
The two strands of the original molecule serve as templates for the two strands of a new DNA molecule, then rewind into an all "old" molecule. After the two complementary copies separate from their templates, they wind together into an all "new" molecule.
Phosphodiester bond
The type of bond that links the nucleotides in DNA or RNA. Joins the phosphate group of one nucleotide to the hydroxyl group on the sugar of another nucleotide
What is the significance of a telomere not having genes?
They act as a buffer to the deletion of genes by shortening. That is, with each replication, a fraction of the telomere repeats is lost but the genes are unaffected.
Four different deoxyribonucleoside triphosphates are used for DNA replication.
They differ by the base they contain, that is, adenine (A), guanine (G), cytosine (C), or thymine (T), and they are given the short names dATP, dGTP, dCTP, and dTTP, where the "d" stands for "deoxyribose."
What did Watson and Crick do?
They figured out the structure of DNA.
What stabilizes the DNA double helix?
Van der Waals forces between stacked bases.
Which features of the DNA molecule did Watson and Crick describe?
Watson and Crick described the right-handed double helix that consists of two sugar—phosphate backbones on the outside and complementary base pairs between the two backbones. A complementary base pair is a purine paired with a pyrimidine, more specifically, an A with a T, and a G with a C. The two strands of DNA are antiparallel. The key dimensions of the molecule are: diameter = 2 nm; 1 base pair = 0.34 nm; 1 turn of the helix = 10 base pairs = 3.4 nm.
Theoretically, could the existence of the transforming principle have been shown with the opposite setup, that is, by injecting mice with heat-killed R cells plus live S cells?
Yes, theoretically this could have been done; that is, the transforming principle is the genetic material. Given that R and S cells are genetically different, the transforming principle released from heat-killed R cells could convert living S cells to R cells. However, only if all S cells in a mouse were converted to R cells would the mouse live. This is unlikely so, practically speaking, this proposed experimental design is not a good one.
During replication,
enzymes known as DNA polymerases assemble complementary polynucleotide chains from individual deoxyribonucleotides.
Each DNA strand has two distinct ends:
the 5' end has an exposed phosphate group attached to the 5' carbon of the sugar, and the 3' end has an exposed hydroxyl group attached to the 3' carbon of the sugar.
Transformation
The conversion of the hereditary type of a cell by the uptake of DNA released by the breakdown of another cell.
Telomere syndromes
Rare, diverse degenerative human disorders that share short telomere length, caused by mutations in telomere maintenance genes.
Telomeres
Simple, tandemly repeated DNA sequences and associated specific proteins tat protect the ends of linear eukaryotic chromosomes.
Repair mechanisms to correct DNA replication errors
Proofreading, mismatch repair, and excision repair.
Double helix model
Model of DNA consisting of two polynucleotide strands twisted around each other.
Nucleoside triphosphate
Nitrogenous base linked to a sugar, which is linked, in turn, to a chain of three phosphate groups.
Purines
Nitrogenous bases built from a pair of fused rings of carbon and nitrogen atoms.
Chromosome duplication in eukaryotes
Nucleosomes first disassemble from the chromosome as the replication fork passes. Then, almost immediately, the newly replicated DNA assembles into nucleosomes. Those nucleosomes are mixtures of parental histones—histones that were in nucleosomes before replication—and newly synthesized histones.
When does the buffering of a telomere end?
Only when the entire telomere is lost.
What did the discovery of the DNA structure mean?
Opened the way to molecular studies of genetics and heredity, leading to our modern understanding of gene structure and action at the molecular level.
Initiation of a New DNA Strand by Synthesis of a Short RNA Primer by Primase, and the Extension of the Primer as DNA by DNA Polymerase
Primase synthesizes a short RNA primer to initiate a new DNA strand. Primase leaves; DNA polymerase takes over. New DNA extended from primer from polymerase. DNA polymerase.
Erroneously attempting to repair normal chromosome ends could lead to:
Programmed cell death, the purposeful destruction of the cell to avoid propagation of cells with DNA damage.
Single-stranded binding proteins (SSBs)
Protein that coats single-stranded segments of DNA, stabilizing the DNA for the replication process.
Of what does telomerase consist?
Proteins and an RNA molecule.
Discontinuous replication
Replication in which a DNA strand is formed in short lengths that are synthesized in the direction opposite of DNA unwinding.
