Read Microsoft Word - DNAStructureReplica-211.doc text version

DNA Structure and Replication - 1 DNA is the genetic molecule of all life. DNA (along with associated proteins) is found in chromosomes. DNA ultimately controls all cell activities. As studied, specific enzymes catalyze the metabolic activities of cells. The instructions for the synthesis of enzymes (and all proteins), as well as RNA molecules (see later) are found in the structure of DNA. We also know that the DNA in each of our cells is identical; DNA molecules duplicate* prior to cell division ensuring that the new cells formed are genetically identical to the original cell.

*Duplicate and replicate are both commonly used for the same process.

We know, too, that g enes, or our inheritable traits, are functional regions of DNA. A gene locus of DNA stores the information that specifies the sequence of amino acids that form a specific polypeptide. The genes, or more precisely, alleles we inherit from our parents determine the polypeptides we synthesize in our cells, which determine the structure and functioning of our cells and tissues. What DNA is and how DNA works is the subject of this section. We will look at the structure and functions of DNA, how the information stored in DNA is used to direct cell activities and how cells regulate the activity of their genetic molecules. We shall also see how DNA duplicates itself prior to cell division. The search for the molecule of inheritance spanned a century from the 1850's to 1953, when Francis Crick and James Watson announced they had a model for the three dimensional structure of DNA. Genetic Material is in the Nucleus DNA was first isolated by F riedrich Miescher in 1868. Miescher identified a phosphorus containing acid in the nuclei of cells that he called n uclein. He also found a basic protein portion in the nucleus, which we today know are the histone proteins. In 1914 R obert F eulgen developed a stain that was selective for this acid material in the nucleus. Feulgen noted that the stained volume of the nuclear material was the same for all body (somatic) cells, but gametes had half as much of this material. He also noted that cells that were about to divide had twice as much nuclear material. He also noted that different species had different volumes of the stained nuclear materials. By the early 1900's the search for the genetic molecule was focused on molecules found in the nucleus, particularly after Morgan's work confirming that genes were on chromosomes. However, it was not until the 1930's when Hammerling did a set of experiments using an alga, Acetabularia, that we confirmed that the information needed to express genetic traits was located in the nucleus. Acetabularia is a single-celled organism, with several morphologically different species, that is large (5cm) and has three morphologically distinct regions ­ a cap, a stalk and a base. The nucleus is in the base. Hammerling transplanted stalks from one species to a second, and the new caps regenerated were dictated by the base, not by the stalk.

DNA Structure and Replication - 2

Hammerling's work was confirmed in 1952 with frog nucleus transplant experiments conducted to determine how long a cell remained totipotent (full genetic competence) during development. Although few frogs developed from these transplant experiments, the same process we use today to clone animals as discussed in our section on cell division, those that did develop contained nuclear components of the transplanted nucleus, not the nuclear components of the donated enucleated egg.

Discovering the Genetic Molecule ­ Protein or Nucleic Acid Although genes, chromosomes and the transmission of genetic information were studied extensively in the first half of the 20th century, the molecular structure of a gene was not known. Despite Feulgen's evidence in 1914 that DNA behaved the way chromosomes do in cell division, no one had actually proven that the genetic material was even in the nucleus when Feulgen did his work and the nucleus contained concentrations of both proteins and nucleic acids.

DNA Structure and Replication - 3 Because of protein's diversity of structures and specificity of functions many scientists believed that the genetic molecule had to be protein. In contrast, DNA is composed of some fairly simple molecules: phosphates, a five-carbon sugar, and four different nucleotides, so the means by which it could serve as the genetic molecule was perplexing. The evidence slowly accumulated that ultimately determined that the genetic molecule had to be DNA rather than protein. The work continued. Evidence #1 In 1928, Fred Griffith was trying to find a vaccine to protect against a pneumoniacausing bacterium, Streptococcus pneumoniae (called Pneumococcus at that time). He isolated two strains of the bacterium, one pathogenic and one not. The pathogenic strain also had a polysaccharide capsule that gave it a smooth (S) appearance in culture. The harmless strain appeared rough (R) in culture. The S form's virulence comes from capsule, which protects it from harmful things in its environment, in this case is the immune system of the host. Griffith injected bacteria of each strain into mice, and observed what happened. Mice injected with S forms died. Mice injected wit R forms lived. He also heated the bacteria to kill them and mice injected with heat-killed S forms lived (as did those injected with heat-killed R forms. But: Mice injected with a mixture of heatkilled S forms and live R forms died, and when necropsied, contained live bacteria with proteins characteristic of the R forms, but the virulent coating (capsule) of the S form.

What did this mean? 1. The production of a capsule is an inheritable trait that distinguishes the R form from the S form of the bacterium. 2. The heat that killed the S cells did not damage the material that had genetic instructions (on how to make a capsule) so these instructions could be incorporated into the living R cells from the environment t ransforming the R cells into virulent S forms. 3. The substance was called a C hemical Transforming Principle. Today, t ransformation is defined as the process by which external DNA is assimilated into a cell changing its genotype and phenotype as discussed briefly in our section on Bacterial gene transfer. Transformation is one of the processes used in

recombinant DNA technologies and a means of genetic change in bacteria (discussed previously.

DNA Structure and Replication - 4 Evidence #2 Starting in the 1930's, a group of microbiologists, headed by Oswald Avery, determined to identify the transforming molecule, surmising that the molecule that caused this inheritable transformation had to be the genetic molecule. Avery's group purified the transforming extracts from the mixed R and S bacteria, achieving 99.98% purity of DNA, RNA (the second nucleic acid) and protein. · Chemical analysis of one purified extract showed it to be the same as DNA. · Using ultracentrifuging, the transforming extract had the same density as DNA. · The purified extract retained its transforming ability when injected into mice along with the non-pathogenic R form Streptococcus pneumoniae. Avery went beyond this step to "rule out" the other extracts (RNA and protein) by repeating Griffith's experiments with the transformation extracts, adding a series of enzymes (from the pancreas) that selectively destroyed DNA, RNA or protein. They performed the following experiments:

1. Mice + RNA-digesting enzyme + heat-killed S + R Dead Mice 2. Mice + Protein-digesting enzyme + heat-killed S + R Dead Mice 3. Mice + DNA-digesting enzyme + heat-killed S + R Live Mice

Avery completed their work by purifying DNA from their sample and with the purified DNA extract got transformation.

DNA Structure and Replication - 5 In their work, published in 1944, Avery's group concluded that DNA was the genetic molecule, or at least the hereditary material for Pneumococcus (Streptococcus pneumoniae). Transformation was prevented only when DNA was destroyed. To be precise, they wrote: "a nuclei acid of the deoxyribose type is the fundamental unit of the transforming principle in Pneumococcus Type III". We know today that the gene that caused the transformation codes for an enzyme that catalyzes the capsule formation. Many scientists still disputed this conclusion, since DNA lacked what was assumed to be the requisite complexity for our genetic material (the structure of DNA was still unknown), and virtually nothing was known about bacterial genetics, so even if this might be true for bacteria, why should we assume it to be the same in other organisms. Evidence # 3 The Viral Confirmation Bacteriophages (viruses that invade bacteria and convert the bacteria into virus making machines) proved to be the means by which the question was finally answered. In 1952, Hershey and Chase (and others) confirmed that DNA was the genetic molecule. Viruses have just a genetic molecule (DNA or sometimes RNA) and a protein protective coat. In 1952 Alfred Hershey and Martha Chase used phages to demonstrate that the genetic molecule was DNA. Proteins contain sulfur, but not phosphorus and DNA contains phosphorus, but not sulfur. Hershey and Chase used radioactive Sulfur (35S) and Phosphorus (32P) to "label" T2 phages. They then tracked the invasion of phages into host bacteria (a strain of E coli) to determine what part of the phage entered the bacterial cells and whether new phages produced had radioactive 35S or 32P. The bacterial hosts had only 32P and the medium contained 35S, proving that only viral DNA entered the bacterial hosts. In addition some of the new generation of phages also had 32P . Hershey and Chase were able to confirm that DNA was the genetic molecule.

Phages on Bacterium

DNA Structure and Replication - 6

Still, the structure of DNA was unknown, so no one had an explanation for how DNA could do its job. Moreover, research on genetics of viruses and bacteria had just commenced and many were skeptical that DNA transformation (called transfection in eukaryotes) could occur in eukaryotes.

As a brief aside: Transfection does occur in eukaryotes and can be demonstrated using a genetic marker, a gene that has an easily observable phenotype (gene expression). Transfection is an important tool in recombinant DNA research and a genetic marker permits researchers to readily identify whether or not DNA has been incorporated into the target cells or organisms, even when the desired gene may not be expressed. Common gene markers are fluorescent genes, which are readily visible in the host, or genes that confer a nutritional need or antibiotic resistance when using cell cultures. Target cells are grown in a nutrient deficient medium or a medium with antibiotics. Only those cells transformed will grow. Vectors (viruses or artificial chromosomes) are typically used for gene transfer in eukaryotic transfection. Recombinant DNA techniques are explored in our section on genetic technology.

Now back to the DNA story:

DNA Structure and Replication - 7 Structural Evidence supporting DNA as the Genetic Molecule Demonstrating that DNA was the genetic molecule was one significant part of the solution. Knowledge of the three dimensional structure of the molecule was also required to understand how DNA works. By 1950 the following was known about the DNA molecule: 1. PA Levene had determined in the 1920's that DNA was a polymer of nucleotides. Each nucleotide contained: · Phosphate (P) · The 5-carbon sugar, deoxyribose · One of four different nitrogen-containing bases Two were double ring purines Adenine Guanine Two were single ring pyrimidines Thymine Cytosine

The sugar phosphate formed a backbone with one of the four bases attached to the side of the sugar. The phosphate attaches to the number 5 carbon and the nitrogen base attaches to the number 1 carbon of the sugar. (Yes, you need to know

this.)

It was also known that nucleic acids were formed by linking the number 3 carbon of one sugar to the phosphate of the second sugar by a p hosphodiester bond. The sugar-phosphate chains form a "backbone" for the nucleic acid with the nitrogen bases attached to the side of each sugar molecule (S-P-S-P-S-P-S-P, etc.). This description, regrettably, can be misleading. When DNA molecules models are assembled, one wants to make the backbone chain first, and then add the nitrogen bases. We need to remember that DNA molecules are assembled nucleotide by nucleotide. But the polymer structure was still unknown.

DNA Structure and Replication - 8

Recall that the 3' carbon of the sugar has a bonding site to attach to a second nucleotide's phosphate (which is bonded to the 5' carbon). The second nucleotide's 3' carbon of its sugar can now bond to the third nucleotide's phosphate, a process that is repeated as the polymer grows. This little detail is important to the structure of DNA. Deoxyribose is a 5carbon sugar. What molecule bonds to which carbon is critical to DNA's structure. And the polarity of the DNA molecule is determined by this precise bonding. 2. In addition, Mirsky had restated Feulgen's work from 1914 that determined the relationship of the volume of DNA in the nucleus for normal body cells, for cells just prior to division and for "germ" cells or gametes. This provided evidence that DNA was the genetic molecule because it corresponded with the behavior of chromosomes in mitosis and meiosis. Erwin Chargaff published nucleotide base composition of several species in 1950. · The amounts of the four nitrogen bases varied in the different species. · But: The amount of Adenine was always the same as Thymine The amount of guanine was always the same as Cytosine This information is known as Chargaff's rules (although not to Chargaff). In addition, it was known that adenine had two potential hydrogen bonding sites, as did thymine, and guanine and cytosine each had three hydrogen bonding sites. Still we did not have a structural model for DNA.

3.

DNA Structure and Replication - 9 4. X-ray diffraction (best done by Rosalind Franklin working in Maurice Wilkins' laboratory at King's College in London) showed that DNA: · was long and thin · had a uniform diameter of 2 nanometers · had a highly repetitive structure with .34 nm between nitrogen bases in the stack · was probably helical in shape, like a circular stairway

Rosalind Franklin had also concluded that the sugar phosphate backbone was on the exterior of the molecule. Watson and Crick From this accumulated information, James Watson and Francis Crick determined the structure of DNA in 1953 in a basement laboratory at the Cavendish College of Cambridge University, after visiting Wilkins' laboratory and seeing Franklin's image and reading some of the data. They published their work in Nature. They surmised (and confirmed): · DNA was probably a double strand (because of the 2 nm diameter). This was interesting because Linus Pauling, who was also working on the structure of DNA, had just written a proposal for DNA having a triple stranded structure. · To maintain the uniform 2 nm diameter, a double-ring base probably would pair with a single-ring base along the length of the molecule. This took some experimenting with possible DNA models they constructed. · This base-pairing, however, was consistent with Chargaff's findings that the amounts of adenine and thymine were the same, and the amounts of guanine and cytosine were the same in species, and strongly suggested that adenine would base pair with thymine and guanine with cytosine. · It was also consistent with knowledge that: Adenine and thymine each had two hydrogen bonding sites. Guanine and cytosine each had three hydrogen bonding sites.

DNA Structure and Replication - 10 · Two strands of nucleotides, with their bases hydrogen-bonded to each other would form a ladder only if the sugar phosphate backbones ran in opposite directions to each other, or anti-parallel to each other, and , twisted to form a double helix that turns to the right. (This is where that 5' and 3' bonding mentioned earlier gets important).

·

Moreover, the base pairs lie flat and stack at 0.34 nm apart because of the hydrophobic interactions. The coiling also results in alternating major and minor grooves in the DNA structure, something that is important for gene transcription and regulation. The outer edges of the nitrogen bases get exposed in the major and minor grooves.

DNA Structure and Replication - 11 The constancy of the c omplementary base pairing is critical to the structure of DNA, as noted by Watson and Crick in their Nature paper when they wrote that the base pairing suggested a way in which the DNA molecule could be copied. In addition, DNA of different species and of different genes shows variation in the sequence of base pairs in the DNA chain, explaining genetic variation. Once the structure of DNA was known, active research could take place on how DNA can account for three functions of our genetic molecules: 1. Storing Genetic Information DNA stores genetic information in its base sequence. Variation in the base sequences of DNA explains genetic variation. 2. Susceptibility to Mutation Changes in the linear base sequence of DNA are mutations. (Mutation is now defined as a change in the DNA. 3. Replication of Genetic Material in Cell Division The complement base pairing of DNA provides a mechanism for DNA replication A fourth function of our genetic molecule, gene expression, was less obvious from the structure of DNA, but was soon established that the linear sequence of nucleotides in the DNA molecule served as a code that specified the sequence of amino acids for protein synthesis. (Gene expression is the subject of our next section.) DNA Replication · DNA is a double stranded molecule. The two chains (or strands) are attached by hydrogen bonds between the complementary nitrogen bases. · The two strands are anti-parallel to each other (run in opposite directions). That is, the 3' carbon of the sugar (the free sugar end) starts one strand and the 5' carbon sugar end (the free phosphate end) starts the other. This is essential for the complementary base pairs to hydrogen bond correctly. · Adenine must bond to thymine, and guanine must bond to cytosine.

Because of the complementary base pairing, if one side of the double stranded molecule is known, we automatically know what the other half is. This model for DNA replication is known as the s emi-conservative model for DNA replication. It was first proposed by Watson and Crick, but was not proven for several years until Stahl and Meselson devised a method using heavy nitrogen (15N). There were three probable ways in which DNA could replicate 1. Conservative: Each replication results in maintaining one original copy and one new copy 2. Dispersive: Each replication results in the original DNA interspersed into the two new copies 3. Semiconservative: Each replication results in one strand of the old and one strand of the new in the two new copies

DNA Structure and Replication - 12

Semiconservative Replication

Conservative Replication

Dispersive Replication

After two rounds of replication, it was clear that DNA replication was semiconservative. The process of DNA Replication (or Duplication) The basic steps of DNA replication are: · The two DNA strands of the parental chromosome must unwind and separate. · Each strand of the parent chromosome then serves as a template for the synthesis of a new strand. DNA is always synthesized in the 5' 3" direction from a 3' 5" template. New nucleotides can only be added to the available -OH bonding site of 3' sugar of the template strand. · New nucleotides form complementary base pairs with their template strand with phosphodiester covalent bonds so that the new polymers formed have a complementary base sequence to the template strands. · The newly synthesized double helix of each parent-daughter combination rewinds to form the DNA chains of a replicated chromosome. Each new DNA molecule is composed of one-half of the parent chromosome and one-half newly synthesized DNA.

DNA Structure and Replication - 13 A few details about DNA Replication: Most of what is presented about DNA replication is the result of extensive study of the bacterium, E. coli. Since bacteria have a single circular chromosome, some details differ; bacteria have a single origin and one replication fork (see later). Eukaryote chromosomes have a number of origins and replication forks on each chromosome. There are also different DNA polymerase molecules involved in eukaryotes than in prokaryotes, but we shall use DNA polymerase "generically" in our discussion. Linear chromosomes also have an issue with complete replication of each strand of the DNA, as we shall discuss, that is not an issue with the circular DNA molecule of bacteria. DNA replication occurs with a protein complex of several enzymes called the replication complex, into which the template strand of DNA fits. Prior to cell division, the double-strand of DNA is unwound at places called o rigins or replication (ori) that have specific DNA sequences, forming r eplication bubbles in the DNA molecule. Initiator proteins bind to the origin to start the process. The enzyme, DNA helicase, facilitates the unwinding of the DNA molecule by binding to and moving along one strand of the DNA while "pushing aside" the second DNA strand. This is an endergonic process requiring ATP. A set of single-strand b inding proteins line up on the strands after DNA helicase unwinds DNA to keep the hydrophobic exposed bases of the strands separated. f Unwinding and replication occurs in both directions as the DNA molecule "f orks" from the origins (where the unwinding starts). These regions are called replication forks and new DNA is replicated behind the fork as the DNA molecule continues to open in both directions.

The complex of proteins involved in DNA replication work collaboratively to complete DNA replication on both strands. Although most illustrations represent each enzyme independently, in E.coli, the replication complex of enzymes is called the replisome, or replication "organelle". It consists of the DNA polymerase dimer and the p rimosome, comprised of primase, helicase and the accessory proteins. In eukaryotes, multiple DNA replication complexes are found with the nuclear membrane matrix.

DNA Structure and Replication - 14 Replication occurs rapidly, adding as many as 500 - 1000 nucleotides per second in prokaryotes. Since DNA is a helix, the uncoiling (rotation caused by helicase) occurs at 100 revolutions per second (DNA coils every 10 nucleotides), causing the DNA to twist and kink, something that is called t orque. (If you have ever tried to prokaryotes) solve the twisting (torque) problem for DNA by cutting the DNA and allowing it to pass through the other strand to minimize supercoiling. The strand is then reconnected after it has been untwisted. Some topoisomerases cut one strand and some cut both strands.

coil a hose around your arm, you've noticed the kinks and twists that happen to the portion of the hose not yet coiled.) Enzymes called t opoisomerases (or DNA gyrase in

In eukaryotic organisms, there are many, many replication bubbles involved in the replication of DNA on each chromosome. However, DNA polymerase is slower in eukaryotes, adding around 50 nucleotides per second in human DNA replication. Each replication bubble forms replication forks at its "origin". As DNA replication progresses, replication bubbles join other units when they meet at adjacent replication forks. As stated, bacteria usually have one origin.

Replication Bubble with Forks

Origins and Replication Forks in Eukaryotes

DNA Structure and Replication - 15

Origin and Replication Fork in Prokaryotes

Each of the two strands of the DNA molecule in the fork serves as a template for the attachment of its complement nucleotides (A-T, C-G, G-C and T-A). This takes place on both sides of the replication forks simultaneously, but in opposite directions. (This is seldom illustrated. Most diagrams show only one section of a

replication fork to try and simplify things a bit.)

There are multiple variations of the enzyme DNA polymerase in both prokaryotes and eukaryotes. The variant responsible for replication is DNA polymerase III in prokaryotes and DNA polymerase or in eukaryotes. DNA polymerase promotes the synthesis of the new DNA strands by recognizing the appropriate complementary base needed and bonding the appropriate phosphorylated nucleotide to the growing DNA molecule. DNA polymerase III is a dimer complex of 10 proteins, and is used to simultaneously replicate both sides of the DNA molecule.

DNA Polymerase III

b2 subunits "sliding clamp"

DNA moving through DNA Polymerase

However, DNA polymerase has two limitations: · First, DNA polymerase must bond to an available 3' sugar. It cannot add nucleotides until there is a double-stranded template starter. The unwound DNA template molecules are single-stranded molecules. DNA polymerase can read the single chain template, but can't bond the nucleotides for the new strand for replication with just a single-stranded template, so DNA polymerases cannot initiate replication. · Second, DNA polymerase only works in one direction and the double-stranded DNA runs in two directions at each replication fork.

DNA Structure and Replication - 16 DNA Polymerase and the Single Chain Template Since DNA polymerase needs to add nucleotides to a pre-existing double chain, it cannot get started by itself. Replication is initiated with a R NA p rimer of about 10 nucleotides, activated by an enzyme called p rimase. Primase catalyzes a short RNA molecule primer that is used to start the synthesis of DNA.

DNA Polymerase and the Anti-parallel Template Strands Once the DNA strand has been "primed" by primase adding the RNA primer, DNA polymerase can go to work adding nucleotides to the 3' open end of the newly forming molecule. The energy for this process is provided by the hydrolysis of the triphosphate nucleotides. Two of the phosphates are removed to provide energy to bond the nucleotides onto the growing DNA polymer.

DNA Structure and Replication - 17 DNA' polymerase's second limitation is that DNA polymerase can only attach a new nucleotide to the exposed -OH group on the 3' end of an existing nucleotide molecule. DNA is always synthesized in the 5' 3" direction from the 3' 5" template. (It helps to remember that the 5' end is the free phosphate (PO43 ) end, and the 3' end is the ­OH end of the sugar.) When DNA helicase initiates the unwinding of the DNA molecule, the two opening strands are in opposite directions at each replication fork: one strand is in a 3' 5' direction and the opposite strand is 5' 3'. This is fine for DNA polymerase to add nucleotides reading the 3' 5' DNA template strand of the original molecule, but not for the second strand, which is running in the 5' 3' opposite direction, hence the complication. The upper 3' end of the original DNA molecule at a replication fork is called the leading (or continuous) strand of the template because DNA polymerase can readily start adding nucleotides to the initial primer at the 3' sugar's ­OH group. The opposite (5' 3") strand, whose nucleotides are oriented in the opposite direction, is called the lagging (or segmented or discontinuous) strand because it does not have an exposed 3' bonding site. It has the 5' (PO4-3) group exposed. The lagging strand has to be uncoiled a greater distance along the DNA template before replication can begin.

Once positioned, a DNA polymerase molecule positions the parent strand of the DNA molecule of the leading strand on both replication forks of a replication bubble into a groove and pulls the DNA through the groove as it directs the synthesis of a new DNA molecule. (Only one fork of the replication bubble is shown in the illustration above.) The DNA synthesis on the leading strand is c ontinuous following helicase. DNA polymerase III add nucleotides to the newly available 3' end.

DNA Structure and Replication - 18 The lagging template strand of DNA is also unzipped in the 5' to 3" direction. The required reading and synthesis direction of the lagging strand is opposite the direction of the unzipping DNA helicase enzyme, and as helicase moves along the double helix the lagging strand's 3' sugar end gets distanced from the fork. So the lagging strand grows differently from the leading strand. New DNA polymerase enzyme molecules attach and work on small portions, called O kazaki fragments, of the l agging strand DNA in a d iscontinuous synthesis. In addition the DNA polymerase functions as a dimer that catalyzes adding nucleotides to both sides of the DNA molecule so the lagging strand has to fold back on itself to "face" the correct 5' to 3' direction to fit into the DNA polymerase sliding rings or clamp. The DNA polymerase clamp stabilizes both DNA polymerase and the template strands so they can remain tightly associated during catalysis.

The Replication Fork

Each new Okazaki fragment (about 100 ­ 200 nucleotides long in eukaryotes, and 1000 or so in prokaryotes) of the lagging strand must be initiated with a primer. DNA polymerase III then attaches to and adds nucleotides to the growing Okazaki segment. Once an Okazaki fragment is synthesized, the RNA primer nucleotides are removed from the fragment and DNA polymerase I replaces them with DNA nucleotides. The enzyme, DNA ligase, joins the Okazaki fragments of the lagging strand together.

DNA Structure and Replication - 19

The loop in the lagging strand provides for DNA replication to take place in the 5' 3' direction on both strands while the replisome complex moves along the fork.

When the DNA polymerase III on the lagging strand reaches the previous Okazaki fragment the template strand and clamp of the DNA polymerase complex are released. DNA polymerase I can now remove the primer from that Okazaki fragment and DNA ligase joins the Okazaki fragments.

The clamp is re-attached to the lagging strand and transfers the strand to DNA polymerase III, creating a new loop in the lagging strand and DNA polymerase adds nucleotides to the next Okazaki fragment until the replication fork reaches the next bubble.

DNA Structure and Replication - 20

Lagging Strand Synthesis Summary

After the DNA is replicated along its entire length, two DNA molecules have been formed, each with one strand of the original and one with new nucleotides. Both DNA molecules are identical to each other and to the original.

DNA Structure and Replication - 21

DNA Replication Summary

DNA Structure and Replication - 22 Primers, DNA polymerase and Telomeres Before we leave our discussion of DNA replication we need to return to one more "problem" related to DNA polymerase's limitations. Recall that DNA is always synthesized in the 5' 3" direction from the 3' 5" template. On the lagging strand, as Okazaki fragments are completed, the primer RNA nucleotides from each Okazaki fragment can be removed and DNA polymerase has the doublestranded 3' end of the previous Okazaki fragment to which it can add the replacement DNA nucleotides until we reach the 5' end of the strand. When the primer of the 5' ends of the new DNA strands are removed, DNA polymerase cannot "finish" those 5' ends. DNA polymerase cannot add nucleotides to a 5' end of a DNA molecule. After each replication, the DNA molecule has a bit of singlestranded DNA at the end. The result is that the single-stranded bit gets removed and the chromosome gets a bit shorter with each replication.

Prokaryotes with their circular DNA molecule can complete the ends of their chromosome, but each of the chromosomes of a Eukaryotic organism shortens with each division. A second issue with eukaryote chromosomes is simply that they do have ends. As mentioned in our section on cell division and cell cycle controls, the metaphase checkpoint involves checking the integrity of the DNA, including finding broken sections. Ends could be mistaken for breaks in the chromosome. Both shortening of chromosomes and mistaking chromosome ends as sections needing repair, hence stopping cell division at the metaphase checkpoint, would have potential impacts on our genetic information if cells did not have a mechanism to resolve this "problem". This mechanism is the t elomere.

DNA Structure and Replication - 23 The ends of eukaryotic chromosomes have special non-gene coding repetitive nucleotide sequences, such as TTAGGG. These regions are called t elomeres. The telomere region gets shorter with each DNA replication. The number of telomere repeats varies from about 100 to 2500, and most cells can divide about 30 times before they run out of telomeres and can no longer replicate DNA without losing codable genes.

Mouse Telomeres (Orange Tips)

To prevent misinterpretation of chromosome ends for damaged DNA the telomere repeats bind special proteins that protect the DNA from cell molecules that detect damaged DNA, hence inhibiting the cell pathways that would destroy damaged cells. Telomeres also "cap" the DNA molecule promoting stability and maintaining homeostasis. In addition, a chromosome lacking telomeres cannot replicate indicating that telomeres also have a role in duplication of the DNA beyond being "disposable" DNA. Some tissues have t elomerases, special RNA nucleoproteins that contain an RNA sequence that catalyzes telomeres using the RNA template at the 3' ends of chromosomes coding only telomere nucleotide sequences. The 5' end can then be expanded using DNA polymerase and DNA ligase.

DNA Structure and Replication - 24 In humans, telomerases are normally found only in tissues that divide frequently, such as cells in bone marrow in humans and those that produce gametes, ensuring that zygotes have a full complement of telomeres. However, about 90% of human cancers have active telomerases. Shortening telomeres in tissues has been proposed to be a factor in aging and lifespan. Cells from large benign tumors often have shorter telomeres and most cells divide no more than 20 ­ 30 times. There is some evidence that stress affects telomeres, too. Telomerases seem to be abundant in some cancer cells, so that rapid reproduction does not result in shortening of telomeres and cell death from lack of needed genes. Research on telomerases and telomeres is of much interest to biologists and it is evident that telomerase and telomeres have more of a role in any cell division than we have do far discovered. Proofreading and Repairing DNA During the DNA replication process, DNA polymerase enzymes also p roofread the DNA and initiate repair of replication errors. If there is a base-pairing error, it deletes the section with the error, and replaces with the correct nucleotides. Occasionally, there is a mismatched DNA pair that is not detected or occurs after replication has finished. Special enzymes search out and repair such errors in a process called m ismatch repair. DNA repair is not restricted to DNA replication error. DNA is easily damaged by chemical and physical agents to which our cells are exposed (a subject discussed in greater detail our mutations section). For example, exposure to UV light can cause DNA damage, which is why those who spend more time in the sun without protection are at greater risk for skin cancer. Cells continually monitor the integrity of their DNA and initiate repair. For some repair, such as the UV thymine damage, there are specialized repair options including one that is catalyzed by an enzyme, p hotolase, which absorbs visible light energy and uses that energy to repair the thymine damage. A more generic excision repair uses a technique of excision and replacement. DNA repairing enzymes called n ucleases recognize the damage, cut out (excise) the damaged or mutated DNA and DNA polymerase and ligase fill in the gap with correct nucleotides, assuming there is an undamaged DNA strand to serve as the template.

DNA Structure and Replication - 25

Even so, some mistakes do not get repaired resulting in mutations. About 1 in 1 billion DNA base pair errors are not caught. Deterioration of DNA replication accuracy is likely a contributing factor in aging and in cancer, particularly when the mutations occur in the genes that code for the error-detecting proteins. DNA Amplification ­ the Polymerase Chain Reaction (PCR) Knowledge of how DNA replicates and how damaged DNA may be repaired led to one of the techniques most commonly used today for studying genes and in genetic technology and DNA research work: Making multiple copies of short DNA sequences using the p olymerase chain reaction (PCR). Researchers most often need multiple copies of DNA (or gene of interest) for their work. The Polymerase Chain Reaction is often the way to obtain adequate quantities of a segment of DNA when the DNA sample is very tiny or may be contaminated. PCR is also valuable when trying to do a detailed analysis of a DNA molecule. This is often the case when one is using DNA materials for potential evidence in forensic investigations, to detect diseases or when one is trying to reconstruct DNA from preserved and fossil materials. We will revisit the use of PCR in

our unit on genetic technologies.

DNA Structure and Replication - 26 Kary Mullis shared the Nobel Prize in chemistry in 1993 for his 1980's development of PCR. PCR uses alternating heating and cooling cycles to first denature and separate a DNA molecule. The single-stranded DNA is cooled and joined with artificially synthesized primers (annealing) that have complementary DNA to the sample. Since PCR requires heating the samples above temperatures that denature proteins, Mullis used a DNA polymerase enzyme isolated from a thermophilic bacterium, Thermus aqauticus (Taq) which is used in the heated cycle to synthesize new double-stranded DNA molecules. In order to do PCR the nucleotide sequence at the 3' ends of the sample must be known in order to synthesize the appropriate primers. The metabolism of the thermophilic bacteria, which was studied by Thomas Brock, is heat--resistant so its enzymes do not denature at high temperatures. The process occurs in a controlled near-neutral pH environment. PCR today is automated so that billions of copies of the gene are made in just a few hours. This is also a method of DNA amplification.

Information

Microsoft Word - DNAStructureReplica-211.doc

26 pages

Report File (DMCA)

Our content is added by our users. We aim to remove reported files within 1 working day. Please use this link to notify us:

Report this file as copyright or inappropriate

508294


You might also be interested in

BETA
Pages vol 17, no 3
Neuroscience Retreat
BIOLOGY