Chapter 6: DNA Replication and Repair

DNA replication

A cell's fundamental process of accurately copying the vast quantity of genetic information carried in its DNA. This must occur before the cell can divide to produce two genetically identical daughter cells.

Telomeres form structures that mark the true ends of a chromosome. These structures allow the cell to distinguish unambiguously between the natural ends of chromosomes and the double stranded DNA breaks that sometimes occur accidentally in the middle of chromosomes. In addition, the repetitive DNA sequences found within telomeres attract other telomere binding proteins that not only physically protect chromosome ends, but help maintain telomere length.

Besides solving the end problem, what else do telomeres do?

repair polymerase that replaces RNA primase with DNA

DNA polymerase I

DNA polymerase that carries out the bulk of replication at the replication fork

DNA polymerase III

DNA polymerase

Enzyme that catalyzes the addition of nucleotides to the 3' end of a growing DNA strand one of the original, parental DNA strands as a template. This enzyme stays associated with the DNA and moves along the template strand stepwise for many cycles of the polymerization reaction. The polymerization reaction involves the formation of a phosphodiester bond between the 3' end of the growing DNA chain and the 5' phosphate group of the incoming nucleotide, which enters as a deoxyribonucleoside triphosphate.

An enzyme called DNA helicase sits at the very front of the replication machine where it uses energy of ATP hydrolysis to propel itself forward, prying apart the double helix as it speeds along the DNA. Single strand DNA binding proteins then latch onto the single stranded DNA exposed by the helicase, preventing the strands from reforming base pairs and keeping them in an elongated form so that they can serve as efficient templates.

How is the DNA double helix continuously pried apart so that the incoming nucleoside triphosphates can form base pairs with each template strand?

For the leading strand, an RNA primer is needed only to start replication at the replication origin. At that point, the DNA polymerase takes over, extending this primer with DNA synthesized in the 5' to 3' direction. On the lagging strand, where DNA synthesis is discontinuous, new primers are continuously needed to keep polymerization going. The movement of the replication fork continuously exposes unpaired bases on the lagging strand, and new RNA primers must be laid down at intervals along the newly exposed single-stranded stretch. DNA polymerase then adds a deoxyribonucleotide to the 3' end of each new primer to produce another Okazaki fragment, and it will continue to elongate this fragment until it runs into the previously synthesized RNA primer.

How many RNA primers are needed?

bacteria: 1 humans: about 10,000 (has much larger genome so greatly shortens time needed to copy)

How many replication origins do bacterial genomes have vs the human genome?

5' to 3'

In which direction is DNA replicated?

thymine dimer

The covalent linkage between two adjacent pyrimidine bases. Can be caused by the ultraviolet radiation in sunlight.

It is provided by the incoming deoxyribonucleoside triphosphate itself: hydrolysis of one of its high-energy phosphate bonds fuels the reaction that links the nucleotide monomer to the chain, releasing pyrophosphate. Pyrophosphate is further hydrolyzed to inorganic phosphate which makes the polymerization reaction effectively irreversible.

Where does the energy for the polymerization reaction of a nucleotide come from?

It is an unintended consequence of the vast number of chemical reactions that occur inside cells. Sometimes it also occurs as a result of mistakes in replication.

Where does the majority of DNA damage come from?

A-T base pairs have fewer hydrogen bonds (2) than G-C base pairs (3). Therefore, DNA rich in A-T base pairs is especially easier to open.

Why are replication origins typically rich in A-T base pairs?

Each strand of a DNA double helix contains a sequence of nucleotides that is exactly complementary to the nucleotide sequence of its partner strand. In other words, if we designate two DNA strands as S and S', strand S can serve as a template for creating a new strand S', while strand S' can serve as a template for making a new strand S. Thus, each separated strand then serves as a template for the production of a new complementary partner strand that is identical to its former partner.

Why can each DNA strand serve as a template?

That's because if a "backward polymerase" were to remove an incorrectly paired nucleotide from the 5' end, it would create a chemical dead end- a strand that could no longer be elongated. (DNA polymerase can only proofread if it can remove a nucleotide from the 3' end). The cumbersome backstitching mechanism in the lagging strand can be seen as a necessary consequence of maintaining this proofreading activity.

Why does DNA polymerase needs to synthesize in the 5' to 3' direction exclusively to be able to proofread?

Because both of the new strand at a replication fork are synthesized in the 5' to 3' direction, the lagging strand of DNA must be made initially as a series of short DNA strands, which are later joined together. To replicate the lagging strand, DNA polymerase uses a backstitching mechanism: it synthesizes short pieces of DNA (called Okazaki fragments) in the 5' to 3' direction and then moves back along the template strand (towards the fork) before synthesizing the next fragment

Why does the lagging strand need to be replicated discontinuously?

Because each parental strand serves as the template for one strand, each of the daughter DNA helices ends up with one of the original strands plus one strand that is completely new

Why is DNA replication said to be semiconservative?

1. Some of the stress is relived by supercoiling. 2. Enzymes called DNA topoisomerases relieve this tension by producing a transient, single-strand nick in the DNA backbone. Stress in relieved by free rotation of the DNA around the phosphodiester bond opposite the single strand break. The enzyme then reseals the nick before falling off the DNA.

Localized unwinding of the DNA double helix by helicase presents a problem. As the helicase moves forward, prying open the double helix, the DNA ahead of the fork gets wound more tightly. This excess twisting in the front of the replication fork creates tension in the DNA that makes unwinding of the double helix increasingly difficult and ultimately keeps replication machinery from moving forward. How is this problem dealt with?

all cells in the body of the multicellular organism that develop from it, including the gametes responsible for the production of the next generation

Mutations that occur in germline cells will be passed on to

The process of DNA synthesis is begun by initiator proteins that bind to specific DNA sequences called replication origins. Here, the initiator proteins pry the two DNA strands apart, breaking the hydrogen bonds between the bases. Although the H bonds collectively make the DNA molecule very stable, individually they are weak. Separating a short length of DNA a few base pairs at a time therefore does not require a large input of energy, and the initiator proteins can readily unzip short regions of the double helix at normal temps.

Since DNA is a very stable molecule, (H bonds), only temperatures approaching boiling water have enough thermal energy to separate the two strands. How are the strands separated to expose nucleotide bases to start the process of replication in the temp of the cell?

sickle-cell anemia

A disease caused as a result of a mutation of a single nucleotide in the human hemoglobin gene. The hemoglobin protein is used to transport oxygen in the blood. Mutations in the hemoglobin gene can produce a protein that is less soluble than normal hemoglobin and forms fibrous intracellular precipitates. Because these cells are more fragile and frequently tear as they travel through the bloodstream, these people have fewer red blood cells than usual.

homologous recombination

A flawless repair mechanism of a double strand break with no loss of genetic information. If a double strand break occurs in a double helix shortly after that stretch of DNA has been replicated, the undamaged copy can serve as a template to guide the repair of both broken strands of DNA. Because the two DNA molecules are homologous, they are identical or nearly identical nucleotide sequences outside the broken region. This often occurs shortly after a cell's DNA has been replicated before cell division, when the duplicated helices are still physically closer to each other.

Xeroderma pigmentosum

A genetic disease in which humans cannot mend the damage done by ultraviolet radiation because they have inherited a defective gene for one of the proteins involved with this repair process. Such individuals develop severe skin legions including skin cancer because the of DNA damage that accumulates in cells exposed to sunlight and the consequent mutations that arise in these cells.

homologous end joining

A repair mechanism used to deal with a double strand break. This is carried out by a specialized group of enzymes that "clean" the broken ends and rejoin them by DNA ligation. This mechanism seals the break quickly, but in cleaning the break to prepare it for ligation, nucleotides are often lost at the site of repair. If this imperfect repair disrupts the activity of a gene, the cell could suffer serious consequences.

depurination

A spontaneous reaction in which a purine base is removed from a nucleotide , giving rise to legions that resemble missing teeth.

deamination

A spontaneous reaction in which an amino group from a cytosine in DNA is lost to form uracil

DNA polymerase has two special qualities that greatly increase the accuracy of DNA replication. 1. First, the enzyme carefully monitors the base-pairing between each incoming nucleoside triphosphate and the template strand. Only when the match is correct does DNA polymerase undergo a small structural arrangement that allows it to catalyze the nucleotide addition reaction. 2. Second, when DNA polymerase does make a rare mistake and adds the wrong nucleotide, it can correct the error through proofreading.

DNA polymerase is so accurate that it only makes about one error in every 10^7 nucleotide pairs it copies. Why is it so accurate?

leading strand

DNA strand that is made continuously

lagging strand

DNA strand that is made discontinuously

In their report, they proposed that DNA's complementary bases paired with one another along the center of the double helix, holding the two strands together. They also suggested that the two strands of the double helix unwind and each strand serves as a template for the synthesis of a complementary daughter strand. In their model, dubbed semiconservative replication, each new DNA molecule consists of one strand derived from the original parent molecule and one newly synthesized strand.

Describe Watson and Crick's models of DNA

In this theory, the parent helix would somehow remain entirely intact after copying, and the daughter molecule would contain two entirely new DNA strands.

Describe the theory of conservative replication

In this theory, DNA replication proceeds through a series of breaks and reunions in which the DNA backbone is broken and the strands are copied in short segments (perhaps only 10 nucleotides at a time) before being rejoined. The resulting copies would be patchwork collections of old and new DNA, each strand containing a mixture of both. No unwinding was necessary.

Describe the theory of dispersive replication

To do this, they took a flask of bacteria that had been grown in heavy nitrogen and transferred the bacteria into a medium containing the light isotope, and allowed division for one hour. This produced a band that is positioned between the heavy and light DNA. These results rule out the conservative model of replication. If replication was conservative, the parental DNA would remain completely heavy, while the daughter DNA would be completely light. This did not distinguish between the semiconservative and dispersive models however.

How did Meselson and Stahl rule out the conservative model?

To do this, they turned up the heat of the experiment. When DNA is subjected to high temperature, the hydrogen bonds holding the helix break and the strands come apart, leaving a collection of single stranded DNAs. When the researchers heated the hybrid molecules before centrifuging, they discovered that one strand of the DNA was heavy, whereas the other was light. This observation rued out the dispersive model; if this model were correct, the resulting strands, each containing a mottled assembly of heavy and light DNA, would have all banded together at intermediate density.

How did Meselson and Stahl rule out the dispersive model and prove the semiconservative model?

At a speed as high as 1000 nucleotides per second

How fast can DNA replicate its DNA?

They started by growing two batches of E. Coli, one in a medium containing a heavy isotope of nitrogen 15N, and the other in a medium containing the normal lighter 14N. The nitrogen in the nutrient medium gets incorporated into the nucleotide bases, and from there, makes its way into the DNA of the organism. After growing bacterial cultures for many generations in either the 15N or 14N containing medium, the researchers had the two flasks of bacteria, one with heavy DNA and one with light DNA. The cells are then broken open, and the DNA is loaded into an ultracentrifuge tube containing cesium chloride salt solution. These tubes are centrifuged at high speed for two days to allow the cesium chloride to form a gradient with low density at the top of the tube and high density at the bottom. As the gradient forms, the DNA will migrate to the region with its density matches that of the salt surrounding it. the heavy and light DNA molecules thus collect in different positions in the tube.

How did Meselson and Stahl step up their experiment?

Their chromosomes are circular

How do bacteria avoid the end replication problem?

Eukaryotes add long, repetitive nucleotide sequences to ends of every chromosome. These sequences are incorporated into structures called telomeres, which attract an enzyme called telomerase to the chromosome ends. Telomerase carries its own RNA template, which it uses to add multiple copies of the same repetitive DNA sequence to the lagging strand template. In many dividing cells, telomeres are continuously replenished and the resulting extended templates can then be copied by conventional DNA replication, ensuring that no peripheral chromosome sequences are lost.

How do eukaryotes avoid the end replication problem?

Proofreading takes place at the same time as DNA synthesis. Before the enzyme adds the next nucleotide to a growing DNA strand, it checks whether the previously added nucleotide is correctly base-paired to the template strand. If so, the enzyme adds the next nucleotide. If not, the polymerase clips off the mispaired nucleotide and tries again. Polymerization and proofreading are tightly coordinated and the two reactions are carried out by different catalytic domains in the same polymerase molecule.

How does DNA polymerase proofread?

An inherited predisposition to certain cancers is caused by mutations in genes that encode mismatch repair proteins. Human cells have two copies of these genes, and individuals who inherit one damaged mismatch repair gene are unaffected until the undamaged copy of the same gene is randomly mutated in a somatic cell. This mutant cell and all of its progeny are then deficient in mismatch repair. They therefore accumulate more rapidly than do normal cells. Because cancers arise from cells that have accumulated multiple mutations, a cell deficient in mismatch repair has a greatly enhanced chance of becoming cancerous.

How does mismatch repair play an important role in preventing cancer?

Cells that divide at a rapid rate throughout the life of the organism keep their telomerase fully active. Many other cell types however, gradually turn down their telomerase activity. After many rounds of cell division, the telomeres in these descendant cells will shrink until they essentially disappear. At this point, these cells will cease from dividing.

How does telomere length change with age?

Whenever the replication machinery makes a copying mistake, it leaves behind a mismatch nucleotide. If left uncorrected, the mismatch will result in a permanent mutation in the next round of DNA replication. In most cases however, a complex of mismatch repair proteins will detect the DNA mismatch, remove the a portion of the DNA strand containing the error, and then synthesize the missing DNA.

How does the DNA mismatch pair repair system work?

nonhomologous end joining or homologous recombination

How does the cell deal with a double strand break?

The double helix provides two copies of the genetic information in each strand if DNA. Thus, if the sequence in one strand is accidentally damaged, information is not lost irretrievably because a backup version of the altered strand remains in the complementary sequence of nucleotides in the undamaged strand. Most DNA damage creates structures that are never encountered in an undamaged DNA strand; thus, the good strand is easily distinguished from the bad.

How does the double helix of DNA help repair mechanisms?

The system recognizes and only removes the newly made DNA. Different cells use different strategies to mark which strand is old/new.

How does the mismatch repair system recognize which of the DNA strands contains the error?

An enzyme called primase makes a short length of RNA using DNA as a template. This short length of RNA (about 10 nucleotides long) is base paired to the template strand and provides a base paired 3' end as a starting point for DNA polymerase. Unlike DNA polymerase, primase can start a new polynucleotide chain by joining together two nucleoside triphosphates without the need for a base paired 3' end as a starting point. This RNA fragment thus serves as a primer for DNA synthesis. Primase is an example of an RNA polymerase, an enzyme that synthesizes RNA using DNA as a template. RNA can complementary base pair with DNA because uracil can form a base pair with adenine.

How does the newly synthesized DNA strand get started?

1. nuclease- degrades the RNA primer 2. repair polymerase- DNA polymerase that replaces the RNA primers with DNA (using the end of the adjacent Okazaki fragment as its primer) 3. DNA ligase- joins the 5' phosphate end of one DNA fragment to the adjacent 3- hydroxyl end of the next by using a molecule of ATP

To produce a continuous DNA strand from the many separate pieces on the lagging strand, enzymes must remove the RNA primer, replace it with DNA, and join the remaining DNA fragments together. What three enzymes are needed?

Two Y-shaped replication forks form at each replication origin. At each fork, a replication machine moves along the DNA, opening up the two strands and using each strand as a template to make a new daughter strand. The two forks move away from the origin in opposite directions, unzipping the DNA double helix and copying DNA as they go. DNA replication (in both bacterial and eukaryotic chromosomes) is bidirectional.

What happens at the replication fork?

DNA damage leads to either the substitution of one nucleotide pair for another as a result of incorrect base-pairing during replication or to deletion of one or more nucleotide pairs in the daughter DNA strand after DNA replication. Some types of DNA damage (thymine dimers) can stall the DNA replication machinery at the site of the damage.

What happens if DNA is left unrepaired?

As the replication fork approaches the end of the chromosome, the leading strand can be replicated all the way down to the chromosome tip. However, the lagging strand cannot. When the final RNA primer on the lagging strand is removed, there is no enzyme that can replace it with DNA. Without a strategy to deal with this problem, the lagging strand would become shorter and shorter with each round of DNA replication. After repeated cell divisions, the chromosomes themselves would shrink.

What is the "end problem" of DNA replication?

1. The damaged DNA is recognized and removed by one of a variety of mechanisms. These involve nucleases, which cleave the covalent bonds that join the damaged nucleotides to the rest of the DNA strand, leaving a small gap on one strand on the double helix. 2. A repair DNA polymerase binds to the 3'-hydroxyl end of the cut DNA strand. The enzyme then fills the gap by making a complementary copy of the information present in the undamaged strand. 3. When the repair DNA polymerase has filled in the gap, a break remains in the sugar phosphate backbone of the repaired strand. This nick in the helix is sealed by DNA ligase.

What is the basic pathway for repairing damage to DNA?

The clamp loader uses the energy of ATP hydrolysis to lock the sliding clamp onto DNA. This loading cycle needs to occur only once per replication cycle on the leading strand. On the lagging strand however, the clamp is removed and then reattached each time an Okazaki fragment is made.

What is the role of the clamp loader?

This protein keeps DNA polymerase firmly attached to the template while it is synthesizing new strands of DNA. Left on their own, most DNA polymerase molecules will synthesize only a short string of nucleotides before falling off the DNA template strand. The sliding clamp forms a ring around the newly formed DNA double helix, and by tightly gripping the DNA polymerase, allows the enzyme to move along the template strand without falling off.

What is the role of the sliding clamp?