Essential Cell Biology Chapter 6 - DNA Replication and Repair

double strand break

-Mishaps at the replication fork, radiation, and various chemical assaults can all fracture DNA - damage is especially difficult to repair

homologous recombination

1. a recombination-specific nuclease chews back the 5ʹ ends of the two broken strands at the break 2. one of the broken 3ʹ ends "invades" the unbroken homologous DNA duplex and searches for a complementary sequence through base- pairing 3. accurate match is made, the invading strand is elongated by a repair DNA polymerase, using the com- plementary undamaged strand as a template 4. After the repair polymerase has passed the point where the break occurred, the newly elongated strand rejoins its original partner, forming base pairs that hold the two strands of the broken double helix together 5. Repair is then completed by additional DNA synthesis at the 3ʹ ends of both strands of the broken double helix (Figure 6-31F), followed by DNA ligation (Figure 6-31G) 6. Resulting in two intact DNA helices, for which the genetic information from one was used as a template to repair the other.

helicase, ATP hydrolysis

A ____________ sits at the very front of the replication machine, where it uses the energy of _________ ___________ to propel itself forward, prying apart the double helix as it speeds along the DNA

Watson and Crick: Semiconservative model (even though those fuuc3rs stole it from Rosalind Franklin

each parent strand serves as a template for the synthesis of a new daughter strand. The first round of replication would produce two hybrid molecules, each containing one strand from the original parent and one newly synthesized strand. A subsequent round of replication would yield two hybrid molecules and two molecules that contain none of the original parent DNA

DNA is said to be semiconservative

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

sliding clamp

forms a ring around the newly formed DNA double helix and, by tightly gripping the polymerase, allows the enzyme to move along the template strand without falling off as it synthesizes new DNA

Nonhomologous end joining

involves hurriedly sticking the bro- ken ends back together, before the DNA fragments drift apart and get lost often results in lost nuceotides

sliding clamp

keeps DNA polymerase firmly attached to the template while it is syn- thesizing new strands of DNA

germ-line cells

mutation in this kind of cell will be passed on to all the cells in the body of the multicellular organism that develop from it, including the gametes responsible for the production of the next generation.

DNA topoisomerases

produces a transient, single-strand nick in the DNA backbone, which temporarily releases the built-up tension; the enzyme then reseals the nick before falling off the DNA

1. Nonhomologous end joining 2. Homologous recombination

what are the 2 strategies used to repair a double strand break?

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, the same enzyme that joins the Okazaki fragments during replication of the lagging DNA strand

what is the 3rd step of repairing DNA?

clamp loader

Assembly of the clamp around DNA requires the activity of another replication protein which hydrolyzes ATP each time it locks a sliding clamp around a newly formed DNA double helix

.the polymerase clips off the mispaired nucleotide and tries again

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 polymerase adds the next nucleotide; if not..........

the ligase enzyme uses a molecule of ATP to activate the 5ʹ phosphate of one fragment converting ATP to ADP (step 1) before forming a new bond with the 3ʹ hydroxyl of the other fragment converting ADP to AMP (step 2).

DNA ligase joins together Okazaki fragments on the lagging strand during DNA synthesis, How?

If the old strand were "repaired" using the new strand that contains a replication error as the template, then the error would become a permanent mutation inthe genome. The old information would be erased in the process. Therefore, if repair enzymes did not distinguish between the two strands, there would be only a 50% chance that any given replication error would be corrected

DNA mismatch repair enzymes preferentially repair bases on the newly synthesized DNA strand, using the old DNA strand as a template. If mismatches were simply repaired without regard for which strand served as template, would this reduce replication errors as effectively? Explain your answer.

replication forks

DNA molecules in the process of being replicated contain Y-shaped junctions called

No- all only ad to the 3' end

Does the cell have two types of DNA polymerase, one for each direction?

adding long, repetitive nucleotide sequences to the ends of every chromosome. These sequences, which are incorporated into structures called telomeres, attract an enzyme called telomerase to the chromosome ends.

Eukaryotes get around the issue of shortening chromosomes by.....

1. DNA helicases 2. single-strand DNA-binding proteins

For DNA replication to occur, the double helix must be continuously pried apart so that the incoming nucleoside triphosphates can form base pairs with each template strand. What 2 types of replication proteins carry out this task?

You cannot transform an individual from one species into another species simply by introducing random changes into the DNA. It is exceedingly unlikely that the 5000 mutations that would accumulate every day in the absence of the DNA repair enzyme would be in the very positions where human and chimpanzee DNA sequences are different. It is very likely that, at such a high mutation frequency, many essential genes would be inactivated, leading to cell death. Furthermore, your body is made up of about 1013 cells. For you to turn into an ape, not justone but many of these cells would need to be changed. And even then, many of these changes would have to occur during development to effect changes in your body plan (making your arms longer than your legs, for example).

Suppose a mutation affects an enzyme that is required to repair the damage to DNA caused by the loss of purine bases. The loss of a purine occurs about 5000 times in the DNA of each of your cells per day. As the average difference in DNA sequence between humans and chimpanzees is about 1%, how long will it take you to turn into an ape? Or would this transformation be unlikely to occur?

1. A nuclease degrades the RNA primer 2. a DNA polymerase called a repair polymerase replaces the RNA primers with DNA by using the end of the Okazaki fragment as its primer 3. and the enzyme DNA ligase joins the 5ʹ-phosphate end of one DNA fragment to the adjacent 3ʹ-hydroxyl end of the next

How is the the RNA primer removed, replaced with DNA, and join the remaining DNA fragments together

A single replication origin, humans have thousands

How many replication origins does a bacterial genome have?

telomeres

In addition to allowing replication of chromosome ends, _____________ form structures that mark the true ends of a chromosome

elongated, repair DNA polymerase

In homologous recombination Once an extensive, accurate match is made, the invading strand is ______________ by a __________ ____________ ____________, using the com- plementary undamaged strand as a template . After the repair polymerase has passed the point where the break occurred, the newly elongated strand rejoins its original partner, forming base pairs that hold the two strands of the broken double helix together

5ʹ ends

In homologous recombination to initiate the repair, a recombination-specific nuclease chews back the ____________ of the two broken strands at the break

inorganic phosphate (Pi)

In replication forks Pyrophosphate is further hydrolyzed to ______________ _______________, which makes the polymerization reaction effectively irreversible

RNA template

Telomerase carries its own _______ __________, which it uses to add multi- ple copies of the same repetitive DNA sequence to the lagging-strand template.

Cell Type, Age

Telomere Length Varies by _____ ______ and with ______

deoxyribonucleoside triphosphate, pyrophosphate

The energy for polymerization (for replication fork) is provided by the incoming ______________ ______________ itself: hydrolysis of one of its high-energy phosphate bonds fuels the reaction that links the nucleotide monomer to the chain, releasing _________________

DNA polymerase.

The movement of a replication fork is driven by the action of the replica- tion machine, at the heart of which is an enzyme called________ ____________

RNA Primers

The movement of the replication fork continually exposes unpaired bases on the lagging-strand template, and new _______ __________ must be laid down at intervals along the newly exposed, single-stranded stretch

phosphodiester bond

The polymerization reaction involves the formation of a _______ ________between the 3ʹ end of the growing DNA chain and the 5ʹ-phosphate group of the incoming nucleotide, which enters the reaction as a deoxy ribonucleoside triphosphate

Initiator Proteins, replication origins

The process of DNA synthesis is begun by ___________ that bind to specific DNA sequences called___________

A.False. Identical DNA polymerase molecules catalyze DNA synthesis on the leading and lagging strands of a bacterial replication fork. The replication fork is asymmetrical because the lagging strand is made in pieces while the leading strand is synthesized continuously. B. False. Okazaki fragments initially contain both RNA primers and DNA, but only the RNA primers are removed by RNA nucleases. C. True. With proofreading, DNA polymerase has an error rate of one mistake in 107 nucleotides polymerized; 99% of its errors are corrected by DNA mismatch repair enzymes, bringing the final error rate to one in 109. D. True. Mutations would accumulate rapidly, inactivating many genes. E.True. If a damaged nucleotide also occurred naturally in DNA, the repair enzyme would have no way of identifying the damage. It would therefore have only a 50% chance of fixing the right strand. F. True. Usually, multiple mutations of specific types need to accumulate in a somatic cell lineage to produce a cancer. A mutation in a gene that codes for a DNA repair enzyme can make a cell more liable to accumulate these mutations, thereby accelerating the onset of cancer.

Which of the following statements are correct? Explain your answers. A. A bacterial replication fork is asymmetrical because it contains two DNA polymerase molecules that are structurally distinct. B. Okazaki fragments are removed by a nuclease that degrades RNA. C. The error rate of DNA replication is reduced both by proofreading by DNA polymerase and by DNA mismatch repair. D. In the absence of DNA repair, genes become less stable. E. None of the aberrant bases formed by deamination occur naturally in DNA. F. Cancer can result from the accumulation of mutations in somatic cells.

RNA

________ acts as a starting point for polymerase, serving as a primer for DNA symthesis

DNA topoisomerases

_________ _____________ relieves tension as DNA is wound too tight

cancer

Nucleotide changes that occur in somatic cells can give rise to variant cells, some of which grow and divide in an uncontrolled fashion at the expense of the other cells in the organ- ism. In the extreme case, an unchecked cell proliferation

A group of proteins creating the replication Machine

Once an initiator protein binds to DNA at a replication origin and locally opens up the double helix, it attracts__________

different

Polymerization and proofreading are tightly coordinated, and the two reactions are carried out by __________catalytic domains in the same polymerase molecule

Same Time

Proofreading takes place at the _______ ________ as DNA synthesis

preventing the strands from re-forming base pairs and keeping them in an elongated form so that they can serve as efficient templates.

Single-strand DNA-binding proteins then latch onto the single-stranded DNA exposed by the helicase,......

the lagging strand cannot

although the leading strand can be replicated all the way to the chromosome......... which would cause the chromosome to shrink because when the final RNA primer on the lagging strand is removed there is no enzyme that can replace it

primase

and the enzyme that synthesizes the RNA primer is

asymmetrical

at each replication fork, one new DNA strand is being made on a template that runs in one direction (3ʹ to 5ʹ), whereas the other new strand is being made on a template that runs in the opposite direction making the replication fork.....

deamination

common reaction is the spontaneous loss of an amino group from a cytosine in DNA to produce the base uracil typically but can do this with other bases

depurination

does not break the DNA phosphodiester backbone but instead removes a purine base from a nucleotide, giving rise to lesions that resemble missing teeth can remove guanine(or adenine) from DNA

DNA ligase links the DNA fragments that are produced on the lagging strand. In the absence of ligase, the newly replicated DNA strands will remain as fragments, but no nucleotides will be missing.

A gene encoding one of the proteins involved in DNA replication has been inactivated by a mutation in a cell. In the absence of this protein, the cell attempts to replicate its DNA. What would happen during the DNA replication process if DNA ligase were missing?

no replication can take place at all. RNA primers will be laid down at the origin of replication.

A gene encoding one of the proteins involved in DNA replication has been inactivated by a mutation in a cell. In the absence of this protein, the cell attempts to replicate its DNA. What would happen during the DNA replication process if DNA polymerase were missing?

the DNA polymerase will stall because it cannot separate the strands of the template DNA ahead of it. Little or no new DNA will be synthesized.

A gene encoding one of the proteins involved in DNA replication has been inactivated by a mutation in a cell. In the absence of this protein, the cell attempts to replicate its DNA. What would happen during the DNA replication process if the DNA helicase were missing?

the RNA fragments will remain covalently attached to the newly replicated DNA fragments. No ligation will take place, because the DNA ligase will not link DNA to RNA. The lagging strand will therefore consist of fragments composed of both RNA and DNA.

A gene encoding one of the proteins involved in DNA replication has been inactivated by a mutation in a cell. In the absence of this protein, the cell attempts to replicate its DNA. What would happen during the DNA replication process if the Nuclease that removes RNA primers were missing?

In the absence of primase, RNA primers cannot be made on either the leading or the lagging strand. DNA replication therefore cannot begin.

A gene encoding one of the proteins involved in DNA replication has been inactivated by a mutation in a cell. In the absence of this protein, the cell attempts to replicate its DNA. What would happen during the DNA replication process if the primase were missing?

the DNA polymerase will frequently fall off the DNA template. In principle, it can rebind and continue, but the continual falling off and rebinding will be so time-consuming that the cell will be unable to divide.

A gene encoding one of the proteins involved in DNA replication has been inactivated by a mutation in a cell. In the absence of this protein, the cell attempts to replicate its DNA. What would happen during the DNA replication process if the sliding clam were missing?

polymerase, 3ʹ-hydroxyl

A repair DNA _____________ binds to the ______________ end of the cut DNA strand. The enzyme then fills in the gap by making a complementary copy of the information present in the undamaged strand. Although they differ from the DNA polymerase that replicates DNA, repair DNA polymerases synthesize DNA strands in the same way. For example, they elongate chains in the 5ʹ-to-3ʹ direction and have the same type of proofreading activity to ensure that the template strand is copied accurately. In many cells, the repair polymerase is the same enzyme that fills in the gaps left after the RNA primers are removed during the normal DNA replication process

bidirectional

DNA replication—in both bacterial and eukaryotic chromosomes is therfore ___________

initiator protiens

DNA sequences at replication origins are recognized by _______________

DNA damage by deamination and depurination reactions occurs spontaneously. This type of damage is not the result of replication errors and is therefore equally likely to occur on either strand. If DNA repair enzymes recognized such damage only on newly synthesized DNA strands, half of the defects would go uncorrected. The statement is therefore incorrect.

Discuss the following statement: "The DNA repair enzymes that fix deamination and depurination damage must preferentially recognize such damage on newly synthesized DNA strands."

Hydrogen bonds

How are DNA strands locked together?

by having a circular DNA

How do bacteria deal with the leading strand problem of potentially shrinking chromosomes?

Backstitching with Okazaki fragments

How do replication forks make up for being asymmetrical?

-Although the hydrogen bonds collec- tively make the DNA helix very stable, individually each hydrogen bond is weak -short length of DNA a few base pairs at a time therefore does not require a large energy input

How does this separation occur at the temperatures found in living cells even though it would normally require boiling temperatures?

thymine dimer

The ultraviolet radiation in sunlight is also damaging to DNA; it promotes covalent linkage between two adjacent pyrimidine bases Two adjacent thymine bases have become covalently attached to each other to form a thymine dimer. Skin cells that are exposed to sunlight are especially susceptible to this type of DNA damag

bacteria, eukaryotes, 3ʹ ends,

Then in homologous recombination, with the help of specialized enzymes (called recA in ________ and Rad52 in _____________), one of the broken ___________ "invades" the unbroken homologous DNA duplex and searches for a complementary sequence through base pairing

DNA polymerase

This enzyme catalyzes the addition of nucleotides to the 3ʹ end of a grow- ing DNA strand, using one of the original, parental DNA strands as a template.

once, Okazaki fragment

This loading needs to occur _________ per replication cycle on the leading strand, on the lagging strand, however, the clamp is removed and then reattached each time a new_____________ _____________ is made

primase

Unlike DNA polymerases I and III, __________ does not proofread its work.

1. 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 rearrangement that allows it to catalyze the nucleotide-addition reaction. 2. when DNA polymerase does make a rare mistake and adds the wrong nucleotide, it can correct the error through an activity called proofreading.

What are the 2 special qualities of DNA polymerase that greatly increase the accuracy of DNA replication?

A-T has fewer than G-C

What base pairing is held together by fewer hydrogen bonds?

initiator proteins

What breaks the hydrogen bonds between DNA strands (unzips the DNA)?

-often occurs shortly after a cell's DNA has been replicated before cell division, when the duplicated hel- ices are still physically close to each other

When does homologous recombination typically occur?

sugar-phosphate, ligase, Okazaki fragments

When the repair DNA polymerase has filled in the gap, a break remains in the _________________ backbone of the repaired strand. This nick in the helix is sealed by DNA _________, the same enzyme that joins the ______________ ______________ during replication of the lagging DNA strand

1. can be useful in many kinds of DNA damage not just double breaks 2. Is crucial for formation of gametes, the crossing over part

What has homologous recombination been useful for in life on earth?

sickle cell anemia

What is an example of a disease that arises from a mutation of a germ line cell?

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 of the DNA double helix.

What is the first step of repairing DNA damage?

A repair DNA polymerase binds to the 3ʹ-hydroxyl end of the cut DNA strand. The enzyme then fills in the gap by making a complementary copy of the information present in the undamaged strand. Although they differ from the DNA polymerase that replicates DNA, repair DNA polymerases synthesize DNA strands in the same way. For example, they elongate chains in the 5ʹ-to-3ʹ direction and have the same type of proofreading activity to ensure that the template strand is copied accurately. In many cells, the repair polymerase is the same enzyme that fills in the gaps left after the RNA primers are removed during the normal DNA replication process

What is the second step of repairing DNA?

DNA polymerase III

the polymerase that carries out the bulk of DNA replication at the forks is known as

DNA polymerase I

the repair polymerase involved in this process with DNA forks is often called