Ch6.doc

CHAPTER 6 DNA REPLICATION, REPAIR, AND RECOMBINATION 

2009 Garland Science Publishing

DNA Replication 6-1

The process of DNA replication requires that each of the parental DNA strands be used as a ___________________ to produce a duplicate of the opposing strand. (a) catalyst (b) competitor (c) template (d) copy

6-2

DNA replication is considered semiconservative because ____________________________. (a) after many rounds of DNA replication, the original DNA double helix is still intact (b) each daughter DNA molecule consists of two new strands copied from the parent DNA molecule (c) each daughter DNA molecule consists of one strand from the parent DNA molecule and one new strand (d) new DNA strands must be copied from a DNA template

6-3

The classic experiments conducted by Meselson and Stahl demonstrated that DNA replication is accomplished by employing a ________________ mechanism. (a) continuous (b) semiconservative (c) dispersive (d) conservative

6-4

If the genome of the bacterium E. coli requires about 20 minutes to replicate itself, how can the genome of the fruit fly Drosophila be replicated in only 3 minutes? (a) The Drosophila genome is smaller than the E. coli genome. (b) Eucaryotic DNA polymerase synthesizes DNA at a much faster rate than procaryotic DNA polymerase. (c) The nuclear membrane keeps the Drosophila DNA concentrated in one place in the cell, which increases the rate of polymerization. (d) Drosophila DNA contains more origins of replication than E. coli DNA.

6-5

Meselson and Stahl grew cells in media that contained different isotopes of nitrogen (15N and 14N) so that the DNA molecules produced from these different isotopes could be distinguished by mass.

A. B. C.

Explain how “light” DNA was separated from “heavy” DNA in the Meselson and Stahl experiments. Describe the three existing models for DNA replication when these studies were begun, and explain how one of them was ruled out definitively by the experiment you described for part A. What experimental result eliminated the dispersive model of DNA replication?

6-6

Indicate whether the following statements are true or false. If a statement is false, explain why it is false. A. When DNA is being replicated inside a cell, local heating occurs, allowing the two strands to separate. B. DNA replication orgins are typically rich in G-C base pairs. C. Meselson and Stahl ruled out the dispersive model for DNA replication. D. DNA replication is a bidirectional process that is initiated at multiple locations along chromosomes in eucaryotic cells.

6-7

Answer the following questions about DNA replication. A. On a DNA strand that is being synthesized, which end is growing—the 3′ end, the 5′ end, or both ends? Explain your answer. B. On a DNA strand that is being used as a template, where is the copying occurring relative to the replication origin—3′ of the origin, 5′, or both?

6-8

How does the total number of replication origins in bacterial cells compare with the number of origins in human cells? (a) 1 versus 100 (b) 5 versus 500 (c) 10 versus 1000 (d) 1 versus 10,000

6-9

The chromatin structure in eucaryotic cells is much more complicated than that observed in procaryotic cells. This is thought to be the reason that DNA replication occurs much faster in procaryotes. How much faster is it? (a) 2× (b) 5× (c) 10× (d) 100×

6-10

DNA polymerase catalyzes the joining of a nucleotide to a growing DNA strand. What prevents this enzyme from catalyzing the reverse reaction? (a) hydrolysis of PPi to Pi + Pi (b) release of PPi from the nucleotide (c) hybridization of the new strand to the template (d) loss of ATP as an energy source

6-11

Figure Q6-11 shows a replication bubble.

Figure Q6-11 A. B. C. D. E. F.

On the figure, indicate where the origin of replication was located (use O). Label the leading-strand template and the lagging-strand template of the righthand fork [R] as X and Y, respectively. Indicate by arrows the direction in which the newly made DNA strands (indicated by dark lines) were synthesized. Number the Okazaki fragments on each strand 1, 2, and 3 in the order in which they were synthesized. Indicate where the most recent DNA synthesis has occurred (use S). Indicate the direction of movement of the replication forks with arrows.

6-12

Which of the following statements about the newly synthesized strand of a human chromosome is true? (a) It was synthesized from a single origin solely by continuous DNA synthesis. (b) It was synthesized from a single origin by a mixture of continuous and discontinuous DNA synthesis. (c) It was synthesized from multiple origins solely by discontinuous DNA synthesis. (d) It was synthesized from multiple origins by a mixture of continuous and discontinuous DNA synthesis.

6-13

You have discovered an “Exo–” mutant form of DNA polymerase in which the 3′-to-5′ exonuclease function has been destroyed but the ability to join nucleotides together is unchanged. Which of the following properties do you expect the mutant polymerase to have? (a) It will polymerize in both the 5′-to-3′ direction and the 3′-to-5′ direction. (b) It will polymerize more slowly than the normal Exo+ polymerase. (c) It will fall off the template more frequently than the normal Exo+ polymerase. (d) It will be more likely to generate mismatched base pairs.

6-14

A molecule of bacterial DNA introduced into a yeast cell is imported into the nucleus but fails to replicate the yeast DNA. Where do you think the block to replication arises? Choose the protein or protein complex below that is most probably responsible for the failure to replicate bacterial DNA. Give an explanation for your answer. (a) primase (b) helicase (c) DNA polymerase (d) initiator proteins

6-15

Most cells in the body of an adult human lack the telomerase enzyme because its gene is turned off and is therefore not expressed. An important step in the conversion of a normal cell into a cancer cell, which circumvents normal growth control, is the resumption of telomerase expression. Explain why telomerase might be necessary for the ability of cancer cells to divide over and over again.

6-16

Which diagram accurately represents the directionality of DNA strands at one side of a replication fork?

6-17

Indicate whether the following statements are true or false. If a statement is false, explain why it is false. A. Primase is needed to initiate DNA replication on both the leading strand and the lagging strand. B. The sliding clamp is loaded once on each DNA strand, where it remains associated until replication is complete. C. Telomerase is a DNA polymerase that carries its own RNA molecule to use as a primer at the end of the lagging strand. D. Primase requires a proofreading function that ensures there are no errors in the RNA primers used for DNA replication.

6-18

Because all DNA polymerases synthesize DNA in the 5′-to-3′ direction, and the parental strands are antiparallel, DNA replication is accomplished with the use of two mechanisms: continuous and discontinuous replication. Indicate whether the following items relate to (1) continuous replication, (2) discontinuous replication, or (3) both modes of replication. ______ primase ______ single-strand binding protein ______ sliding clamp ______ RNA primers ______ leading strand ______ lagging strand ______ Okazaki fragments ______ DNA helicase ______ DNA ligase

6-19

The synthesis of DNA in living systems occurs in the 5′-to-3′ direction. However, scientists synthesize short DNA sequences needed for their experiments on an instrument dedicated to this task. A. The chemical synthesis of DNA by this instrument proceeds from the 3′-to-5′ direction. Draw a diagram to show how this is possible and explain the process. B. Although 3′-to-5′ synthesis of DNA is chemically possible, it does not occur in living systems. Why not?

6-20

DNA polymerases are processive, which means that they remain tightly associated with the template strand while moving rapidly and adding nucleotides to the growing daughter strand. Which piece of the replication machinery accounts for this characteristic? (a) helicase (b) sliding clamp (c) single-strand binding protein (d) primase

6-21

Use the components in the list below to label the diagram of a replication fork in Figure Q6-21. A. DNA polymerase B. single-strand binding protein C. Okazaki fragment D. primase E. sliding clamp F. RNA primer G. DNA helicase

Figure Q6-21 6-22

Researchers have isolated a mutant strain of E. coli that carries a temperature-sensitive variant of the enzyme DNA ligase. At the permissive temperature, the mutant cells grow just as well as the wild-type cells. At the nonpermissive temperature, all of the cells in the culture tube die within 2 hours. DNA from mutant cells grown at the nonpermissive temperature for 30 minutes is compared with the DNA isolated from cells grown at the permissive temperature. The results are shown in Figure Q6-22, where DNA molecules have been separated by size by means of electrophoresis (P, permissive; NP, nonpermissive). Explain the appearance of a distinct band with a size of 200 base pairs (bp) in the sample collected at the nonpermissive temperature.

Figure Q6-22 6-23

Telomeres serve as caps at the ends of linear chromosomes. Which of the following is not true regarding the replication of telomeric sequences? (a) The lagging strand telomeres are not completely replicated by DNA polymerase. (b) Telomeres are made of repeating sequences. (c) Additional repeated sequences are added to the template strand. (d) The leading strand doubles back on itself to form a primer for the lagging strand.

DNA Repair 6-24

Sickle-cell anemia is an example of an inherited disease. Individuals with this disorder have misshapen (sickle-shaped) red blood cells caused by a change in the sequence of the β-globin gene. What is the nature of the change? (a) chromosome loss (b) base-pair change

(c) (d)

gene duplication base-pair insertion

6-25

Even though DNA polymerase has a proofreading function, it still introduces errors in the newly synthesized strand at a rate of 1 per 107 nucleotides. To what degree does the mismatch repair system decrease the error rate arising from DNA replication? (a) 2-fold (b) 5-fold (c) 10-fold (d) 100-fold

6-26

Which of the choices below represents the correct way to repair the mismatch shown in Figure Q6-26?

Figure Q6-26

6-27

A mismatched base pair causes a distortion in the DNA backbone. If this were the only indication of an error in replication, the overall rate of mutation would be much higher. Explain why.

6-28

Beside the distortion in the DNA backbone caused by a mismatched base pair, what additional mark is there on eucaryotic DNA to indicate which strand needs to be repaired? (a) a nick in the template strand

(b) (c) (d)

a chemical modification of the new strand a nick in the new strand a sequence gap in the new strand

6-29

A pregnant mouse is exposed to high levels of a chemical. Many of the mice in her litter are deformed, but when they are interbred with each other, all their offspring are normal. Which two of the following statements could explain these results? (a) In the deformed mice, somatic cells but not germ cells were mutated. (b) The original mouse’s germ cells were mutated. (c) In the deformed mice, germ cells but not somatic cells were mutated. (d) The toxic chemical affects development but is not mutagenic.

6-30

The repair of mismatched base pairs or damaged nucleotides in a DNA strand requires a multistep process. Which choice below describes the known sequence of events in this process? (a) DNA damage is recognized, the newly synthesized strand is identified by an existing nick in the backbone, a segment of the new strand is removed by repair proteins, the gap is filled by DNA polymerase, and the strand is sealed by DNA ligase. (b) DNA repair polymerase simultaneously removes bases ahead of it and polymerizes the correct sequence behind it as it moves along the template. DNA ligase seals the nicks in the repaired strand. (c) DNA damage is recognized, the newly synthesized strand is identified by an existing nick in the backbone, a segment of the new strand is removed by an exonuclease, and the gap is repaired by DNA ligase. (d) A nick in the DNA is recognized, DNA repair proteins switch out the wrong base and insert the correct base, and DNA ligase seals the nick.

6-31

You are examining the DNA sequences that code for the enzyme phosphofructokinase in skinks and Komodo dragons. You notice that the coding sequence that actually directs the sequence of amino acids in the enzyme is very similar in the two organisms but that the surrounding sequences vary quite a bit. What is the most likely explanation for this? (a) Coding sequences are repaired more efficiently. (b) Coding sequences are replicated more accurately. (c) Coding sequences are packaged more tightly in the chromosomes to protect them from DNA damage. (d) Mutations in coding sequences are more likely to be deleterious to the organism than mutations in noncoding sequences.

6-32

Sometimes chemical damage to DNA can occur just before DNA replication begins, not giving the repair system enough time to correct the error before the DNA is duplicated. This gives rise to mutation. If the cytosine in the sequence TCAT is deaminated and not repaired, which of the following is the point mutation you would observe after this segment has undergone two rounds of DNA replication? (a) TTAT (b) TUAT

(c) (d)

TGAT TAAT

6-33

During DNA replication in a bacterium, a C is accidentally incorporated instead of an A into one newly synthesized DNA strand. Imagine that this error was not corrected and that it has no effect on the ability of the progeny to grow and reproduce. A. After this original bacterium has divided once, what proportion of its progeny would you expect to contain the mutation? B. What proportion of its progeny would you expect to contain the mutation after three more rounds of DNA replication and cell division?

6-34

Sometimes chemical damage to DNA can occur just before DNA replication begins, not giving the repair system enough time to correct the error before the DNA is duplicated. This gives rise to mutation. If the adenosine in the sequence TCAT is depurinated and not repaired, which of the following is the point mutation you would observe after this segment has undergone two rounds of DNA replication? (a) TCGT (b) TAT (c) TCT (d) TGTT

6-35

Which of the following statements is not an accurate statement about thymidine dimers? (a) Thymidine dimers can cause the DNA replication machinery to stall. (b) Thymidine dimers are covalent links between thymidines on opposite DNA strands. (c) Prolonged exposure to sunlight causes thymidine dimers to form. (d) Repair proteins recognize thymidine dimers as a distortion in the DNA backbone.

6-36

Indicate whether the following statements are true or false. If a statement is false, explain why it is false. A. Ionizing radiation and oxidative damage can cause DNA double-strand breaks. B. After damaged DNA has been repaired, nicks in the phosphate backbone are maintained as a way to identify the strand that was repaired. C. Depurination of DNA is a rare event that is caused by ultraviolet irradiation. D. Nonhomologous end joining is a mechanism that ensures that DNA double-strand breaks are repaired with a high degree of fidelity to the original DNA sequence.

6-37

Several members of the same family were diagnosed with the same kind of cancer when they were unusually young. Which one of the following is the most likely explanation for this phenomenon? It is possible that the individuals with the cancer have _______________________. (a) inherited a cancer-causing gene that suffered a mutstion in an ancestor’s somatic cells (b) inherited a mutation in a gene required for DNA synthesis (c) inherited a mutation in a gene required for mismatch repair (d) inherited a mutation in a gene required for the synthesis of purine nucleotides

6-38

You have made a collection of mutant fruit flies that are defective in various aspects of DNA repair. You test each mutant for its hypersensitivity to three DNA-damaging agents: sunlight, nitrous acid (which causes deamination of cytosine), and formic acid (which causes depurination). The results are summarized in Table Q6-38, where a “yes” indicates that the mutant is more sensitive than a normal fly, and blanks indicate normal sensitivity.

Table Q6-38 A. B.

Which mutant is most likely to be defective in the DNA repair polymerase? What aspect of repair is most likely to be affected in the other mutants?

Homologous Recombination and Mobile Genetic Elements and Viruses 6-39

Homologous recombination is an important mechanism in which organisms use a “backup” copy of the DNA as a template to fix double-strand breaks without loss of genetic information. Which of the following is not necessary for homologous recombination to occur? (a) 3′ DNA strand overhangs (b) 5′ DNA strand overhangs (c) a long stretch of sequence similarity (d) nucleases

6-40

In addition to the repair of DNA double-strand breaks, homologous recombination is a mechanism for generating genetic diversity by swapping segments of parental chromosomes. During which process does swapping occur? (a) DNA replication (b) DNA repair (c) meiosis (d) transposition

6-41

Recombination has occurred between the chromosome segments shown in Figure Q6-41. The genes A and B, and the recessive alleles a and b, are used as markers on the maternal and paternal chromosomes, respectively. After alignment and homologous recombination, the specific arrangements of A, B, a, and b have changed.

Figure Q6-41 Which of the choices below correctly indicates the gene combination from the replication products of the maternal chromosome? (a) (b) (c) (d) 6-42

AB and aB ab and Ab AB and Ab aB and Ab

The events listed below are all necessary for homologous recombination to occur properly: A. Holliday junction cut and ligated B. strand invasion C. DNA synthesis D. DNA ligation E. double-strand break F. nucleases create uneven strands Which of the following is the correct order of events during homologous recombination? (a) E, B, F, D, C, A (b) B, E, F, D, C, A (c) C, E, F, B, D, A (d) E, F, B, C, D, A

6-43

Homologous recombination is initiated by double-strand breaks (DSBs) in a chromosome. DSBs arise from DNA damage caused by harmful chemicals or by radiation (for example x-rays). During meiosis, the specialized cell division that produces gametes (sperm and eggs) for sexual reproduction, the cells intentionally cause DSBs so as to stimulate crossover homologous recombination. If there is not at least one occurrence of crossing-over within each pair of homologous chromosomes during meiosis, those noncrossover chromosomes will not segregate properly.

Figure Q6-43 A.

B. C.

Consider the copy of chromosome 3 that you received from your mother. Is it identical to the chromosome 3 that she received from her mother (her maternal chromosome) or identical to the chromosome 3 she received from her father (her paternal chromosome), or neither? Explain. Starting with the representation in Figure Q6-43 of the double-stranded maternal and paternal chromosomes found in your mother, draw two possible chromosomes you may have received from your mother. What does this indicate about your resemblance to your grandfather and grandmother?

6-44

Mobile genetic elements are sometimes called “jumping genes,” because they move from place to place throughout the genome. The exact mechanism by which they achieve this mobility depends on the genes contained within the mobile element. Which of the following mobile genetic elements carry both a transposase gene and a reverse transcriptase gene? (a) L1 (b) B1 (c) Alu (d) Tn3

6-45

Which of the following is true of a retrovirus but not of the Alu retrotransposon? (a) It requires cellular enzymes to make copies. (b) It can be inserted into the genome. (c) It can be excised and moved to a new location in the genome. (d) It encodes its own reverse transcriptase.

6-46

Which of the following DNA sequences is not commonly carried on mobile genetic elements? You may choose more than one option. (a) transposase gene (b) Holliday junction (c) recognition site for transposase (d) antibiotic resistance gene

6-47

HIV is a human retrovirus that integrates into the host cell’s genome and will eventually replicate, produce viral proteins, and ultimately escape the host cell. Which of the following proteins is not encoded in the HIV genome? (a) reverse transcriptase (b) envelope protein (c) RNA polymerase (d) capsid protein

6-48

Some retrotransposons and retroviruses integrate preferentially into regions of the chromosome that are packaged in euchromatin and are also located outside the coding regions of genes that contain information for making a protein. Why might these mobile genetic elements have evolved this strategy?