SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. The Experiment
  4. Correct Answers
  5. Explanations
  6. Reference

Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [α-32P]ATP, [γ-32P]ATP, DNA ligase, polynucleotide kinase, DNA polymerase, terminal transferase, DNA helicase, UV-irradiation, pyrimidine dimer, base modifications, mismatch, polyacrylamide gel electrophoresis, autoradiography, molecular hybridization, satellite DNA, nucleosomes. © 2012 by The International Union of Biochemistry and Molecular Biology, 41(1):52–54, 2013


Abbreviations
UV

ultraviolet

MCQ

multiple-choice question

The Experiment

  1. Top of page
  2. Abstract
  3. The Experiment
  4. Correct Answers
  5. Explanations
  6. Reference

Proteins involved in repairing DNA damage are vital for all cells: without them the rate of mutations would increase leading to impaired cell functions and inheritance of mutations to daughter cells. In humans, germline mutations and somatic mutations escaping DNA repair may lead to inherited diseases and malignant cancers, respectively. Understanding DNA repair mechanisms is thus essential in the prevention of diseases caused by DNA damage.

The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells [1]. A 148 basepair long restriction endonuclease fragment prepared from Escherichia coli DNA was labeled at its 5′-end with a radioactive phosphate group.

Five-choice Completion

(This type of question consists of a question or incomplete statement followed by five suggested answers or completions. Select the one best answer.)

1.____ Which of the following possibilities can be used for such labeling of the fragments?

A. [α-32P]ATP + DNA ligase

B. [γ-32P]ATP + polynucleotide kinase

C. [γ-32P]ATP + DNA polymerase

D. [α-32P]ATP + terminal transferase

E. [γ-32P]ATP + DNA helicase

Two end-labeled DNA fragments (called “probes” throughout this test) were prepared: one of the probes was left intact (f148*), while the other probe sample was irradiated with a strong ultraviolet (UV) light (UV-f148*).

Five-choice Completion

(This type of question consists of a question or incomplete statement followed by five suggested answers or completions. Select the one best answer.)

2.____ What is the most important consequence of UV-irradiation?

A. Formation of pyrimidine dimers

B. Base oxidation

C. Base methylation

D. Mismatch formation

E. Base deamination

The f148* probe (Samples 1–7 in Fig. 1) and the UV-f148* probe (Samples 8–14) were then incubated alone (Samples 1–8), in the presence of a human tumor cell line extract (Samples 2–4 and 9–11) or yeast cell extract (Samples 5–7 and 12–14). Various amounts of unlabelled undamaged f148 DNA (“cold” f148) was also added to some of the mixtures (Samples 3, 4, 6, 7, 10, 11, 13, 14).

image

Figure 1. Analysis of protein binding to undamaged and UV-irradiated DNA (for experimental details see the text; H, human; Y, yeast; the arrow indicates the direction of electrophoresis).

Download figure to PowerPoint

Five-choice Completion

(This type of question consists of a question or incomplete statement followed by five suggested answers or completions. Select the one best answer.)

3.____ What was the aim of adding the “cold” DNA fragment?

A. To reduce non-specific protein binding to the probes

B. To enhance non-specific protein binding to the probes

C. To reduce specific protein binding to the probes

D. To enhance specific protein binding to the probes

E. None of the above

The products of in vitro reactions were resolved by electrophoresis in a polyacrylamide gel that did not disrupt protein–DNA interactions. The gel was dried and analyzed by autoradiography. Study the results in Fig. 1 and solve the following multiple-choice questions (MCQs).

Five-choice Completion

(This type of question consists of a question or incomplete statement followed by five suggested answers or completions. Select the one best answer.)

4.____ What conclusions can be drawn from comparing Samples 2 and 9?

A. UV-irradiation induces DNA-binding proteins in human cells

B. Human cells contain proteins that recognize and bind to pyrimidine dimers

C. Probe molecules hybridized to cellular DNA

D. A and B

E. A, B, and C

5.____ What conclusions can be drawn from comparing Samples 9 and 11?

A. Proteins in bands H1 and H2 recognize undamaged DNA

B. Proteins in bands H1 and H2 recognize damaged DNA

C. f148 competes with UV-f148* for the binding proteins

D. A and C

E. B and C

Experiment Analysis

(The following statements are related to the information presented in the description of the experiment. Based on the information given, select

A: if the statement is supported by the information given;

B: if the statement is contradicted by the information given;

C: if the statement is neither supported nor contradicted by the information given.)

6.____ The UV-f148* probe is able to bind more than one protein molecule.

7.____ The binding proteins recognize pyrimidine dimers in specific DNA sequences.

8.____ Human tumor cell and yeast cell proteins bind to different regions of the UV-f148* fragment.

9.____ The binding proteins recognize satellite DNA.

10.____ UV-damage recognizing proteins are identical in human and yeast cells.

11.____ Under the conditions used nucleosomes were formed on f148*.

12.____ Band X corresponds to free probe molecules.

Quantitative Comparison

(In this type of question paired statements describe two entities that are to be compared in a quantitative sense. Select:

A: if A is greater than B;

B: if B is greater than A;

C: if the two are equal or very nearly equal.)

13.____ A: The size of the H1 complex

B: The size of the H2 complex

14.____ A: Protein/DNA ratio in the Y1 complex

B: Protein/DNA ratio in the Y2 complex

Correct Answers

  1. Top of page
  2. Abstract
  3. The Experiment
  4. Correct Answers
  5. Explanations
  6. Reference
1. B8. C
2. A9. B
3. C10. B
4. B11. B
5. B12. A
6. A13. A
7. C14. A

Explanations

  1. Top of page
  2. Abstract
  3. The Experiment
  4. Correct Answers
  5. Explanations
  6. Reference

The technique used in this study to identify pyrimidine dimer binding proteins (MCQ 2: A) is called electrophoretic mobility shift assay (or gel retardation assay): end-labeled DNA fragments (MCQ 1: B) are mixed with protein extracts and then electrophoresed under non-denaturing conditions. Protein–DNA complexes migrate slower than free probe molecules (compare the positions of band X to bands H1, H2, Y1 and Y2, MCQ 12: A; MCQ 13: A; MCQ 14: A). Protein binding takes place on the UV-irradiated probe molecules only (MCQ 4: B). This observation is further supported by the fact that an excess of undamaged “cold” probe was unable to compete with the UV-irradiated probe for protein binding (MCQ 3: C; MCQ 5: B). The appearance of bands at various positions indicates that more than one protein molecules bind to the probe (MCQ 6: A) and that these proteins are of different size in yeast and in human cells (MCQ 10: B). Sequence specificity of protein binding was not addressed in this experiment (MCQ 7: C; MCQ 8: C). The E. coli genome does not contain satellite DNA (MCQ 9: B). Since Samples 2–7 in Fig. 1 do not show band shifting, histones did not bind to the probe under the experimental conditions (MCQ 11: B).

Reference

  1. Top of page
  2. Abstract
  3. The Experiment
  4. Correct Answers
  5. Explanations
  6. Reference
  • 1
    M. Patterson, G. Chu(1989)Evidence that xeroderma pigmentosum cells from complementation group E are deficient in a homolog of yeast photolyase,Mol. Cell. Biol.9,51055112.