14.1: Historical Basis of Modern Understanding
Modern understandings of DNA have evolved from the discovery of nucleic acid to the development of the double-helix model. In the 1860s, Friedrich Miescher, a physician by profession, was the first person to isolate phosphate-rich chemicals from white blood cells or leukocytes. He named these chemicals (which would eventually be known as RNA and DNA) nuclein because they were isolated from the nuclei of the cells.
If DNA of a particular species was analyzed and it was found that it contains 27 percent A, what would be the percentage of C?
- 27 percent
- 30 percent
- 23 percent
- 54 percent
The experiments by Hershey and Chase helped confirm that DNA was the hereditary material on the basis of the finding that:
- radioactive phage were found in the pellet
- radioactive cells were found in the supernatant
- radioactive sulfur was found inside the cell
- radioactive phosphorus was found in the cell
Explain Griffith's transformation experiments. What did he conclude from them?
Live R cells acquired genetic information from the heat-killed S cells that “transformed” the R cells into S cells.
Why were radioactive sulfur and phosphorous used to label bacteriophage in Hershey and Chase's experiments?
Sulfur is an element found in proteins and phosphorus is a component of nucleic acids.
14.2: DNA Structure and Sequencing
The building blocks of DNA are nucleotides. The important components of the nucleotide are a nitrogenous base, deoxyribose (5-carbon sugar), and a phosphate group. The nucleotide is named depending on the nitrogenous base. The nitrogenous base can be a purine such as adenine (A) and guanine (G), or a pyrimidine such as cytosine (C) and thymine (T).
DNA double helix does not have which of the following?
- antiparallel configuration
- complementary base pairing
- major and minor grooves
In eukaryotes, what is the DNA wrapped around?
- single-stranded binding proteins
- sliding clamp
Provide a brief summary of the Sanger sequencing method.
The template DNA strand is mixed with a DNA polymerase, a primer, the 4 deoxynucleotides, and a limiting concentration of 4 dideoxynucleotides. DNA polymerase synthesizes a strand complementary to the template. Incorporation of ddNTPs at different locations results in DNA fragments that have terminated at every possible base in the template. These fragments are separated by gel electrophoresis and visualized by a laser detector to determine the sequence of bases.
Describe the structure and complementary base pairing of DNA.
DNA has two strands in anti-parallel orientation. The sugar-phosphate linkages form a backbone on the outside, and the bases are paired on the inside: A with T, and G with C, like rungs on a spiral ladder.
14.3: Basics of DNA Replication
The elucidation of the structure of the double helix provided a hint as to how DNA divides and makes copies of itself. This model suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied. What was not clear was how the replication took place. There were three models suggested: conservative, semi-conservative, and dispersive.
Meselson and Stahl's experiments proved that DNA replicates by which mode?
- none of the above
If the sequence of the 5'-3' strand is AATGCTAC, then the complementary sequence has the following sequence:
How did the scientific community learn that DNA replication takes place in a semi-conservative fashion?
Meselson’s experiments with E. coli grown in 15N deduced this finding.
14.4: DNA Replication in Prokaryotes
DNA replication has been extremely well studied in prokaryotes primarily because of the small size of the genome and the mutants that are available. E. coli has 4.6 million base pairs in a single circular chromosome and all of it gets replicated in approximately 42 minutes, starting from a single origin of replication and proceeding around the circle in both directions. This means that approximately 1000 nucleotides are added per second. The process is quite rapid and occurs without many mistake
Which of the following components is not involved during the formation of the replication fork?
- single-strand binding proteins
- origin of replication
Which of the following does the enzyme primase synthesize?
- DNA primer
- RNA primer
- Okazaki fragments
- phosphodiester linkage
In which direction does DNA replication take place?
DNA replication is bidirectional and discontinuous; explain your understanding of those concepts.
At an origin of replication, two replication forks are formed that are extended in two directions. On the lagging strand, Okazaki fragments are formed in a discontinuous manner.
What are Okazaki fragments and how they are formed?
Short DNA fragments are formed on the lagging strand synthesized in a direction away from the replication fork. These are synthesized by DNA pol.
If the rate of replication in a particular prokaryote is 900 nucleotides per second, how long would it take 1.2 million base pair genomes to make two copies?
1333 seconds or 22.2 minutes.
Explain the events taking place at the replication fork. If the gene for helicase is mutated, what part of replication will be affected?
At the replication fork, the events taking place are helicase action, binding of single-strand binding proteins, primer synthesis, and synthesis of new strands. If there is a mutated helicase gene, the replication fork will not be extended.
What is the role of a primer in DNA replication? What would happen if you forgot to add a primer in a tube containing the reaction mix for a DNA sequencing reaction?
Primer provides a 3'-OH group for DNA pol to start adding nucleotides. There would be no reaction in the tube without a primer, and no bands would be visible on the electrophoresis.
14.5: DNA Replication in Eukaryotes
Eukaryotic genomes are much more complex and larger in size than prokaryotic genomes. The human genome has three billion base pairs per haploid set of chromosomes, and 6 billion base pairs are replicated during the S phase of the cell cycle. There are multiple origins of replication on the eukaryotic chromosome; humans can have up to 100,000 origins of replication
The ends of the linear chromosomes are maintained by
- DNA pol
How do the linear chromosomes in eukaryotes ensure that its ends are replicated completely?
Telomerase has an inbuilt RNA template that extends the 3' end, so primer is synthesized and extended. Thus, the ends are protected.
14.6: DNA Repair
DNA replication is a highly accurate process, but mistakes can occasionally occur, such as a DNA polymerase inserting a wrong base. Uncorrected mistakes may sometimes lead to serious consequences, such as cancer. Repair mechanisms correct the mistakes. In rare cases, mistakes are not corrected, leading to mutations; in other cases, repair enzymes are themselves mutated or defective.
During proofreading, which of the following enzymes reads the DNA?
- DNA pol
The initial mechanism for repairing nucleotide errors in DNA is ________.
- mismatch repair
- DNA polymerase proofreading
- nucleotide excision repair
- thymine dimers
What is the consequence of mutation of a mismatch repair enzyme? How will this affect the function of a gene?
Mutations are not repaired, as in the case of xeroderma pigmentosa. Gene function may be affected or it may not be expressed.