Are genes encoded by protein or DNA? Would you find proteins or DNA in chromosomes?

• Genes are encoded by DNA.
• You would find both.

Describe the experiment of Hershey and Chase.

• The Hershey-Chase experiment was attempting to figure out whether genes contained DNA or proteins.
• They used a virus called T2 which infects cells by attaching to them and injecting its genes to the interior of the cell, leaving behind its protein coat. They radioactively labeled one population with phosphorus and one population with sulfur. After the viruses infected E. coli cells, they sheared the capsids off by agitating them. When the samples were spun in a centrifuge, the small phage capsids remained in the solution while the cells formed a pellet at the bottom of the centrifuge tube. They found that all the radioactive protein was outside the cell in the solution while almost all the radioactive DNA was inside the cells.

Explain the reasoning behind the labeling in the Hershey and Chase experiment.

The reasoning behind the labeling in their experiment was that DNA contains phosphorus but not sulfur and proteins contain sulfur but not phosphorus.

Illustrate the major features of DNA’s primary and secondary structure.

• The primary structure consists of a backbone made up of sugar and phosphate groups of deoxyribonucleotides and a series of bases that project from the backbone.
• The secondary structure is formed by hydrogen bonding between complementary nitrogenous bases.

Explain the concept of DNA polarity.

• Each strand has directionality of polarity, the two strands line up in opposite directions
• One strand has an exposed hydroxyl group on a 3’ carbon of a deoxyribose
• One strand has an exposed phosphate group on a 5’ carbon

Describe the DNA synthesis reaction catalyzed by DNA polymerase.

What does it mean to say that DNA is synthesized in the 5’ to 3’ direction?

DNA polymerase adds deoxyribonucleotides to the 3’ end of a growing DNA chain therefore DNA is always synthesized in the 5’ to 3’ direction.

Why is an RNA primer required during DNA synthesis?

What enzyme synthesizes the primer?

• Since DNA polymerase cannot begin synthesizing from scratch, the RNA primer places a short complementary strand that allows DNA polymerase to attach new nucleotides to the free 3’ OH group.
• It is synthesized by primase.

List the proteins required for DNA synthesis in E. coli , and describe the function of each. Imagine a collection of mutant cells, each with a mutation rendering one of these proteins non-functional. What would happen at the replication fork in each of these mutants?

• DNA polymerase III requires a single strand DNA and a 3’ end to extend from and synthesizes in the 5’ to 3’ direction
• Both leading and lagging strand will not be synthesized
• Topoisomerase relieves twisting forces
• A supercoil with too much tension will occur and DNA will break
• Helicase opens double helix
• There would not be a replication fork
• SSBPs (single stranded binding proteins) stabilize single strands by attaching to them and preventing them from binding back
• Strands will bind back together on helicase and replication fork will not exist
• Primase synthesizes a short strand of RNA that acts as a primer for DNA polymerase
• DNA polymerase doesn’t have the OH on the 3’ end of the primer to start synthesizing
• DNA polymerase I removes RNA primer and replaces it with DNA
• Lagging strand will not be synthesized fully because RNA primers aren’t being removed
• DNA ligase catalyzes the formation of phosphodiester bonds between okazaki fragments
• It will result in fragmented strands

The error rate during DNA replication averages less than one mistake per billion nucleotides (error rate < 1 x 10 -9 ). How is this extremely low error rate achieved?

What happens when a DNA polymerase inserts the wrong base into a sequence?

• DNA polymerase III proofreads so if the wrong base is added during DNA synthesis, the enzyme pauses because geometry differs from correct base pairing and a portion of DNA polymerase III called epsilon unit acts as an exonuclease and removes deoxyribonucleotides form the ends of the DNA strand.

When during the cell cycle does mismatch repair occur?

Mismatch repair occurs in the S phase.

Why is it important for E. coli cells to distinguish the “old” strand from the “new” (i.e., newly synthesized) strand? How do they do so?

• It is important to distinguish the old strand from the new because the old strand is the correct template and the new strand is the strand with the error.
• Chemical marks (methylated bases) on the older strand allow the enzymes to distinguish the old strand from the new strand which has not gained the marks yet

How can DNA be damaged?

• DNA can be damaged by sunlight, x-rays, and many chemicals.
• UV light can cause covalent bonding between adjacent pyrimidine bases in the same strand causing a kink that prevents DNA polymerase from replicating the DNA and causes the cell to die.

Explain the mechanism by which damaged nucleotides are recognized and repaired.

• Nucleotide excision repair fixes errors such as thymine dimers and other types of damage that distort the DNA helix
• First, an enzyme recognizes the kink in the DNA double helix and another enzyme removes the section of single stranded DNA containing the defective sequence
• The intact DNA strand serves as a template for the synthesis of a corrected strand and the 3’ hydroxyl of the DNA strand next to the gap serves as a primer
• DNA ligase links the newly synthesized DNA to the original undamaged DNA

How could defects in DNA repair pathways increase the risk of cancer?

• If DNA damage in the genes involved in the cell cycle go unrepaired, mutations that may allow the cell to grow in an uncontrolled manner will result.
• If the mutation rate in a cell increases due to defects in DNA repair, mutations that trigger cancer become more likely.

DNA polymerase cannot copy the end of the lagging strand of a linear chromosome. Why not?

• DNA polymerase cannot copy the end of the lagging strand of a linear chromosome because the RNA primer is removed.

How do some cells avoid the problem of telomere shortening?

• Telomerase fixes this issue by binding to the overhanging section of single-stranded DNA and extending the parent DNA by adding deoxyribonucleotides. Primase, DNA polymerase and ligase synthesize the lagging strand in the 5’ to 3’ direction.
• Active in gametes and some cancer cells