DNA Replication Flashcards
What are the two critical functions that DNA must satisfy to act as genetic material?
- It must maintain all the information necessary to direct and regulate all the aspects of biological function (development, cell growth, neurobiology, reproduction, etc).
- The information that DNA carries must be faithfully inherited as it is passed from parent to offspring.
Give the four basic steps/states of DNA replication.
- Original double helix.
- Strands separate.
- Complementary bases align opposite templates.
- Enzymes link sugar-phosphate elements of aligned nucleotides into a continuous new strand.
Give a brief overview of the semiconservative model for DNA replication.
In this model, which was favored by Watson and Crick, the parental DNA molecule acts as a template for the building of the entire daughter strand.
Give a brief overview of the conservative model for DNA replication.
In this model, histones bind to DNA to expose the strands for replication. Then, in the first generation, there is an entirely new chromosome where both strands of the duplex are essentially generated from the parental duplex. Subsequent generations continue this process.
Give a brief overview of the dispersive model for DNA replication.
The DNA backbone is broken up about every 10-20 base pairs. Short stretches of DNA are replicated using those break points to provide both access for the machinery as well as access to the parental template. This is the so-called “salt and pepper” approach.
Describe the Meselson-Stahl experiment that proved the semiconservative model of DNA replication in prokaryotes.
E. coli was grown in two different mediums: N14 and N15 (isotopes of nitrogen). Since DNA incorporates nitrogen into its structure, DNA of different densities was generated. Using the technique of equilibrium density centrifugation and cesium chloride, a density gradient was created. If you put a protein into the density gradient, it will migrate to the point that corresponds with its exact density, and this fact was used to distinguish the N14 and N15. The N15 DNA (the heavy DNA) was transferred to the N14 medium and allowed to replicate. Then the changes through the generations could be tracked.
How was the semiconservative model of DNA replication for eukaryotic DNA proved?
In the experiment that demonstrated this, cell cultures were allowed to divide in the presence of an analog of thymidine known as bromodeoxyuridine (BrdU). After one round of replication, both chromatids will contain one strand that has the parental thymidine and the other will incorporate BrdU into its structure. Continued replication in BrdU-containing medium leads to one chromatid of each chromosome containing thymidine. Fluorescence can be used to label the thymidine-containing chromatids.
Describe the origin of DNA replication in bacteria.
The origin of replication in E coli is defined by a specific sequence known as the oriC sequence. This is where proteins act to start the process bidirectionally. In E. coli, dnaA encodes a replication initiator protein that recruits other proteins to help separate the two strands and begin the process of DNA replication. These origins are typically rich in A-T.
Why does eukaryotic DNA take longer to replicate than prokaryotic DNA, and how does it still accomplish replication efficiently?
Eukaryotic DNA is tightly packaged in chromatin. Eukaryotic DNA replication is still efficient, however, because it uses multiple origin sites of replication that are not activated at once. Different regions of each chromosome are replicated in a reproducible order in which regions with less condensed chromatin are replicated early and regions with highly condensed chromatin are replicated late.
What is the function of topoisomerases (gyrases)?
As the helicase unwinds DNA at the replication fork, the DNA ahead must rotate and build up twists. Topoisomerases solve this by cutting DNA backbones to remove knots and other entanglements. They remove the supercoiling and other types of highly structured twists that might be present in the DNA while it is still bound in the duplex form. Essentially, it changes the topology of DNA (hence the name topoisomerase).
What is the function of helicases?
Helicases degrade helices.
What is the function of single strand binding proteins?
DNA wants to take on the duplex helical form, so when it is replicated and split into two strands, something must stabilize the intermediate single strand. Single strand binding proteins do this by associating with DNA and allowing it to stay stable as a single-stranded molecule.
What direction does a DNA polymerase move along the DNA template?
DNA polymerase moves 3’ to 5’ along the template to synthesize a new strand that grows 5’ to 3’. One of the new strands grows toward the RF whilst another grows away from the RF.
What drives the reaction of DNA polymerase action?
The hydrolysis of high energy pyrophosphate on dNTP drives the reaction.
Why are there more proteins involved in the lagging strand than the leading strand?
The Okazaki fragments on the lagging strand lead to the need for more proteins.
What is primase, and what is its role in DNA replication?
Primase is an RNA polymerase that is responsible for building the essential RNA primer that provides the 3’ hydroxyl group that is required for DNA polymerase.
What is a replisome, and what is its function?
The replisome is a DNA polymerase III holoenzyme complex that incorporates components from the polymerase III structure along with other non-enzymatic components.
What is the role of clamp-loading proteins?
These proteins are used to initially load the clamp, recognizing the junction between template and RNA primers.
On the lagging strand, why does the template sometimes have to temporarily dissociate from the polymerase?
On the lagging strand, the template sometimes has to temporarily dissociate from the polymerase to allow it to skip over those RNA primases and the primers that they are making.
What is the role of clamp proteins?
They form a sliding clamp around DNA, helping the DNA polymerase maintain contact with its template and thereby assisting with processivity. The inner face of the clamp enables DNA to be threaded through it. Once the polymerase reaches the end of the template or detects double stranded DNA, the sliding clamp undergoes a conformation change which releases the DNA polymerase.
Which type of DNA polymerase is responsible for the majority of the DNA synthesis?
Polymerase III.
