Ch. 20 Flashcards
What are the 3 proposed hypotheses for DNA replication? Which one is correct?
- Conservative replication
- Dispersive replication
- Semiconservative replication
What experiment proved that DNA replication was conservative?
Meselson–Stahl experiment
What is the origin of replication?
DNA sequence at which DNA replication initiates
What is the replication fork?
Region of the chromosome that is being unwound during DNA synthesis
What direction is DNA synthesized in?
5’ to 3’
How are both strands of DNA synthesized at the same time if DNA synthesis can only go in the 5’ to 3’ direction?
- One strand is synthesized continuously
- Other strand is synthesized in Okazaki fragments
What are Okazaki fragments?
Short fragments of DNA that form the lagging strand during DNA synthesis
What is the leading strand? What is the lagging strand?
- Leading: synthesized continuously in the 5′ to 3′ direction
- Lagging: synthesized discontinuously; generates Okazaki fragments
What is discontinuous synthesis?
Mechanism used by cells to synthesize the lagging strand during DNA replication
How was discontinuous synthesis discovered?
Pulse-chase experiment
- Identify the most recently synthesized DNA during the replication process
Explain pulse-labeling and pulse-chase.
Pulse-labeling (DNA example):
- Provide replicating cells with a small amount of 3H-thymidine for a short period of time (hence called “pulse”)
- Isolate and separate biomolecules
Pulse-chase (protein example:
- Pulse with radioactive [S35]-Met
- Chase with cold or non-radioactive Met
What is DNA polymerase?
Group of enzymes that catalyze the synthesis of new DNA molecules
What does DNA polymerase need in order to bind to ssDNA and start synthesis?
Primer
What is a primer?
Provides the free 3′-OH needed for DNA polymerase to initiate synthesis
- RNA primers must later be removed and replaced with DNA to complete the replication process
How does DNA polymerase facilitate correct Watson-Crick base pairing?
Shape of the DNA polymerase active site will only fit the correct pairs
What is processivity?
Average number of nucleotides added before a polymerase dissociates
- DNA polymerase has a very high processivity
What are the 3 major DNA polymerases in prokaryotes?
Pol I, II, and III
What are the major functions of Pol I?
Proofreading, repair, primer removal
What are the major functions of Pol III?
Main polymerizing enzyme
What are the major functions of Pol II?
Repair
What is the Klenow fragment?
Fragment of Pol I that contains 3’ –> 5’ exonuclease activities
- 68 kDa
What is the β clamp?
DNA replication fork protein that facilitates Pol III attachment to DNA
- Increases processivity
What is a replisome?
The complete complex of proteins and enzymes required to replicate DNA
What are the main classes of proteins in the replisome?
- Helicases
- Topoisomerases (gyrases)
- SSB: Single Stranded DNA Binding proteins
- Primases
- DNA ligases
What is the function of helicase?
Unwinds DNA ahead of DNA polymerase at the replication fork
- Specific to the reaction they catalyze
- 6 super families
- Involved in DNA repair and replication
- ATP-dependent
What is the function of topoisomerase (gyrase)?
Relieves the torsional strain generated by helicase as it unwinds the DNA double helix
- Induces negative supercoiling or relaxes positive supercoils
What is the function of SSB?
Prevents reannealing of dsDNA until DNA synthesis is complete
What is the function of primase?
Synthesizes RNA primers needed for discontinuous DNA synthesis
What is the function of ligase?
Seals nicks
What is the structure of the replication fork?
What happens after the replication fork is formed?
- dsDNA is converted into ssDNA.
- RNA primer is added
- RNA primer is extended
Explain the trombone model of DNA synthesis.
The lagging strand DNA template is alternately bound and released by the Pol III core complex as each Okazaki fragment is synthesized
- Complex to visualize the coordinated DNA synthesis on the leading and lagging strand templates by two Pol III core complexes
- This coordinated synthesis is mediated by the clamp loader complex
How does termination of DNA synthesis result in 2 identical daughter DNA?
DNA synthesis is initiated at oriC and proceeds bidirectionally until the DNA replication forks each reach halfway around the genome at a sequence called the termination region
What is the function of reverse transcriptase?
Converts RNA to DNA
- Used in replication of HIV (RNA virus) replication
- Highly prone to error (responsible for hypermutability of the virus)
- No proofreading function
- Similar structure to DNA Pol I
- Also has the ability to cleave the RNA genome
What is the function of telomerase?
Adds telomeric DNA sequences to the ends of chromosomal DNA
- Limited to undifferentiated embryonic stem cells, male germ cells, and activated lymphocytes in humans
How does telomerase affect aging?
- Decreased levels of telomerase are associated with telomere shortening
- Once the telomeres have reached a minimum size, the cells stop dividing and become senescent
- Telomere shortening is thought to play a role in aging
What are the 10 types of DNA damage in eukaryotic cells?
What are somatic mutations?
Changes to the DNA sequence within the genome of a cell other than a germ cell
- Not genetically inherited
- Result in decreased cell viability
- Some somatic mutations can alter genes that are regulators of cell division, which leads to uncontrolled cell growth
Explain mismatch repair.
Mismatched base pairs are removed and replaced by the correct base pair
- MutH (eukaryotes)
- MutS and MutL (prokaryotes; ATP dependent)
Explain base excision repair.
Removes damaged bases creating an apurinic site, which is followed by repair synthesis
Explain nucleotide excision repair.
Removes damaged bases and then repairs the region with a DNA repair replication process
Explain BRCA mutations.
BRCA = Breast cancer susceptibility proteins
- BRCA1 and BRCA2
- Tumor suppressors found in many cells
- Mutations to their genes give rise to breast, ovarian, colon, and pancreatic cancer
Individuals with a mutation in either BRCA1 or BRCA2 genes have a 55% to 80% chance of developing breast or ovarian cancer by the age of 70
- Due to a defective DNA repair mechanism
