Flashcards in 8.8.16 Lecture Deck (45):
What are the correct Watson-Crick base pairings for DNA?
Describe the structure of DNA.
DNA is a double-stranded helix. Each strand runs from the 5' to the 3' end. The strands are aligned in an anti-parallel fashion.
What are the names and roles of the two strands in DNA replication?
1. Template strand - strand used to create a copy
2. Primer strand - begins the process of DNA synthesis
DNA polymerase requires a ___.
What engages in a nucleophilic attack on the 3' end of the primer strand?
The 5' triphosphate of the incoming deoxyribonucleoside triphosphate.
DNA is always synthesized in the ___ direction.
5' to 3'
Describe the two strands formed in DNA synthesis.
1. Leading strand: continuous 5' to 3' synthesis
2. Lagging strand: Okazaki fragments in the 5' to 3' direction
DNA replication is bi-directional. Describe this process.
1. Replication begins at an origin of replication.
2. The DNA helix is opened locally.
3. RNA primer is synthesized on each strand.
4. Leading-strand DNA synthesis begins.
5. RNA primers start lagging strand synthesis
In total, this leads to 2 forks from each origin - each with a leading strand and a lagging strand.
What are the four important characteristics governing nucleic acid synthesis?
1. Pre-existing nucleic acid strand is copied by the rules of Watson-Crick base pairing
2. Nucleic acid strands grow in the 5' to 3' direction only
3. Polymerases synthesize nucleic acids.
4. Duplex DNA synthesis requires a special growing fork because the strands are antiparallel.
What are the six steps of DNA replication?
4. Unidirectional fork movement
Where does initiation begin?
At origins of replication
Describe the process of initiation of DNA replication.
1. Initiator proteins bind to the origin and destabilize the AT-rich sequence. These proteins bind to and bend the DNA to loosen the double strand tightness.
2. DNA helicase binds to the helicase-loading protein and is loaded onto the DNA. When activated, helicases unzip the protein.
3. DNA primase is loaded (synthesizes RNA)
4. RNA primer synthesis enables DNA polymerase to start leading-strand synthesis.
5. Loading of 2 additional DNA polymerases allows lagging-strand synthesis to begin.
6. This process results in two replication forks moving in opposite directions.
___ is an allosteric motor protein that unwinds DNA.
How much energy is needed for helicase to unzip one nucleotide?
1 molecule of ATP
What do single-strand binding proteins (SSBs) do?
SSBs prevent reannealing without preventing base pairing.
What happens without SSBs?
Single-stranded regions of DNA template can pair with one another, blocking replication.
SSBs bind ___ and straighten the backbone of the DNA chain while leaving the bases open and accessible.
DNA synthesis requires an ___.
What does the sliding clamp do?
A regulated sliding clamp permits processive DNA synthesis on the leading strand and rapid reassembly of the replication complex on the lagging strand.
How does the sliding clamp function?
1. A clamp loader binds to the sliding clamp and opens it using ATP.
2. The sliding clamp slips around the DNA.
3. ATP hydrolysis locks the sliding clamp around DNA and releases the clamp loader.
4. DNA polymerase binds to the sliding clamp.
Describe the process of addition and removal of RNA primer.
1. DNA primase synthesizes a new RNA primer.
2. DNA polymerase adds to the new RNA primer to start a new Okazaki fragment.
3. DNA polymerase finishes the DNA fragment.
4. DNA polymerase bumps into the old primer, activating RNAse, which erases the primer and replaces it with DNA.
5. DNA ligase seals the nick and joins the new fragment to the growing chain.
What does ligase do?
Ligase synthesizes the phosphodiester bond that links Okazaki fragments.
How does ligase work?
Ligase uses ATP to add a phosphate group to the 5' P. The 3' OH bonds to this new P. AMP is released.
DNA polymerase ___ as it synthesizes DNA.
How does DNA polymerase proofread as it synthesizes DNA?
3' to 5' exonuclease activity attached to DNA polymerase chews back to create a base-paired 3'-OH end on the primer strand.
What three things give rise to high-fidelity DNA synthesis?
1. 5' to 3' polymerization
2. 3' to 5' exonucleolytic proofreading
3. Strand-directed mismatch repair
Germline mutations affecting the ___ proofreading domains predispose people to colorectal adenomas and carcinomas.
Which strand is more dependent on SSD binding proteins?
Why does the lagging strand have to fold back?
The replication proteins act together as one large multienzyme complex.
What are the two major winding problems that occur during DNA replication?
Torsional stress and superhelicity
What reliever supercoils and untangles DNA during replication?
What does DNA topoisomerase I do?
Relieves supercoiling ahead of the replication fork
How does DNA topoisomerase I function?
1. DNA topoisomerase I has tyrosine at its active site. It covalently attaches to a DNA phosphate, breaking a phosphodiester linkage in one DNA strand.
2. The two ends of the DNA double helix can now rotate relative to each other, relieving accumulated strain.
3. The original phosphodiester bond energy is stored in the phophotyrosine linkage, making the reaction reversible.
4. Spontaneous reformation of the phosphodiester bond regenerates both the DNA helix and the DNA topoisomerase.
What does DNA topoisomerase II do?
Untangles DNA after it is replicated.
How does DNA topoisomerase II function?
1. Two circular DNA double helices are interlocked. Type II DNA topoisomerase makes a reversible covalent attachment to opposite strands, interrupting one helix and forming a protein gate.
3. The topoisomerase gate opens and shuts to let a second helix pass.
4. Reversal of the covalent attachment of the topoisomerase restores an intact double helix.
Why are DNA topoisomerase II inhibitors effective cancer drugs?
Increase stability of topo-DNA cleavage complexes, leading to high levels of transient DNA breaks. These become permanent when replication machinery or helicases attempt to traverse the DNA. This leads to cell death.
What is the mechanism that ensures each origin of replication is activated only once per cycle?
1. In G1, origin recognition complexes (ORCs) bind to the origin, along with the helicase to form a pre-replicative complex.
2. In S, the helicase and ORC are phosphorylated by kinases, leading to displacement of ORC and recruitment of DNA polymerase and other replication protein. The ORC rebinds and synthesis begins.
3. In G2, the replication is completed.
Describe how patterns of histone modification can be inherited.
H3-H4 tetramers are distributed to the daughter DNA randomly, with roughly equal numbers inherited by each daughter. H2A-H2B dimers are released from the DNA as the replication fork passes. Reader-writer complexes recognize the same modifications they catalyze and fill in the blanks.
What are the three major ways eukaryotic chromosomal DNA replication differs from prokaryotic DNA replication?
1. Compartmentalized within the nucleus, partitioning it from the site of synthesis of replication proteins and precursors and from extracellular stimuli that may trigger initiation of synthesis.
2. Begins at multiple origins, activated throughout S-phase in a precise and temporally regulated manner
3. The nucleosomal proteins must be duplicated along with the DNA to maintain proper chromosomal organization.
What is telomerase?
A riboprotein containing an RNA template for synthesizing the G-rich telomere sequence
Telomerase is primarily expressed in what three types of cells?
Germ, stem, and cancer cells
Why is telomerase needed?
The 3' end of the template DNA strand has no room for a primase to synthesize a primer.
What does telomerase do?
Elongates the 3' end
Telomere shortening can trigger...
...replicative senescence in human cells that don't express telomerase.