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DNA Replication, Repair, and Recombination
Terms in this set (73)
Survival of an organism depends on ________ _______________
change in DNA sequence due to a mistake when the DNA is copied or as a result of environmental factors
_____________ is the source of all variation.
What does short term survival depend on? What does long term survival depend on?
Short term - preventing changes in DNA
Long term - requires that DNA be changeable over many generations to allow for evolutionary adaptation
Mutations are always bad (T/F). Why?
False - necessary for long term survival
What causes sickle cell disease? What is this an example of?
Due to a mutation in Hb, ß-subunit. Can cause polymerization and cause RBC to form a sickle cell shape to cause an occlusion. This helps patients have resistance to malaria. An example of long term survival.
What are germ cells?
cells that transmit genetic information from parent to offspring
What are somatic cells?
cells that form the body of the organism
What is the mutation rate permissible for life?
1 mutation in 10 billion nucleotides per cell division
Name and describe the experiment that proved DNA replication was semi-conservative. What was the technique they used?
Meselson and Stahl 1958
Grew bacteria in heavy Nitrogen (15N) and centrifuged it from bacteria. They removed the heavy nitrogen and added light nitrogen (14N) and only let one proliferation happen (one generation to occur). They saw that they ultimately had one band in the middle and cancelled out the conservative hypothesis. Allowed one more replication cycle to occur - saw a light band and an intermediate band. This proved the semiconservative model.
Used the equilibrium density gradient using cesium chloride to separate different sized DNA strands. DNA will form a band where the buoyant density is equal to the CsCl solution.
What is the name of the enzyme that catalyzes the addition of a deoxyribonucleotide to the 3'-OH group on the end of a polynucleotide chin?
What is the name of the newly synthesized strand?
What needs to happen to propel the reaction of DNA synthesis?
coupling of an energetically favorable reaction: hydrolysis of a triphosphate
Where does DNA replication occur?
origin of replication
What (and how) unwinds the DNA helix at the replication fork? Which direction does it work in?
Works by being propelled by the hydrolysis of ATP - binds to a single stranded DNA and when encounters a double helix, pry it open
Two types - work in both directions 5' -> 3' and 3' -> 5'
What else helps unwind the double helix? What is their specific role?
Single-stranded DNA binding proteins (helix destabilizing proteins)
stabilize the unwound single stranded conformation, prevent the formation of short hairpin helices. they demonstrate cooperative binding. Allow for the DNA bases to remain exposed in this protein-DNA complex for DNA polymerase to work. Binds to the sugar-phosphate backbone.
The ___________ _______________ has an asymmetric structure.
Due to polarity of leading versus lagging strand
Okazaki fragments are produced in the ______________ strand.
What is DNA primase and what does it do?
Adds 10 complementary ribonucleotides (primer) onto the lagging strand template so that it has a 3'-OH for DNA polymerase to add nucleotides. Synthesis of the Okazaki fragments is marked by the RNA primer then the RNA is removed and replaced with RNA.
Why is RNA used in the primer sequence instead of DNA for synthesis of Okazaki fragments?
This is because any enzyme that primes the synthesis of Okazaki fragments will make a relative inaccurate copy. Using RNA rather than DNA allows ribo-nucleotides in the primer to automatically mark these sequences as "suspect copy" to be efficiently removed and replaced by proofreading mechanisms.
There is also limited availability of DNA to be used as primers.
What is the enzyme that pastes the Okazaki fragments together and replaces the RNA primer? Is this a -∆G? How many ATP are involved?
Not a -∆G rxn, has to be coupled with hydrolysis of 1 ATP molecule thus making this an overall, favorable reaction.
Describe how DNA is replicated by semi-conservative templated polymerization.
Happens at replication forks at origin of replication, DNA helicase unwinds DNA at origin of replication. DNA is unwound by helicase (in both the 5'à3' and 3'à5' direction) and unwound DNA is stabilized by the SSB proteins (helix-destabilizing proteins). DNA polymerase then begins to synthesize the new DNA strand by adding them in a 5' à 3' direction. There is both lagging and a leading strand. In the lagging strand, Okazaki fragments are made with the use of DNA primase that uses short RNA molecules (RNA primers) for DNA polymerase to add nucleotides to. DNA ligase pastes the Okazaki fragments together.
What is PCNA?
PCNA (Proliferating Cell Nuclear Antigen) or sliding clamp that "clamps" DNA polymerase to DNA when moving but releases it when it runs into a double stranded region.
What is the clamp loader? What is the mechanism?
assembles PCNA around the DNA by ATP hydrolysis.
1. the clamp loader binds to a free clamp molecule
2. recognizes the DNA region that is double stranded
3. tightens the clamp through ATP hydrolysis and locks PCNA into place, clamp loader is released
4. DNA polymerase binds to sliding clamp
sliding clamp acts as a _________ and clamp loader acts as a _____________.
tether ; lock
What are the roles of topoisomerases?
they relieve tension in front of the replication fork and solves the DNA tangling problem
What does topoisomerase I do? describe mechanism.
produces a transient single stranded break on the phosphodiester backbone. this allows for two sections of DNA to rotate freely relative to each other. Uses the phosphodiester bond across the nick to work as a swivel point. Nick is rapidly removed after tension is relieved.
What does topoisomerase II do? describe mechanism.
produces a transient double stranded break and relieves tension in the case of supercoiling (two helices or more wrapped around each other).
1. makes a reversible covalent attachment to the two opposite strands of one of the double helices creating a double stranded break - forming a DNA gate
2. causes the second nearby double helix to pass through
3. reseals the break and dissociates from the DNA
needs to hydrolyze ATP to release and reset the enzyme after each cycle.
What is the first mechanism in DNA polymerase proofreading?
5' -> 3' polymerization Double-checking proof reading mechanism.
Right before a new nucleotide is covalently added, DNA polymerase undergoes a conformational change tightening around the newly added nucleotide. If the incorrect base pairing occurs, the conformational change takes longer and the incorrectly paired nucleotide has more time to diffuse away.
What is the second mechanism in DNA polymerase proofreading?
3' -> 5' exonucleolytic proofreading
Takes place immediately after incorporation fo an incorrect nucleotide. An exonuclease region of the DNA polymerase removes (clips off) the incorrect 3'-OH end. Improper 3'-OH from the wrong nucleotide are not as effective as templates to add from DNA polymerase.
What is the third mechanism in DNA polymerase proofreading?
Strand directed mismatch pair
MutS detects distortion of the DNA helix from misfit non complementary base (looks for a kink)
MutL will scan the strand for a nick (only found in new strand) and once it is found, this triggers degradation and removes nucleotides all the way up until the mismatch (kink).
Which one needs the use of ATP hydrolysis - topoisomerase I or II?
What are the two active sites on DNA polymerase?
1. Polymerase domain - adding more bases to the primer strand. Double checking proofreading mechanism.
2. Exonuclease domain - allows for an incorrect nucleotide to be removed right after incorporation.
How does the eukaryotic cell distinguish the old strand from the newly synthesized one during strand-directed mismatch repair proofreading?
Only the newly synthesized strands will have a nick
DNA synthesis begins at ______ _______ that attract the help of special ___________ ____________
replication origins; initiator proteins
How do would you identify an origin or replication in bacteria?
Regions rich with Adenine and Thymines because they have only 2 H-bonds connecting the bases
What is the mechanism in which E. coli controls DNA replication? Where does it control replication?
Controls replication at initiation and this is a highly regulated step in DNA replication.
1. initiator proteins bind in multiple copies to specific DNA sites at replication origin - wrapping DNA around the proteins to form a large protein-DNA complex that destabilizes the adjacent double helix
2. this complex attracts two DNA helicases each bound to a helicase loader and placed wound adjacent DNA single strands whose bases have been exposed by assembly of initiatory protein-DNA complex
3. once helicases are loaded, the loaders dissociate and the helicases begin to unwind the DNA exposing enough single stranded DNA for DNA primase to synthesize the first RNA primers
4. continue to synthesize DNA until all of the DNA template downstream of fork is replicated
Eukaryotic origins of replication contain (three things)?
1. binding site for a large multi-subunit initiator protein (ORC)
2. stretch of DNA that is able to be "melted" (A-T rich)
3. a binding site for proteins that facilitate ORC binding by adjusting chromatin structure
Eukaryotic organisms only have one origin of replication (T/F). Why is this?
False - they have approximately 30,000-50,000.
Different cell types use different sets of origins - allows to control which genes are being expressed
Describe the experiment that was done in 1960s using 3H-Thymidine
Human cells growing in culture with highly radioactive thymidine was used to label DNA that was synthesized.
The cells were then gently lysed and the DNA was stretched on the surface of a glass slide coated with photographic emulsion.
Development of emulsion reveals the pattern of labeled DNA through autoradiography.
Emulsion revealed that there were multiple replication forks thus, multiple origin of replication sites. They were also able to quantify how fast replication was taking place which was 50 nucleotides per second.
In eukaryotes, DNA is only replicated in the _____________. Heterochromatin is replicated _________ in the _________. Euchromatin is replicated _________ in the _________.
In eukaryotes, DNA is only replicated in the S phase. Heterochromatin is replicated early in the S phase. Euchromatin is replicated late in the S phase.
How does our cell ensure that an ORC only gets used once during cell cycle?
During G1 phase, a pre-replicative complex forms and this consists of replicative helicases that is placed next to the ORC (ORC + helicase = pre-replicative helicase) G1 -> S phase
At the beginning of S phase, kinase phosphorylates the helicase and ORC
- phosphorylation of helicase = activates
- phosphorylation of ORC = deactivates (unable to accept another helicase)
Activated helicase displaces the inactivated ORC (falls off)
A new prereplicative complex cannot form until the next G1 phase which allows time for ORC to be dephosphorylated
Chromosomal duplication requires not only the replication of DNA but also ......
assembly of new chromosomal proteins onto the DNA behind the replication fork
- large amount of histone protein - to make new nucleosomes in each cell)
Histones are synthesized continuously (T/F)
False - mainly made in the DS phase when the level of histone mRNA increases fifty fold
What complex destabilizes the DNA-histone interactions and allows DNA polymerase to replicate DNA wound tightly by histones?
chromatin remodeling complex - can remove histones so that DNA replication can occur. This aids the replication fork to go through the nucleosome.
Describe the production of new histones.
1. chromatin remodeling complex transiently displaces the histones into an H3-H4 tetramer and H2A-H2B tetramer (breaks apart dimers ahead of replication fork)
2. The H2A-H2B tetramer is released completely while the H3-H4 tetramer remains loosely attached to the DNA and is distributed randomly to one of the daughter strands.
3. Newly made H3-H4 tetramers bind to the other DNA strand
4. Old and new H2A-H2B tetramers bind to the H3-H4 DNA bound tetramers
Histone chaperones facilitate this process by helping rebuild the nucleosome histone back into an octamer. (NAP1 and CAF1)
Histone chaperones are directed to newly replicated NDA through interaction with the eukaryotic sliding clamp PCNA. These clamps are left behind moving replication forks and remain on the DNA long enough for the histone chaperons to complete their tasks.
Histone chaperones are also known as ...
Chromatin assembly factors
NAP1 and CAF1
Histone chaperones are recruited by ______
PCNA - eukaryotic sliding clamp
Telomere sequences are recognized by __________ that attract _______ which...
Telomere sequences are recognized by specific DNA binding proteins that attract telomerase which replenishes these sequences each time a cell divides
Describe how telomerase works and what enzyme it is similar to.
Telomerase replenishes telomere DNA sequence by recognizing the tip of an existing telomere DNA repeat sequence and elongating it in the 5' to 3' direction using an RNA template that is a component of the enzyme itself to synthesize new copies of the repeat.
The enzymatic portion of telomerase is similar to reverse transcriptase (proteins that synthesize DNA using an RNA template) however telomerase RNA also contributes functional groups to make catalysis more efficient.
What component is used to extend the incomplete daughter strand at the chromosome end after extension of the parental strand by telomerase?
DNA polymerase alpha
This type of polymerase carries a DNA primase as one of its subunits so it does not need to recruit DNA primase for the mechanism.
Telomerase is fully active in our cells (T/F).
In cancer, it is fully ON
Name the two mechanisms that prevent the repair of telomere regions?
A special nuclease chews back the 5' end of a telomere and attracts a group of proteins that acts as a protective chromosome cap to hide telomeres from cell's damage detectors that continuously monitor DNA.
Where the protruding telomere loops back and tucks itself into the duplex DNA of the telomere repeat sequence. T-loops are regulated by shelterin and provide additional protection for the ends of telomeres.
What happens to our telomeres with age? What is replicative cell senescence?
Telomerase is not active in every cell of our bodies thus overall, our telomeres shorten with each cell division. This regulates the lifetime of a cell lineage.
After many cell generations, descendant cells that lack telomeres will activate a DNA-damage response and cause cells to stop dividing. This is known as replicative cell senescence. This prevents the cells from proliferating uncontrollable and protects us from cancer.
What are two cell types that retain full telomerase activity besides cancer cells?
hematopoietic (bone marrow) stem cells and gastrointestinal stem cells (gut lining
What is dyskeratosis congenita? Include symptoms
Is a disease in which individuals have one functional and one nonfunctional telomerase gene.
1. shortened telomeres
2. progressive bone marrow failure
3. epidermal abnormalities of the skin, hair follicles, and nails
Can an individual survive with two non functional telomerase genes?
No this is lethal.
An individual can survive with only one non-functional telomerase gene but will have severe complication - dyskeratosis congenita.
Name and describe three ways in which DNA can spontaneously become damaged.
1. oxidative - happens in mitochondria making ATP
2. hydrolytic - when exposed to chemicals in environment
What is depurination?
hydrolysis of N-glycosyl linkages to deoxyribose which releases guanosine or adenosine from the backbone.
Loose A or G
What is deamination?
converts C to U (removed the amino group and replaces it with a carbonyl)
What are thymine dimers? Why do they occur?
covalent linkages between two adjacent pyrimidine (T or C) to form dimers (i.e. Thymine dimers).
occurs by UV radiation from the sun
Out of depurination, deamination, and thymine dimers - which are the most common form of DNA damage?
What are the two most common pathways in DNA damage repair?
1. base excision repair
2. nucleotide excision repair
What allows for the DNA helix to be readily repaired?
due to double helical structure of DNA has two separate copies of all the genetic information.
when one strand is damaged, the complementary strand retains an intact copy of the same information
Describe mechanism of base excision repair (what enzymes are involved?)
involves DNA glycosylases each of which can recognize a specific type of altered bases in DNA and catalyze its hydrolytic removal
key step: enzyme-mediated "flipping out" of the altered nucleotide from the helix which allows the DNA glycosylase to probe all faces of the bases for damage
then, the missing tooth created by DNA glycosylase is recognized by an enzyme AP endonuclease which cuts the phosphodiester backbone along with the help of a phosphodiesterase (cleaves nucleotides.
DNA polymerase repairs the helix and adds correct nucleotides, DNA ligase seals the nick.
DNA glycosylase - recognize
AP endonuclease and phosphodiesterase - remove
DNA polymerase and ligase - repair
Describe mechanism of nucleotide excision repair.
repair damage caused by any large change in DNA "bulky lesions"
1. Large multi-enzyme complex scans DNA for a distortion in double helix (rather than only a specific base change)
2. Once a lesion is found, it cleaves the phosphodiester backbone of the abnormal strand on both sides of the distortion.
3. DNA helicase peels away the single strand oligonucleotide containing the lesion
4. large gap produced in the DNA helix is repaired by DNA polymerase and DNA ligase
What is Cockayne Syndrome?
What are translesion polymerases and what are they used for?
How are double-stranded breaks repaired (name the two mechanisms)
1. non-homologous end joining
2. homologous recombination
How are bulky lesions made in DNA?
Lesions can be created by the covalent reaction of DNA bases with HC as well as the various pyrimidine dimers. Normally caused by methylation to cysteine or adenine.
DNA primase only adds an RNA primer to the lagging strand (T/F)
False - adds to both (adds it more to lagging)
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