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80 Cards in this Set
- Front
- Back
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What are the 4 steps of DNA replication?
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1. Separatn of complementary strands at the Origin.
2. Replication fork formation (via primers, Okazaki fragms) 3. Chain elongation 4. Removal of primers |
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What direction is DNA always synthesized in?
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5' -> 3'
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What 4 rules should I ALWAYS REMEMBER re: DNA replication?
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1. Synthesis is 5' -> 3'
2. Synth is semiconservative 3. Synth is semidiscontinuous 4. ALWAYS REQUIRES A PRIMER |
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What experiment demonstrated that DNA synthesis is semiconservative?
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Meselsohn-Stahl experiment incorporating heavy N to differentiate daughter strands
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What would conservative replication result in?
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1 heavy band, 1 light band
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What wouldn nonconservative replication result in?
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a broad band between heavy and light b/c DNA is 50/50 parent and daughter.
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Why is a 3'OH primer so vital in DNA replication?
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Because it's needed to attack the alpha phosphate of the incoming nucleotide.
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How many primers are needed
-On the leading strand? -On the lagging strand? |
Leading: only one
Lagging: numerous - the primers are called okazaki fragments. |
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What enzyme is responsible for creating RNA primers on the lagging strand?
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Primase
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What enzyme elongates the RNA primers?
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DNA polymerase III
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What enzyme separates the strands of DNA for replication?
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Helicase
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What do ssbp's do?
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Single stranded binding proteins - keep the strands from reannealing
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What enzyme removes RNA primers from the lagging strand?
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DNA Pol I - it's 5'->3' exonuclease activity
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What enzyme seals the nicks between okazaki fragments?
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Ligase
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What is a major difference between Prok/Euk DNA replication?
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Prok: only one replication origin
Euk: multip origins exist |
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What is the function of DNA gyrase in DNA replication?
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Introduction of negative supercoils to neutralize the torque caused by helicase unwinding - ONLY IN PROK
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What is PROCESSIVITY?
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The # of catalytic events by DNA Pol III before dissn from the template.
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Which DNA polymerase is more processive?
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DNA Pol III - synthesizes >500,000 nt before dissn; DNA Pol I only does 3-200
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Which DNA Pol has more exonuclease activity?
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DNA Pol 1 - because it has to remove the primers of okazaki fragments.
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Why doesn't DNA Pol III have 5'-3' exonuclease activity?
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It doesn't have to excise RNA primers. DNA polymerization is continuous.
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What is the 3'-5' exonuclease activity of DNA Pols I and III for?
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Proofreading for mutations.
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What do cells have more (conc) of at any one time; DNA Pol I or III?
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DNA Pol I
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What would seperating the DNA Pol I into fragments do? What would each fragment be capable of doing?
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Create 2 fragments:
small: 5'-3' exonuclease large: proofreader 3'-5' exonuclease and polymerase S |
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Which subunit accounts for DNA Pol III's high processivity?
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the Beta Subunit - the CLAMP
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How does the Beta clamp of DNA Pol III interact with DNA?
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It is topologically bound to the DNA template, completely encircling it.
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What is necessary for the Beta subunit to clamp onto the DNA template during replication?
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ATP hydrolysis
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What problem is encountered at the end of DNA transcription?
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The end problem.
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What is the end problem?
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The fact that once DNA Pol 1 has removed the last primer, lagging strand is then that much shorter than leading.
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What solves the end problem?
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Telomerases
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What is the duty of telomerases?
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Synthesis of tandem repeats of GGGGTT.
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What type of enzymes are telomerases?
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Reverse transcriptases
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How are telomerases able to synthesize DNA w/out primers?
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They carry their own RNA template.
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Which strand does telomerase extend?
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The leading strand.
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So how is the end problem on the lagging strand solved?
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DNA synthesis occurs as normal, but it overlaps because the primase operates on the nongenomic extension of the leading strand.
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What are 3 ways in which DNA mutations can arise?
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1. Misincorp of nt's
2. Nonenzymatic (inherent) chemical instability of bases 3. Environmental agents (chemicals) |
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How can misincorporation of nt's just spontaneously occur?
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By tautomeric shifts that cause non-watson/crick base pairing.
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What does deamination of Cytosine result in?
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Formation of uracil.
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How is mutation from cytosine deamination classified?
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As a non-enzymatic spontaneous chemical instability.
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What is another mutation in this category? Which is more common?
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-Productn of apurinic residues by N-glycosidic bond hydrolysis.
-This is more common; 5000 bp/day mutate. -Only 100bp/day mutate by cytosine deamination. |
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What causes chemically induced deamination of bases?
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Nitrous acid precursors:
-Sodium nitrate -Sodium nitrite -Nitrosamine |
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How many chemically induced mutations via nitrous acid precursors occur p. day?
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100/day
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What are the 4 common environmentally induced mutations?
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1. Deamination
2. Alkylation 3. Oxidation 4. Ionization (via radiation) |
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What are 4 common alkylating agents?
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1. Nitrogen mustards
2. Aflatoxin 3. Benzo(a)pyrene 4. AAF |
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How does alkylation occur?
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Via methylation of the O6 on guanine.
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What does 06-methylguanine base pair with instead of cytosine?
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Thymine.
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How do O6-methylguanosine mutations get fixed into the genome?
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If not fixed immediately, subsequent replication cycles will incorporate the thymine which bp's instead of cytosine.
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How does UV light cause gene mutations?
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It causes pyrimidines to form dimers - thymine dimers.
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How do thymine dimers affect DNA?
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It makes them form kinks.
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What are Intercalating agents?
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Agents that cause insertion or deletion of base pairs, resulting in frameshift mutation.
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Name 2 intercalating agents and descrie their general structure:
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Acridines, ethidium bromide.
-Planar/flat aromatic molecules. |
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What are the 2 names for mutations that occur?
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-Transition
-Transversion |
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What is a transition?
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Substitution of one purine or pyrimidine for another of the same.
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What is a transversion?
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Conversion of purine to pyrimidine or vice versa.
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What test detects mutagens?
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The ames test.
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How is it that when DNA is newly replicated and mutation occurs, mismatch repair takes place on the correct strand?
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Old DNA template is methylated, and there's a window where the new daughter strand is not. Allows for differentiation.
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Describe DNA mismatch repair.
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Nonmethylated DNA is nicked when mismatch is detected by DNA Pol III; exonuclease cleaves new strand, and DNA Pol III fills in the correct bases.
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What are the 3 components of the mismatch repair mchinery?
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-DNA Helicase II
-Exonuclease -SSB |
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Where on DNA does methylation occur?
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At N6 of adenines in 5'-3' GATC sequences.
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Review; what does deamination of cytosine form?
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URacil
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Is uracil normally in DNA?
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No; so it's recognized as foreign by DNA glycosylase.
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How does DNA glycosylase respond to uracil present in DNA?
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It hydrolyzes the glycosidic bond, cleaving the dmgd base.
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What results from DNA glycosylase cleaving a base?
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An apurinic or apyridimic site is created, and recognized then by an endonuclease.
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How does repair of the base excision site occur (3 steps)?
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-Endonuclease AP creates nick
-DNA Pol 1 uses 5'-3' exonuclease to remove damaged portion, replace w/ correct. -DNA ligase seals nick |
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Does base excision repair only occur via DNA glycosylases for deaminated cytosines?
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No; this was just one example of a specific glycosylase in the overall family.
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How does base-excision repair in humans compair to E. coli as we just looked at?
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Pretty similar.
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What is a disease in humans in which the machinery for base-excision repair is mutant?
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Xeroderma pigmentosum.
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What is XP?
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A rare skin disease characterized by extreme sensitivity to UV light.
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Why are patients with XP so sensitive to uv light?
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Because the base-excision repair for the thymine dimers that RESULT from uv light cannot occur.
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List 3 types of DNA lesions caused by ionizing radiation and oxidative reactions:
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-Double-stranded breaks
-Double-strand crosslinks -Lesions in single strands |
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What type of repair is used to fix the 3 previous types of damage?
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Recombinational Repair
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What is the general basis of recombinational repair?
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Repair occurs by using information from a homologous sister chromosome.
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List the 5 main phases of the cell cycle:
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M - Mitosis/cytokinesis yields 2 daughter cells.
G0 - terminally differentiated cells withdraw from cycle G1 - RNA and protein synthesis; no DNA synthesis. S - Synthesis of DNA, RNA, and protein. G2 - CRITICAL POINT in assessing DNA Replication completion. |
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So where does all the machinery for replication and regulation occur?
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S and G2
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What is the G1-S restriction point?
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A critical step where specific signals are monitored by checkpoint proteins before actual replication will proceed.
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How do errors at these critical checkpoints cause cancer?
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If checkpoint proteins are mutated, DNA errors will PROPAGATE inst. of being held in check.
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What specifically controls checkpoint pathways?
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Tumor suppressors, aka recessive oncogenes.
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Why are tumor SUPPRESSORS also called recessive oncogenes?
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Because tumor formation generally requires loss of BOTH copies of the tumor suppressor.
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What protein is the typical tumor suppressor protein we always hear about?
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p53
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What does mutated p53 cause?
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Accumulation of chromosomal lesions.
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What disease is caused by mutations in the p53 gene?
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Li-Fraumeni syndrome - 90% develop cancer by age 70!
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