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80 Cards in this Set

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What are the 4 steps of DNA replication?
1. Separatn of complementary strands at the Origin.
2. Replication fork formation (via primers, Okazaki fragms)
3. Chain elongation
4. Removal of primers
What direction is DNA always synthesized in?
5' -> 3'
What 4 rules should I ALWAYS REMEMBER re: DNA replication?
1. Synthesis is 5' -> 3'
2. Synth is semiconservative
3. Synth is semidiscontinuous
4. ALWAYS REQUIRES A PRIMER
What experiment demonstrated that DNA synthesis is semiconservative?
Meselsohn-Stahl experiment incorporating heavy N to differentiate daughter strands
What would conservative replication result in?
1 heavy band, 1 light band
What wouldn nonconservative replication result in?
a broad band between heavy and light b/c DNA is 50/50 parent and daughter.
Why is a 3'OH primer so vital in DNA replication?
Because it's needed to attack the alpha phosphate of the incoming nucleotide.
How many primers are needed
-On the leading strand?
-On the lagging strand?
Leading: only one
Lagging: numerous - the primers are called okazaki fragments.
What enzyme is responsible for creating RNA primers on the lagging strand?
Primase
What enzyme elongates the RNA primers?
DNA polymerase III
What enzyme separates the strands of DNA for replication?
Helicase
What do ssbp's do?
Single stranded binding proteins - keep the strands from reannealing
What enzyme removes RNA primers from the lagging strand?
DNA Pol I - it's 5'->3' exonuclease activity
What enzyme seals the nicks between okazaki fragments?
Ligase
What is a major difference between Prok/Euk DNA replication?
Prok: only one replication origin

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