DNA Replication And Repair Flashcards
(68 cards)
1
Q
Replication of DNA manner
A
- semi-conservative
- each strand serves as a template for a new strand
2
Q
Meselson and Stahl experiment
A
- Bacterial DNA was labeled with heavy isotope 15 N as source for nitrogen for several generations
- hybrid DNA of 14 N and 15 N was observed leading to the semi-conservative consensus
3
Q
Origin of replication
A
- point of initiation of DNA replication
- eukaryotic chromosomes have 1000-2000 separate origins of replication per chromosome
4
Q
Bidirectional replication
A
- two replication forks (sites where DNA replication will occur) proceed in opposite directions from the origin of replication
- evidence for bidirectional replication is the theta structure of radioactively labeled DNA (E. Cole chromosome) during replication
5
Q
DNA replication direction
A
-proceeds only in 5’ to 3’ direction
6
Q
Primers
A
- DNA replication is initiated from pre-existing primers
- they are short sections of RNA which are complementary to the template strand and contains a free 3’ OH group
7
Q
Leading strand replication
A
-is continuous (the new DNA is synthesized uninterruptedly from a single RNA primer)
8
Q
Lagging strand replication
A
- proceeds from multiple primers and results in forming short DNA sequences which are eventually joined
- synthesis is discontinuous
9
Q
Supercoiling
A
-DNA molecules during replication creates torsional strain which must be removed for replication to proceed
10
Q
DNA polymerase catalyze what rxn?
A
(DNMP)n +dNTP—>(dNMP)n+1 +PPi—>2 Pi
-only deoxyribonucleotide triphosphates can serve as substrates
11
Q
Template and primers of pro and eukaryotic cells
A
-both strands of the parent DNA molecule serves as template and small fragments of RNA serves as primers
12
Q
RNA polymerase (primase)
A
- necessary for the synthesis of the RNA primers required for DNA replication
- requires a free 3’OH terminal from which to start
- adds an oligonucleotide from 10-60 bases to serve as primers
- more primers required for lagging strands
13
Q
Okazaki fragments
A
- shoot sections of primer RNA plus DNA which forms on the lagging strand
- humans have shorter Okazaki fragments than bacteria
14
Q
Helicases
A
- carry out unwinding of DNA
- bind to ss-DNA and require ATP which is hydrolyzed in order to drive enzyme function
15
Q
Helicases in E. coli
A
-rep protein and the proteins dnaB+dnaC which are part of the replisome
16
Q
Effects of supercoiling
A
- closed loops of DNA can increase or decrease torsional strain on the molecule by varying the amount of supercoiling
- supercoils are introduced into DNA when a closed circular duplex is twisted around a central axis
- unwinding of DNA creates positive supercoiling which must be removed by topoisomerases
17
Q
Topoisomerases
A
- catalyze interconversion of different topological isomers of DNA
- relieve tension ahead of the replication fork which is introduced via unwinding of the DNA strands
18
Q
Type I DNA topoisomerase
A
- makes a break or nick in one strand of DNA helix and passes the other strand through the break to relax the supercoil
- break is then resealed by the enzyme
- does not require ATP
19
Q
Type II topoisomerases
A
- aka DNA gyrase
- produce an enzyme-bridged break in both strands of DNA
- another region of duplex DNA is passed through the gap by the enzyme, thus two supercoils are removed in one step
- requires ATP
- break is then rejoined
20
Q
Topoisomerase II and drugs
A
- important anti-cancer drugs such as adriamycin and etoposide
- Ciprofloxacin (Cipro) is a widely used antibiotic which is active against gyrase
- antifungal, antiparasitic and antiviral agents are being directed at topoisomerases
- targets gram + and - , little resistance
21
Q
Single stranded binding proteins (SSB)
A
- keep the separated strands as single strands
- displaced and reused during replication
- affinity for ss is 1000x greater than ds
- proteins bound to ss-DNA would be protected from ss specific nucleases
22
Q
DNA polymerase III and DNA polymerase I
A
- major enzyme involved in DNA replication, responsiable to growing DNA strand until 5’ ribonucleotide of the primer of the previously synthesized precursor fragments is reached and can go no further
- DNA polymerase I takes over since it acts as an exonuclease and removes the RNA primer as it lays down deoxyribonucleotides in the same place
23
Q
DNA polymerase III is processive
A
- once DNA polymerase III is bound to the template it probably never dissociates until the entire chromosome has been replicated
- DNA polymerase is active as a holoenzyme composed of 7 subunits
24
Q
How many DNA polymerase function at a time?
A
-two DNA polymerase molecules function at each replication fork, this 4 in a replication bubble
25
Enzymatic activities of DNA polymerase I
1) it possesses a 5'->3' exonuclease activity and starts cutting out the RNA primer one nucleotide at a time
2) as it removes primer is fills in with the dNTP matching the exposed DNA template
3) possesses a 3'->5' exonuclease activity whose main function is editing or proofreading (recognizes improper hydrogen bonding), since it will cleave off any unpaired 3' terminal nucleotide
26
Replication error rate
- wrong base is incorporated about once in 10,000 elongation steps (10-4 error rate)
- error rate of exonuclease activity of DNA polymerase I is about 10-3
- combined error rate is 10-7, once in every 10 million bases the wrong one will end up being incorporated into to the new DNA molecule
27
DNA Ligase
- seals the nick b/t the fragments of newly synthesized DNA (necessary for both DNA replication and DNA repair)
- E. coli ligase requires NAD for activity, eukaryotes require ATP
28
DNA ligase catalyze?
- synthesis of a phosphodiester bond b/t a 3' OH and a 5' phosphate group, as long as both groups are termini of adjacent-base paired deoxynucleotides
- enzyme cannot bridge a gap (the enzyme cannot fill in a missing nucleotide)
29
Replisome
-large protein complex that carries out DNA replication, starting at the replication origin
30
DNA polymerase alpha
| Location and function
- nuclear
- synthesis and priming of lagging strand
* formation and extension of RNA primers
31
DNA polymerase beta
| Location and function?
- Nuclear
| - DNA repair
32
DNA polymerase gamma
| Location and function?
- mitochondrial
| - replicates mitochondrial DNA
33
DNA polymerase delta
| Location and function?
- nuclear
- synthesis of leading strand
- DNA polymerase III?
34
DNA polymerase epsilon
| Location and function?
- nuclear
| - DNA repair
35
How are histones dissociated from the nucleosome during DNA synthesis?
- weakened through acetylation and phosphorylation
| - makes the histones more negative and weakens their association with DNA
36
Number of Initiation sites in eukaryotes
- perhaps 2000 per chromosome rather than one
| - eukaryotes replicate at about 500-5000 base pairs per minute
37
Origin recognition complex (ORC)
- to activate an origin, ORC must bind at origins of replication sequences
- Helicase and SSB proteins prepare the region for insertion of DNA polymerase-primase
* licensing factor must also be bound near each origin to ensure the origin is used only once per cell cycle
38
PCNA (proliferating cell nuclear antigen) function?
- serves as a clamp which is assembled with DNA pol gamma and epsilon to ensure processivity.
- antibodies to PCNA are used clinically to examine the degree of cell proliferation in a tissue sample
39
Pol alpha and RNase H
- RNA primers are synthesized and extended by pol alpha
- RNA primers are removed by RNAse H (RNA primer is attached to DNA so will only remove RNA) and the gaps filled through further DNA synthesis
40
Old and New histones?
- old histone octamers are not disassembled
| - newly synthesized histone octamers associate with one branch only of the replication fork
41
How does a cell decide to begin DNA replication (in S-phase)?
-proteins called cyclins regulate key steps in the cell cycle, including initiation of DNA synthesis in S-phase
42
Cyclins
-control cyclin-dependent kinases (CDKs) at various times in the cell cycle
43
Cyclins A and E
-synthesized to control the onset of S-phase DNA synthesis by activating kinase CdK2
44
CdK2-cyclin E/A
-complex phosphorylates pRb (retinoblastoma protein) causing dissociation of hyperphosphorylated pRb to activate E2F transcription factor
45
E2F
-turns on many genes to activate DNA synthesis such as DNA pol alpha
46
Inhibitors of DNA replication role?
- block activation of cyclic-dependent kinases (CdKs)
| - ex. Radiation
47
Physical agents that can cause DNA damage
-high temperatures, radiation of different wavelengths but particularly short-wave (240-300nm) ultraviolet (UVB) and x-rays
48
Chemical agents that cause DNA damage?
-methylating agents, nitrous acid, nitrosamines, acridine dyes
49
How do DNA damaging agents act?
- by altering the structure of DNA and causing disruption or normal hydrogen bonding of complementary base pairs
- some cause breaks in phosphate backbone
- cause a spectrum of different damages to DNA
50
Altered bases DNA damage
-thymine dimer is best know example
2 types?
- pyramidine dimers
| - deamination
51
Pyrimidine dimers
Mechanism?
Consequences?
- covalent linkage of two polynucleotide chains of DNA
- ultraviolet radiation is a common cause of dimer formation
- hydrogen bonding of the thymine to their paired adenines is disrupted and results in inhibition of advance of the replication fork
52
Deamination
-chemically induced or spontaneous loss of an amino group results in conversion of cytosine to uracil or conversion of adenine to hypoxanthine
53
Depurination
- spontaneous loss of a purine
| - occurs at a rate of about 10,000 purines per day per cell->called an apurinic site in DNA
54
Strand breaks
| Ss and ds?
- ss- chemical and radiation (DNA ligase can repair)
| - ds- chemical-particularly anticancer drugs (more lethal than single strand breaks)
55
Photoreactivation
- mechanism only operates on pyrimidine dimers
| - E. coli enzyme which catalyzes photoreversal of pyrimidine dimers is called photolyase. (Does not exist in mammals)
56
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Excision repair (molecular scissors)
-what does it require?
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- repair pathway for removal of bulky chemical modifications of DNA and pyrimidine dimers
- requires: an endonuclease to nick the DNA (cut), an enzyme (polymerase) to replace the damaged section of DNA (patch) and a DNA ligase to form phosphodiester bond (seal)
57
When spontaneous deamination of cytosine to uracil, what is the enzyme?
- Uracil DNA glysocylase->hydrolyzes the bond between uracil and deoxyribose resulting in removal of uracil from the DNA
- results in a apyrimidinic site in the DNA and subsequent recognition by a specific endonuclease.
- small gap is made in the damaged strand by endonuclease which is repaired by DNA polymerase and ligase
58
SOS repair
- post replication repair
- is error prone
- last ditch effort
- induced in response to high DNA damage levels
59
Xeroderma pigmentosum (XP)
- increased sensitivity to sunlight
- due to defects in the repair of ultraviolet light-induced damage to the DNA
- eight different genetic loci of the disease have been identified
60
XP variants
- individuals who poses clinical symptoms of xeroderma pigmentosum but who have normal excision repair activity
- have mutations in repair DNA polymerase n
61
Xeroderma Pigmentosum
Sensitivity?
Cancer?
Symptoms?
- ultraviolet radiation
- skin carcinomas, melanomas
- skin and eye photosensitivity
62
Ataxia telangiectasia
Sensitivity?
Cancer?
Symptoms?
- Gamma radiation
- lymphomas
- ataxia, dilation of blood vessels in skin, chromosome
63
Fanconi's anemia
Sensitivity?
Cancer?
Symptoms?
- cross linking agents
- leukemias
- hypoplastic pancytopenia, congenital anomalies
64
Bloom's Syndrome
Sensitivity?
Cancer?
Symptoms?
- ultraviolet
- leukemia's
- photosensitivity
65
Cockayne's syndrome
Sensitivity?
Cancer?
Symptoms?
- ultraviolet
- various tumors
- neurological defects, dwarfism
66
Cockayne's Syndrome (CS)
Lacking?
Mutated protein?
- lack transcription helicases used in repair during gene transcription (known as transcription coupled repair)
- mutated CSB protein does not allow DNA damaged genes to be repaired during transcription (causes loss of mRNA production, more severe phenotype than XP)
67
Loss of DNA repair pathway
- may underlie tumor formation in HNPCC (hereditary nonpolyposis colorectal cancer)
- culprit genes are inc=valves in mismatch repair and mutation in these genes could predispose an individual to this type of cancer
* most commonly inherited genetic diseases and this genetic defect accounts for around 10% of colorectal cancer cases
68
Hereditary breast and ovarian cancers
- BRCA1 and BRCA2 genes are linked
| - code for recombination repair proteins linked to Fanconi's Anemia type
