Chapter 22- DNA Replication, Repair, and Mutagenesis Flashcards
(106 cards)
1
Q
E.Coli DNA polymerase I has what activity?
A
3’ to 5’ exonuclease activity (proof reading and editing activity)
5’ to 3’ exonuclease activity (DNA repair)
5’ to 3’ polymerase activity (nick translation)
2
Q
What is nick translation?
A
Under conditions where displacement reaction does not occur the 5’ to 3’ exonuclease acts on the strand that would be displaced, removing one downstream nucleotide for each nucleotide added to the 3’ side of the nick. The polynucleotide of the nick moves along the strand
3
Q
The chief DNA-replicating enzyme of e.coli is?
A
DNA polymerase III
4
Q
E. Coli DNA polymerase III simplest form consists of what polypeptides?
A
alpha, epsilon, and theta
5
Q
What function does the alpha subunit of E.Coli DNA polymerase III have?
A
catalytic polymerase function
6
Q
What function does the epsilon subunit of E.Coli DNA polymerase III have?
A
3’ to 5’ exonuclease activity (proofreading ability)
7
Q
What function does the theta subunit of E.Coli DNA polymerase III have?
A
unknown, but can stabilize epsilon subunits to promote epsilon 3’ to 5’ exonuclease activity and required for high fidelity of DNA replication
8
Q
How many different subunits does a holoenzyme consist of?
A
at least 10 subunits
9
Q
How can E. coli DNA polymerase III quickly replicate it’s genome?
A
E. Coli DNA polymerase can replicate and entire genome without dissociating making replication faster
10
Q
What are the eukaryotic polymerases?
A
alpha, beta, gamma, delta, epsilon
11
Q
Where is eukaryotic polymerase alpha located in cell?
Is it associated with primase?
Does it have 3’ exonuclease activity?
What is it’s sensitivity to aphidicolin?
A
Located in Nucleus
Yes associated with primase
No 3’ exonuclease
High sensitivity to aphidicolin
12
Q
What is the biological activity of eukaryotic polymerase alpha?
A
replication (primase activity, replication initiator)
13
Q
Where is eukaryotic polymerase beta located in cell?
Is it associated with primase?
Does it have 3’ exonuclease activity?
What is it’s sensitivity to aphidicolin?
A
Located in Nucleus
Not associated with primase
No 3’ exonuclease
Low sensitivity to aphidicolin
14
Q
What is the biological activity of eukaryotic polymerase beta?
A
DNA repair (base excision)
15
Q
Where is eukaryotic polymerase gamma located in cell?
Is it associated with primase?
Does it have 3’ exonuclease activity?
What is it’s sensitivity to aphidicolin?
A
Located in Mitochondria
Not associated with primase
Yes, has 3’ exonuclease
Low sensitivity to aphidicolin
16
Q
What is the biological activity of eukaryotic polymerase gamma?
A
Mitochondrial DNA replication
17
Q
Where is eukaryotic polymerase delta located in cell?
Is it associated with primase?
Does it have 3’ exonuclease activity?
What is it’s sensitivity to aphidicolin?
A
Located in Nucleus
Not associated with primase
Yes, has 3’ exonuclease
High sensitivity to aphidicolin
18
Q
What is the biological activity of eukaryotic polymerase delta?
A
Replication (Main polymerase at the leading and lagging strand)
19
Q
Where is eukaryotic polymerase epsilon located in cell?
Is it associated with primase?
Does it have 3’ exonuclease activity?
What is it’s sensitivity to aphidicolin?
A
Located in Nucleus
Not associated with primase
Yes, has 3’ exonuclease
Low sensitivity to aphidicolin
20
Q
What is the biological activity of eukaryotic polymerase epsilon?
A
Replication (Leading and lagging strand)
21
Q
What is reverse transcriptase?
A
RNA-directed DNA polymerase
22
Q
What are the three enzymatic activities of reverse transcriptase?
A
- RNA-directed DNA Polymerase Activity
- RNase H Activity: an exonuclease that specifically degrades RNA chains in DNA:RNA hybrids
- DNA-directed DNA Polymerase Activity: Replicates the ssDNA to form dsDNA
23
Q
What inhibits HIV reverse transcriptase activity?
A
AZT (3’-azido-2’,3’-dideoxythymidine) inhibits DNA synthesis
24
Q
What are characteristics of HIV reverse transcriptase?
A
polymerase and RNase H activity
High Error rate: 1 per 2000 to 4000 nucleotides
25
What enzymes are involved in unwinding the DNA helix and what is their function?
DNA Gyrase: ATP-dependent negative supercoiling
| Helicases: ATP-dependent unwinding of DNA double helices
26
What enzyme is required for synthesis of DNA primer by RNA
Primase: a specific RNA polymerase (60 kD), primers 5bp long
27
What enzyme seals nicks in dsDNA?
DNA Ligase: joining Okazaki fragments together, ATP-dependent
28
Steps in initiation of E. Coli DNA replication
occurs at specific site (origin/oriC) in E. coli -> dnaA gene product binds at origin -> causes local denaturation of DNA -> two replisomes assemble at this site and replication proceeds bidirectionally
29
Steps in Termination of E. Coli DNA replication
occurs at special site opposite origin on E. coli -> chromosome contains binding sites, a large region (350 kb) flanked by 6 nearly identical 23 bp terminator sites for terminator utilization substance (tus) -> binding of Rho to her site on DNA inhibits helicase and prevents replisome from passing through
30
Steps in elongation of E. Coli DNA leading strand synthesis
The first RNA primer is synthesized by primase. DNA polymerase III can then synthesize DNA progressively until it reaches the terminus
31
Steps in elongation of E. Coli DNA lagging strand synthesis
Each Okazaki fragment must be synthesized and joined to those made previously. The RNA primers with each Okazaki fragment must be removed by the 5’ to 3’ exonuclease activity of DNA polymerase I. The nick between Okazaki fragments after the RNA primers removed are sealed by DNA ligase
32
What factor ensure fidelity of replication?
1. Balanced levels of dNTP's.
2. Two-stage nucleotide incorporation.
- ----Watson-Crick base pairing (to the template base) and stacking (on the primer terminus).
- ----Hydrolytic editing of errors (3' 5' exonuclease).
3. The incorporation accuracy is about 1 error/106 nucleotides.
4. Error prone RNA primers are removed by highly accurate Pol I enzyme.
5. Repair enzymes keep DNA under constant surveillance.
33
What is the phage DNA replication system?
Rolling Circle Replication
34
What are characteristics of rolling circle replication?
1. Synthesis of primer is not necessary
2. The leading strand is covalently linked to template
3. Replication can continue many rounds generating concatameric branch
4. Template for leading strand never separates form the circular part of the molecule
35
What are special considerations in replicating eukaryotic DNA?
Multiple initiation sites
Histone complexes
Telomere structures
36
In eukaryotes when does replication occur?
During S phase of cell cycle
37
What occurs in G1 to initiate replication?
external signal i.e. growth factor bind to receptor (tyrosine kinase etc.) -> send signal to nucleus -> synthesis of cyclin D proteins
38
How does cell cycle progress from G1 to S?
cyclin D-CDK complex phosphorylates RB protein -> allows transcription factor to dissociate from RB -> TF active and synthesizes S phase proteins (e.g. beta subunit of DNA polymerase II and thymidylate synthase)
39
What does p53 do?
DNA damage sensed by ATM and ATR -> inhibits MDM2-p53 complex -> active p53 -> increased transcription and translation of WAF1 gene -> produces p21 -> p21 inhibits cyclin-CDK complex formation -> RB remains dephosphorylated and TF inactive -> cell cycle arrest -> no DNA synthesis (senescence), p53 can repair, or apoptosis
40
Describe the cell cycle check point G2 to M
if incomplete DNA replication -> tyrosine kinase inactive
| If DNA replication ok -> active tyrosine kinase -> active M-phase kinase (CDK2/cyclin B) -> mitosis
41
What happens with histones and replication?
Histones retained by the leading strand-barely dissociate
After replication, the lagging strand is bare while new histones are made and assembled
Histones have greater affinity for dsDNA
42
What stretch of RNA sequence does telomerase carry?
AUCCCAAUC
43
Telomeres contain repeats of what?
Thymidine and Guanosine (e.g. TGGGGTTG)
44
Why would a chromosome shorten?
RNA primers at 5' ends of chromosomes are removed by Pol alpha but Pol alpha cannot fill the gap because there is no 3' OH group -> each replication would shorten the chromosome
45
How do eukaryotic germline cells solve the problem of shortened chromosomes?
telomerase -> extends the 5' end of telomeres
46
What eukaryotic cells have telomerase activity?
Germline (stem cells), long life span/immortal
| Somatic cells lack telomerase activity->chromosome shortening -> senescence -> cell death
47
What enzyme induces negative supercoils ahead of the replication fork in prokaryotes and eukaryotes?
Prokaryotes: Gyrase
Eukaryotes: nucleosome unwinding (topoisomerase)
48
What enzyme unwinds dsDNA-positive supercoils in prokaryotes and eukaryotes?
Prokaryotes: helicase
Eukaryotes: helicase
49
What enzyme adds RNA primers at start of Okazaki fragment at 5' end in prokaryotes and eukaryotes?
Prokaryotes: primase
Eukaryotes: primase subunit of polymerase alpha
50
What enzyme polymerizes leading and lagging strands to 5' to 3' in prokaryotes and eukaryotes?
Prokaryotes: DNA polymerase III
Eukaryotes: DNA polymerase delta (main), alpha initiator, some epsilon
51
What enzyme exonuclease remove primers and fills DNA gaps in prokaryotes and eukaryotes?
Prokaryotes: DNA polymerase I
Eukaryotes: DNA polymerase beta and some alpha
52
What enzyme extends 3' ends of DNA strands. Fills in the complementary strand of telomere in prokaryotes and eukaryotes?
Prokaryotes: circular DNA- not necessary
Eukaryotes: Telomerase, DNA pol alpha
53
What enzyme links Okazaki fragments in prokaryotes and eukaryotes?
Prokaryotes: Ligase
Eukaryotes: Ligase
54
What are the three main classes of DNA inhibitor?
1. Ones that prevent or reduce the synthesis of precursors (bases, nucleotides)
2. Ones that affect either the template or priming ability of the growing strand
3. Ones that act directly on polymerases or other enzymes needed for replication
55
What is the purpose of inhibitors of DNA replication?
used in lab and in the treatment of bacterial, viral, and neoplastic diseases
56
What are four intercalating agents?
acridines, ethidium bromide, anthracyclines, and actinomycin D
57
How do intercalating agents inhibit DNA synthesis?
induce unwinding DNA, lengthening DNA, and stiffening of double helix structure -> viscosity increases, buoyant density decreases, thermal denaturation temp increases, distortion of sugar backbone -> inhibits DNA-binding proteins such as polymerases, topoisomerases -> induce multiple mutations
58
Bleomycin, Zinostatin
Agents that bind to DNA covalently and cause chain breakage
59
Interstrand cross-linkers
alkyl sulphonates, anthramycin, mitomycin, nitrogen mustards
60
Coordination compounds that binds to guanine and inhibits DNA expression
Platinum and gold
61
cordycepin
Agents preventing extension of growing chain: 2’,3’-dideoxyribonucleosides
62
Agents acting on DNA polymerases:
acyclovir, aphidicolin, 2’-dideoxyazidocytidine
63
Acyclovir
inhibits DNA pol of Herpes simplex virus
64
Aphidicolin
inhibits pol α, pol δ (not β, γ)
65
inhibitor of bacterial primase
2’-dideoxyazidocytidine
66
effective inhibitors of DNA gyrase in bacteria
Coumermycin, novobiocin, oxolinic acid, nalidixic acid
67
Name a Topoisomerase I inhibitor and what do they do?
Camptothecins, Drug interaction produces double strand breaks in DNA that are irreversible and can lead to cell death
68
Camptothecins are used to treat what?
effective in treating lung, ovarian and colorectal cancers, also used in the treatment of myelomonocytic syndromes, chronic myelomonocytic leukemia (CMML), acute leukemia, multiple myeloma
69
Genetic stability is achieved with what two factors?
1. Highly Accurate DNA Replication System
| 2. DNA Repair System when DNA is damaged
70
What are consequences of a somatic mutation?
Cell Changes (cell death/disease/survival& division)= mostly deleterious for the affected individual
71
What are consequences of a germline mutation?
heritable diseases, or stable changes which lead to Evolution, new species=essential for long-term survival of life
72
What is a mutation?
refers to any change in the genetic material or base sequence of DNA
73
A mutation is established after how many DNA replications (cell divisions)?
2
74
A base substitution mutation can be
transversion (between purine and pyrimidine) or transition (between burin and purine or pyrimidine and pyrimidine)
75
What are consequences of substitution mutations?
silent, missense, or nonsense mutation
76
What are small scale mutations?
substitutions, deletions, or insertion
77
What are large scale mutations?
translocations, inversions, deletion, nondisjunction
78
Frameshift mutations may cause
deletions
79
Disease examples of deletions causing frameshift mutations?
CFTR gene, factor IX gene, APC gene
80
What is the Ames test?
low cost test for carcinogenesis -> the bacteria you are testing caries a mutant Histidine gene -> can't produce histidine -> placed in reservoir that lacks histidine -> if bacteria survives and begins producing histidine means it reversed the mutation -> the bacteria is therefore carcinogenic however, this test does not always catch all carcinogenic bacteria
81
Types of DNA damage
1. Base Loss- bas lost but sugar phosphate backbone intact
2. Base modification- cytosine -> deamination -> uracil -> methylated -> thymine or from ROS , ionizing radiation, photodamage
3. Replication Errors during DNA replications-mismatch, insertion, deletion
4. Inter-strand crosslinks by bifunctional alkylating agents, UV or ionizing radiations
5. DNA-Protein crosslinks: bifunctional alkylating agents, UV or ionizing radiations can also create crosslinks between DNA strands and protein molecules
6. Strand Breaks: Ionizing radiation can generate both single-strand nicks and double-strand breaks
82
Three broad categories of DNA repair
1. Direct reversal of damage
2. Excision of damaged region, followed by precise replacement
3. Damage tolerance: attempts to minimize the effects of damage that has not been repaired
83
Examples of direct reversal of damage DNA repair mechanisms
1. Photolyases are found in Bacteria, fungi, plants, and many vertebrates, but not in placental mammals -> enzymatic cleavage of a T, C, and CT dimers
2. O6-methyl-guanine-DNA methyltransferase which transfer O6-methyl to Cys of enzyme, permanent damage to Enzyme
3. DNA ligase: sealing nicks
84
Base excision repair names and basic mechanisms
1. mismatch repair- MMR repairs mismatches in newly synthesized DNA strands, replaces non-metylated base because parent strand GATC has methylated A
2. base excision repair- remove incorrect bases (U) or damaged bases (3-methylated A)
3. nucleotide excision repair
85
Mechanism of mismatch repair
1. MutS: scans DNA & binds to mismatched bases
2. MutL: attaches & links MutH & MutS
3. MutH: an endonuclease, recognizes methylated A and nicks the opposite strand
4. A Helicase (UvrD) unwinds DNA from nick-past the mismatch
5. An Exonuclease cuts away single bases from nick
6. SSB binds and DNA pol III fills the site
7. DNA ligase seals the gap
86
Mechanism of base excision repair
1. Removal of the incorrect base by an appropriate DNA N-glycosylase to create an AP site
2. Nicking of the damaged DNA strand by AP endonuclease upstream of the AP site, thus creating a 3’ OH terminus adjacent to the AP site
3. Extension of the 3’-OH terminus by a DNA pol, DNA ligase seals the nick
87
Mechanism of nucleotide excision repair
1. Damage Recognition by UvrA, UvrB
2. Binding of a multi-protein complex at the damaged site (UvrA dissociates from UvrB forms complex with UvrC at damaged site)
3. Double incision of the damaged strand several nucleotides away from the damaged site, on both the 5’ and 3’ sides (UvrB complex cleaves phosphodiester bonds)
4. Removal of the damage-containing oligonucleotides from between the two nicks
5. Filling in of the resulting gap by a DNA polymerase
6. Ligation by DNA ligase
88
In excision repair what protein binds to damaged DNA in humans and E. Coli?
Humans: XPA or XPE
| E. Coli: UvrA/UvrB
89
In excision repair what protein functions as helicase in humans and E. Coli?
Humans: XPB or XPE
| E. Coli: UvrD
90
In excision repair what protein functions as DNA damage sensor in humans?
Humans: XPC
91
In excision repair what protein works with ERRCI to cut DNA in humans and E. Coli?
Humans: XPF or XPG
| E. Coli: UvrB/UvrC
92
Examples of Damage tolerance repair mechanisms
1. Homologus recombination repair-double strand break: sister strand exchange
2. Homologus recombination repair-double strand break: non-homologue end joining (NHEJ)
3. SOS repair-error prone process
93
sister strand exchange
Rad51 protein searches homologous copy of the damaged DNA on the sister chromatid -> DNA synthesis -> ligation
94
non-homologue end joining (NHEJ)
non homologous end joining: heterodimer recognizes ds break damage and recruits protein kinases, which promote ligase reactions
95
SOS repair
SOS repair occurs ONLY when cells are overwhelmed by UV damage - this allows the cell to survive but at the cost of mutagenesis.
Causes polymerase to continue past thymine dimer inserted bases without proof reading
96
Xeroderma pigmentosum
Xeroderma Pigmentosum (XP): mutations in 7 genes (XPA-XPG) that encode the UV excision system (NER): severe light sensitivity, pigmentation irregularities, skin cancer, other forms of cancer, neurological defects
97
Ataxia telangiectasia
no cell cycle arrest because of either ATM, p53, GADD45 genetic visored (autosomal recessive)
Patient will have increased sensitivity to ionizing radiation, neurological, balance, sinus, respiratory, dilated blood vessels in eyes and surface of skin, and immune system abnormalities
98
Hereditary nonpolyposis colon cancer (HNPCC)
Mutations (alterations) of one of five genes ( ch 2- hMSH2, hPMS1, MSH6 or ch 3- hMLH1 or ch 7- hPMS2) are now known to be responsible for most cases of HNPCC
99
Fanconi’s anemia:
a lethal aplastic anemia, due to defective DNA repair on interstrand cross-links or x-ray induced DNA damage
100
Hutchinson-Gilford progeria Syndrome:
an extremely rare genetic disease that accelerates the aging process to about seven times the normal rate. Because of this accelerated aging, a child of ten years will have similar respiratory, cardiovascular, and arthritic conditions that a 70-year-old would have.
Less severe “ Werner syndrome” possibly due to helicase mutations
101
Bloom’s syndrome:
the Enzyme BLM helicase 3 is partially defective, genetic instability in the form of increased frequencies of breaks and interchanges
102
Cockayne’s syndrome:
profound growth retardation, retinopathy, cataracts, large sunken eyes and thin prominent nose. These patients have prematurely aged appearance. Defective in UV-induced DNA repair system
103
Retinoblastoma:
Autosomal dominant inheritance caused by mutation in the tumor-suppressor retinoblastoma gene (RB) on chromosome 13q.
104
Telomerase is what kind of polymerase?
RNA dependent DNA polymerase
105
Primase is what kind of polymerase?
DNA dependent RNA polymerase
106
Reverse transcriptase is what kind of polymerase?
RNA-dependent DNA polymerase
