DNA replication Flashcards
1
Q
B-DNA
A
- “normal” DNA
- Right handed
- Pitch: 35 A (10.4 bp)
2
Q
A-DNA
A
- Right handed
- shorter/wider than B-DNA
- Pitch: 25.3 A
3
Q
Z-DNA
A
- Left handed
- Tall, narrow
- Pitch: 45.6 A
4
Q
Supercoiling
A
- DNA in the cell must be organized
- Allows for packing of large DNA molecules within the cells
- Allows for access of proteins to read the information in DNA sequence
- Many circular DNA are supercoiled
5
Q
L
A
- linking number
- number of times strands go around each other; number of times plane is crossed
6
Q
T
A
- twist
- number of turns of double helix
7
Q
W
A
- writhe
- number of times the double helix wraps around itself
8
Q
L,T,W association in closed system
A
- L=T+W
9
Q
Relaxed DNA
A
- W =0
- L=T=# of bp divided by 10.4
10
Q
(negatively) supercoiled DNA
A
- W ≠ 0 ⇒ compacted DNA
11
Q
How to change linking # of circular or closed DNA
A
- Cut, twist, reattach
12
Q
Function of topoisomerases
A
- change the linking number
13
Q
Type I topoisomerases
A
- Add negative supercoils
- Cleave 1 strand
14
Q
Type II topoisomerases
A
- Remove negative supercoils
- Cleave 2 strands
15
Q
OriC
A
- In E coli: one origin of replication/chromosome
- 245 bp
- highly conserved sequence elements
- Binding site for initiator protein: DnaA
16
Q
ARS
A
- In eukaryotes
- Approx. 400 well defined origins
- Entire genome replicated 1X/cycle
- Regulation due to cyclin proteins and cyclin-dependent kinases (CDKs)
- Cyclins are ubiquinated for proteolytic destruction at the end of the M (mitosis) phase
17
Q
OriC and ARS similarities
A
- A,T rich
- easy to “melt” DNA into single strands
18
Q
Helicase
A
- Melts double stranded DNA
- Use ATP
19
Q
Primase
A
- Make RNA primers
- RNA primers are added first to replicating strand; later removed
20
Q
DNA polymerase I
A
- Replaces RNA primer with DNA
- 5’ to 3’ exonuclease activity
- Not ideal for replication
- Slow, low processivity
21
Q
DNA polymerase III
A
- Principal replication polymerase
22
Q
Ligase
A
- Seals “knicks”
- Puts Okazaki fragments together
23
Q
Telomerase
A
- replicates telomeres
24
Q
Topoisomerase I
A
- Behind replication fork (on 2 daughter DNA)
25
Topoisomerase II
* Ahead of replication fork (on parent DNA)
26
Leading strand synthesis
* Replicated continuously
* DNA polymerase 𝛅
* DNA polym III in prokaryotes
27
Lagging strand synthesis (what and proteins)
* Generated in small steps
* Okazaki fragments
* Priamse: makes 15 bp RNA primer for each okazaki fragment
* DNA polym. III
* Does all leading strand synthesis
* Makes DNA from from 1 primer to the next on lagging strand
* DNA polym. I
* Replaces RNA in primer with DNA
* Ligase
* Seals knicks
* Single stranded binding proteins
* Stabilize single stranded DNA
* Prevent strands from sticking back together
28
Subunits
* 20 individual peptides combined
* In pol III: 10 types of subunits
29
Homotetramer
* 4 identical subunits
* “Same” “four”
30
Heteropentamer
* 2 or more different monomers
* “Different” “five”
31
Holoenzyme
* does most DNA synthesis activity
* proofreading capabilities that correct replication mistakes by means of exonuclease activity working 3'→5'
* high processivity (i.e. the number of nucleotides added per binding event)
32
Central structure
* hold 2 holoenzymes together and allows them to move together
33
a subunit
* catalytic subunit
* Has a groove for DNA to slide along and active site where nucleotides are added
34
e subunit
* proofreading subunit
* Just behind a; removes incorrect nucleotides
35
b subunit
* processivity subunit/beta-clamp
* Forms “donut” around DNA to prevent pol III from falling off
36
What is crossing over?
* The exchange of genetic material between homologous chromosomes that results in recombinant chromosomes during sexual reproduction
37
When does crossing over occur?
* Prophase I (meiosis)
38
Quartenary stucture of pre-recombinase
* homotetramer
39
How does Cre recombinase work?
* catalyzes site specific recombination between 2 DNA recognition sites
* cyclic recombination
* @ LoxP sites (8 bp sequence with 34 bp palindromic sequences on either side)
40
What do tyrosine resiudes do in cre recombinase?
* nucleophilic attack on phosphate bonds of DNA
41
How is cre recombinase similar to topoisomerase I?
* forms covalent bonds between hydrogen bonds and tyrosine residues (how it controls strand breaks and combine DNA)
42
What is a mutation?
* alteration in DNA structure that produce permanent changes in the genetic information encoded therein
43
When do mutations take place?
* during cell division (replication)
* environmental factors
* UV
* chemical
* viruses
44
DNA damage vs mutation
* mutation: affects nucleotide sequence (can't be fixed)
* Damage: damage to bonds, etc; abnormalities in DNA
* can be fixed
45
3 causes/types of DNA damage and the mutations they cause
* UV light
* pyrimidine dimers
* Chemical damage
* point mutation
* Intercalating agents (insertion of molecule between DNA planes)
* frameshift mutation
46
3 steps of DNA repair
1. Abnormal DNA is recognized
2. abnormal DNA is removed (upstream and downstream)
3. normal DNA is synthesized
47
3 enzymes of DNA repair
1. polymerase III
2. Ligase
48
What is an Ames test?
* Indicates mutagenic potential of a compound
* Add compound to plate of salmonella
* see if it grows in His free medium
* Colonies (+test) indicates the compound mutated the salmonella, restored ability to synthesize His
49
Use of liver cells in Ames test
* Can treat the compound with liver cells to turn it into a mutagen, to see if the body processes it into a mutagen
50
RNA polymerase: quartenary structure in prokaryotes
* 5 subunits (pentamer)
* a: (2) assembly and binding to UP (upstream promoter) elements
* B: main catalytic subunit
* B': responsible for DNA binding
* o: directs enzyme to promoter
* w: protect polymerase from denaturation
51
What does RNA polymerase I transcribe in eukaryotes?
* pre rRNA (turns into rRNA)
52
What does RNA polymerase II transcribe in eukaryotes?
* pre mRNA
* some snRNAs
53
What does RNA polymerase III transcribe in eukaryotes?
* pre tRNAs
* 5S rRNA
* some snRNAs
54
Initiation
RNA polymerase binds to promoter on DNA (by transcription factors in eukaryotes)
55
Elongation
* Elongation factors enhance activity of RNA polymerase
* RNA polymerase reads template strand
* Goes in 3’ to 5’ direction
56
Termination
* In bacteria: stops due to hairpin loop
* In eukaryotes:cleavage of the new transcript followed by template-independent addition of adenines at its new 3' end, in a process called polyadenylation
* Pol II released
57
Template strand
* Non-coding
* template for RNA polymerase
* Antisense
58
Coding strand
* Non-template strand
* has same sequence as RNA transcript
* Sense
59
Promoter
region of DNA that initiates transcription of a particular gene
60
+1
* 1 bp away from promoter
* On coding strand
61
Why different sigma subunits in prokaryotes
* bind different promoters
* transcribe unique set of genes
62
What do transcription factors do?
proteins that bind DNA and regulate gene expression by promoting or suppressing transcription
63
How are transcription factors named?
* by what they do
64
RNA Pol vs DNA Pol
* One produces RNA, one produces DNA
* RNA poly does not require primer
* DNA faster than RNA pol
*
65
Protein dependent termination
* C/A rich sequence called rut site
* process continues until termination site reached
* rho protein is a helicase, binds to rut site
66
Protein independent termination
* 3 U's near 3' end of transcript
* self-complementary regions in transcript form hairpin 15-20 before 3' end
* makes RNA poly pause, then dissociate
* C/G rich
67
RNA Processing steps
* Splicing out introns, leaving exons (pro/euk; mRNA)
* Addition of 5' cap (euk mRNA)
* Addition of 3' polyA tail (euk mRNA)
* Clevage
* Base modification
* RNA editing
68
rRNA processing
* in pro/euk
* created from longer pre-rRNA by cleavage/methylation
69
tRNA processing
* formed from pre-tRNA by cleavage, base modification, splicing
70
Consensus sequence for polyadenylation
* @ -10 and -30
* betweem -40 and -60
71
snRNA
* snRNP (small nuclear ribonuclear proteins) RNAs
* Part of spliceosome
72
Spliceosome
* U1 and U2 snRNPs bind intron's ends (at 5' and 3' ends)
* U2-6 bind along with other proteins to splice intron
73
Tetrahymena
self splicing group I intron
74
Two ways a cell can control which genes are expressed
* Increase the affinity of the promotor for RNA polymerase
* Make promotor physically more accessible → controlling DNA structure
75
How do activators/repressors regulate transcription
* bind promoter/release from promoter to allow or prevent transcription of genes
76
Operon
* functioning unit of DNA containing cluster of genes under the control of one promoter
* Found in prokaryotes
77
Chromatin
* Tightly wound complex of DNA and histones to allow for control of DNA/gene expression
* Remodeling only happens in eukaryotes
78
What are histones?
* proteins found in nuclei that package and order the DNA into structural units called nucleosomes
79
Histone structure
* core: 2(H2A, H2B, H3, H4)
* H1: links nucleosomes to create chromatin
80
Histone modifications
* methylation
* acetylation
* Affect charge (DNA binding) or sturcture/condensation to activate or repress transcription
81
Enhancer
* region of DNA bound by proteins that activate transcription of a gene
82
Silencer
DNA sequence capable of binding repressors
83
Location of silencer/repressor
* Can be anywhere, but usually upstream of target gene
84
Location of activator/enhancer
* could be up or downstream of gene
* do not act on promoter region
85
tRNA
86
What do aminoacyl-tRNA synthetases do?
* enzyme that attaches the appropriate amino acid onto its tRNA
* By esterification of a specific cognate amino acid
87
How do aminoacyl-tRNA synthetases determine genetic code?
* Adds charged a.a. onto growing polypeptie chain
* tRNA reads genetic code and adds new a.a according to genetic code
88
Second genetic code
* matching each a.a with correct tRNA (must be specific for each other) can be viewed as second genetic code
* the "code" is in the molecular recognition of a specific tRNA molecule by a specific synthetase
89
Wobble
* pairing between two nucleotides in RNA molecules that does not follow Watson-Crick base pair rules
* allows some base pairs to bind more than one codon (3rd bp of codon)
90
Ribosome structure
small and large subunits
91
Ribosome small subunit
* decoding center which monitors the complementarity of mRNA and tRNA
* has APE sites for adding polypeptides
92
Ribosome large subunit
* contains active site (creates peptide bonds when proteins are synthesized)
93
The directions in which mRNA is translated and proteins are synthesized
* 5’ to 3’
* N to C terminus
94
3 steps of translation
* Initiation
* Elongation
* Termination
95
Initiation (translation)
* mRNA and aminoacylated tRNA bind to ribosome
* IF-1, IF-2, IF-3 (initiation factors)
* FMet (forylmethothionine)
* GTP
96
Elongation (translation)
* Cycles of aminoacyl-tRNA binding and peptide bond formation…until a STOP codon is reached
* EF-TU (elongation factor Tu)
* GTP
97
Termination (translation)
* mRNA and protein dissociate, ribosome recycled
* RF-1, RF-2, RF-3 (release factors)
98
Prokaryotes (translation)
* Shine-Dalgarno sequence
* Must be located for initiation
99
Eukaryotes (translation)
* Scanning
* mRNA loops through ribosome
* Binds 5’ cap
100
MET vs FMET
* FMET binds P site along with initiating AUG
* MET in eukaryotes
* FMET in prokaryotes
101
EF-Tu
* Aminoacyl tRNA binds to a complex of elongation factor Tu that also carries GTP
102
EF-G
* translocase
* prokaryotic elongation factor with GTPase
* catalyze the coordinated movement of tRNA and mRNA through the ribosome
* Moves tRNA along to next site
103
3 stop codons
* UAA
* UGA
* UAG
* Recognized by termination (release) factors/ribosome
