DNA Replication Flashcards
1
Q
Central Dogma?
A
flow of genetic info goes from DNA to RNA to Protein
DNA to DNA (DNA synthesis)
DNA to RNA (Transcription)
RNA to Protein (Translation)
Transcribe= different form of the same language
Translate= different language
2
Q
What are some exceptions to the central dogma?
A
- Reverse transcription (retroviruses)
- Non coding RNA
- RNA editing
- RNA replication to RNA (virus)
3
Q
What makes up the structure of DNA?
A
Sugar
Nitrogenous base
Phosphate group
4
Q
What is the structure of the sugar in DNA?
A
5 carbon pentose ring called ribose (RNA) or deoxyribose (DNA)
5
Q
What carbon is the nitrogenous base attached to in the 5 carbon sugar of DNA?
A
1’ carbon via an N-glycosidic bond, same carbon as sugar-sugar bonds
6
Q
What is difference at the 2’ carbon between DNA and RNA? Why is this significant?
A
DNA has H and RNA has OH
The absence of O makes DNA more stable, less reactive than RNA, so it is a better place to store genetic info
7
Q
What does the 3’ carbon have attached in DNA/RNA?
A
OH
8
Q
What does the 5’ carbon have attached in DNA/RNA?
A
1 to 3 phosphate groups
9
Q
What are the two types of nitrogenous bases?
A
purines (two rings)- Adenine, Guanine
pyrimidines (one ring)- Cytosine, Uracil (RNA), Thymine
10
Q
What is the difference between thymine and uracil?
A
thymine contains a methyl group on the double bonded C=C2
11
Q
How can cytosines next to guanines on the same strand of DNA (CpG) be modified? What does this modification do?
A
enzymatic addition of a methyl group
- changes how proteins interact with DNA
- influences its structure indirectly
- plays a role in gene expression
12
Q
Deamination?
A
spontaneous removal of an amine group (NH2) from a nucleotide, common type of DNA damage
13
Q
Deamination of cytosine=?
A
Uracil, easily detected
14
Q
Deamination of adenine=?
A
Hypoxanthine, easily detected
15
Q
Deaminated methyl-cytosine=?
A
Thymine, not easily detected or repaired
16
Q
Deaminated guanine =?
A
xanthine
17
Q
Another name for nucleotide?
A
nucleoside triphosphate
18
Q
Nucleotides are the subunits of ______.
A
nucleic acids
19
Q
What causes the negative charge in a nucleotide? Why is the negative charge in DNA significant?
A
phosphate (PO4^3-)
- not soluble in lipids, stays in nucleus
- interacts with histones for compact storage
20
Q
AMP?
ADP?
ATP?
where do they attach?
A
monophospate
diphosphate
triphosphate
joined to C5 hydroxyl of ribose or deoxyribose
21
Q
What type of linkage is used to attach phosphate groups to the 5’ carbon?
A
ester linkage
22
Q
How are the phosphates named that are attached to the sugar? Which phosphate is included in nucleic acids?
A
- alpha (closest), beta, gamma
- only alpha is included, hydrolysis of the other two provide energy for the process
23
Q
What type of bond joins nucleotides together?
A
phosphodiester linkage connects the 5’ phosphate and the 3’ OH
24
Q
What makes a DNA strand polar?
A
5’ PO4
3’ OH
25
DNA is synthesized from ___ to ____.
5'
3'
26
How are anti parallel strands of DNA primarily held together?
H bonds between nitrogenous bases
27
Which base pairs are held together tighter? (AT or GC). Why?
GC because they are held together by 3 H bonds whereas AT is held together by 2 H bonds
-purines always pair with pyrimidines
28
What is the backbone of DNA strands composed of?
sugar and phosphate
29
In an alpha helix, where do the bases lie in relation to the axis of symmetry?
perpendicular
30
What is the purpose of the alpha helix? How many bp/turn?
it compacts DNA
10 bp/turn
31
Where are the major and minor grooves located in an alpha helix? What is significant about them?
major- between turns
minor- between strands
-this is where proteins interact with DNA
32
What is significant about DNA having complimentary base pairs and antiparallel structure?
Allows both strands to be used as templates for DNA synthesis, which ensures that each daughter cell gets the same genetic information.
Allows DNA repair to occur, since the damaged strand can be repaired by using the complimentary sequence as a template.
33
What is chromatin?
DNA + proteins
34
What is the difference between Euchromatin and Heterochromatin?
Euchromatin- less condensed, transcriptionally active, on chromosome arms, unique sequences, many genes, throughout S phase, crossing over common
Hetero- inactive, more condensed, sometimes facultative, always constitutive, at centromeres, telomeres, repeated sequences, few genes, late S phase, crossing over uncommon
35
What is the size of the nucleus?
5-8 micrometers diameter
36
What happens during Mitosis to chromatin?
becomes very condensed, chromosomes become visible
37
Where is the site for Ribosome synthesis is the nucleus?
Nucleoli
38
How many base pairs are packed into the Nucleus?
3 billion in an organized way for replication and transcription, compaction ration of 10,000 to 1
39
What are histones?
small positively charged proteins
40
What is the nucleosome core composed of?
H2A, H2B, H3, H4
-2 of each to make an octamer
41
How does DNA pack around histones?
wraps twice around octamer to make a nucleosome bead
42
What is linker DNA?
about 50 base pairs of DNA that separate the nucleosomes
43
What is the role of H1?
bind linker DNA
44
How much do nucleosomes decreases the length of DNA by?
2/3
45
What is the nucleofilament?
30 nm fibers of packed nucleosomes in a 2 start helix consisting of 2 strands of nucleosomes stacked like coins
46
What is the order of compaction from DNA to chromosome?
1. DNA
2. Nucleosome beads- histone plus DNA
3. 30 nm fiber nucleofilament
4. nucleofilament is coiled and anchored to scaffold protein
5. Chromosome
47
How do histones hold onto DNA?
positively charged tails of nucleosomal histone proteins that contain lysine amino acids interact with the negatively charged phosphate groups of DNA
48
What post translational modification occurs to weaken histone interaction with DNA?
acetylation of tails weakens interaction with DNA and allows some transcription factors to bind DNA
49
What is the role of histone acetyl transferases (HAT)?
- add acetyl groups to loosen interaction with DNA, this favors Euchromatin
- positively acting transcription factors or proteins recruited often have HAT activity
50
What is the role of histone deacetylases (HDAC)?
- remove acetyl groups to increase interaction with DNA, favors heterochromatin
- transcriptional repressors often recruit proteins with HDAC activity
51
What are other post translational modifications of histones and their role?
-phosphorylation, methylation, ubiquitination
- make up a complex histone code, which influences chromatin structure and gene expression
- affect histone interaction with DNA, histones, proteins
52
What phase of the cell cycle does DNA replication occur?
S (synthetic) phase- discrete phase
-replicate 3 billion bp in 6-8 hours
53
What regulates passage between phases of the cell cycle?
cyclins and associated kinases (cyclin dependent kinases- CDK)
54
What controls checkpoints in the cell cycle?
tumor suppressors (p53, pRB)
-keeps cell cycle from moving forward
55
Role of proto oncogenes? abnormal?
- send positive signals for continued cycling (c-myc, Ras)
| - abnormal = cancer
56
What happens in the cell cycle if DNA damage is detected? what if it cannot be repaired?
- cell cycle is halted
- DNA repair pathways are initiated
-if damage cannot be repaired, cell undergoes apoptosis or programmed cell death
57
What is licensing in the cell cycle? When does it occur?
- In late G1 and S phase, licensing ensures that all regions or origins of DNA are replicated completely, but only once per cycle
- when the origin fires in S phase, the license is removed, since only licensed origins can replicate, this ensures that the same are is not replicated twice
58
What regions of the genome are replicated early in S phase? later in S phase?
actively transcribed regions are replicated early, where untranscribed regions are replicated later
59
What are the steps of the cell cycle?
```
G1- cell growth and metabolism
Go- non dividing phase in G1
G1/S check- cell is committed to dividing
S- DNA replication
G2- cell prepares for Mitosis
G2/M check- cell can divide
Mitosis/Cytokinesis- cell divides
```
60
What does semiconservative mean?
-each of the two new daughter helices contains one stand of parental DNA and one strand of new DNA
- both parent strands are used as templates
- both daughter cells get the same genetic info
61
Explain the origins of replication in Eukaryotes? Forks? Bubbles?
- there are multiple origins of replication along a chromosome and replication is bidirectional, this allows the process to be more efficient and faster
- replication forks- regions of transition between unwound parent duplex and newly replicated daughter DNA
- replication bubbles- unwound region
62
What are origins rich in (AT or GC)? Why?
AT rich because they are easier to split
-euchromatin origins are first
63
DNA polymerase works in what direction?
synthesizes 5' to 3'
64
Describe the process of initiation in Replication?
1. large protein complex called Origin Recognition Complex (ORC) assembles at the origin throughout the cell cycle, cannot initiate
2. Cdc6 and Cdt1 recruit Mcm helices in late G1 phase to form a pre-replicative complex (pre-RC). Now the origin is licensed to replicate
3. At the beginning of S phase, S-CDK phosphorylates Mcm and ORC which disassembles the pre-RC and initiates DNA replication
4. Phosphorylated Mcm forms the replication bubble and allows the pre initiation complex to form, which contains DNA polymerase and other proteins. Mcm goes with the replication forks to help separate the strands
5. ORC binds the origins of both daughter DNAs, and stays phosphorylated until G1 so that it cannot bind Cdc6 and form the pre-RC until the next S phase
65
What happens if Cdt1 and Cdc6 are over expressed?
re-replication = cancerous cells
66
What is the role of helicase enzymes such as Mcm?
move along one strand of DNA and change the conformation to bind the double strand, then use ATP to separate the two strands and return to their original conformation
67
Role of single stranded binding proteins (RPA)?
bind to the single stranded regions of the replication bubble and prevent the two strands from reannealing to each other or base pairing with themselves (hairpins, DNA pol passes them, could cause deletion)
68
Role of Type 1 Topoisomerase?
make reversible nicks in the DNA ahead of the replication fork, pass the unbroken strand through the gap and then reseal the gap, prevents supercoiling
69
Role of Type II Topoisomerase?
make double stranded breaks in the DNA, allow uncoiling, and then re-ligate, prevents supercoiling
70
Why do some cancer drugs target Topoisomerase?
if Topoisomerase is not working due to the drug, the DNA will supercoil, causing double strand breaks in the DNA that cannot be repaired, leading to apoptosis
71
Process of DNA synthesis, adding dNTPs?
1. New DNA is synthesized from dNTPs
2. In replication, the 3' OH group of the last nucleotide on the strand attacks the 5' phosphate group of the incoming dNTP
3. Two phosphates are cleaved off by hydrolysis for energy
4. phosphodiester bond forms between the the two nucleotides
72
What direction does the DNA polymerase move?
it moves 3' to 5'
synthesizes 5' to 3'
73
What provides the first 3' OH in DNA synthesis for the DNA polymerase to work with?
RNA primer made by Primase (RNA polymerase)
74
Role of 3' to 5' exonuclease activity of DNA polymerase?
allows it to proofread by excising the last nucleotide added if it doesn't base pair correctly with the template, very accurate
75
How is the leading strand copied?
continuously from the origin, in the same direction as the replication fork
76
How is the lagging strand copied?
discontinuously in small fragments beginning in the replication fork and moving toward the origin, a new primer must be generated the beginning of each okasaki fragments
77
What removes RNA primers?
RNA hydrolyses or RNAses
78
Role of DNA ligase?
uses ATP to make the final phosphodiester bond to seal the nicks and join the fragments together
79
8 Steps of DNA replication?
1. Origin + ORC + Cdc6 + Cdt1 + Mcm = pre-RC, license is created, phosphorylated in S phase
2. Helicases unwind the strand, bubble forms
3. Single stranded binding protein (RPA) keeps parental strand from reannealing
4. Topoisomerase prevents supercoiling
5. Primase makes RNA primers
6. DNA pol synthesizes 5' to 3'
7. RNAse removes primers, gaps filled by DNA pol
8. DNA ligase seals the nicks
-at least 30 different proteins are involved
80
What is the role of proliferating cell nuclear antigen (PCNA)?
sliding clamp that increases processivity of DNA polymerase (helps it stay on DNA)
-it is also involved in DNA repair, chromatin remodeling, cell cycle regulation
81
What three DNA polymerases do most of the work in replication?
alpha
delta
epsilon
82
What is epigenetics?
the study of heritable changes in gene expression that occur without changes in DNA primary sequence
83
Epigenetic inheritance vs genetic heritance?
Genetic- DNA sequence change is passed to the next generation
Epigenetic- chromatin change is multiplied in somatic cells (mitotically heritable) within the same organism, but is not passed on to the next generation
84
What are some epigenetic signals?
-histone modifications and DNA methylation, these patterns are maintained during DNA replication
85
How do epigenetic signals influence gene expression?
by regulating the accessibility of the DNA to transcription factors through effects on chromatin structure, affect tightness
- most are erased during production of germ cells
- can be influenced by intra and extracellular environment
86
Where does DNA methylation occur?
on cytosines next to guanines on the same strand of DNA (CpG sequences), 5-methylcytosine is created
87
Why is DNA methylation important?
- associated with mitotically heritable gene inactivation, inhibits transcription
- important for normal development, X chromosome inactivation, imprinting-one allele shut off
- maintains chromosomal stability by keeping repetitive sequences in non coding regions in a repressed state
88
During the course of evolution, what have the CpG sequences mutated into?
TpG due to CpG being methylated and deaminated
-remaining CpG are not evenly distributed in the genome
89
What are CpG islands?
C-G rich areas, often at the 5' end of genes (housekeeping genes that constitutively expressed), normally not methylated
90
How did CpG islands form?
- most CpG sequences were methylated in the germ line except for areas with closed chromatin
- the closed chromatin were maintained because they weren't methylated
91
How are CpG that are methylated passed on to the next generation?
DNA maintenance methylase (DMNT1) recognizes hemi-methylated sites and it methylates the newly synthesized strand
92
T/F
The histone code is maintained during DNA replication.
True
93
How are histone tails modified? What are modifications?
acetylation
methylation
phosphorylation
ubiquitination
modifications- epigenetic signal that helps regulate gene expression
94
How is the histone code modified during replication?
1. During replication, histones are distributed between daughter DNA, leaving gaps
2. newly synthesized histone proteins bind the gaps and are modified by Reader Writer Remodeling Complexes (RWRC) to match the ones already bound
-other chromatin proteins also get distributed the same way to maintain structure
95
Why can't the 3' end of the parental DNA be replicated completely?
the removal of the RNA primer from the end of the lagging strand leads to loss of some DNA
96
What protects the end of chromosomes?
Telomeres
97
Functions of Telomeres?
1. protect ends of chromosomes from degradation and fusion
| 2. distinguish ends of intact chromosomes from broken ones
98
Senescence?
- Loss of telomeric DNA, fewer proteins can bind, risk that telomeres will not be able to protect end of chromosomes, triggers cell cycle checkpoint mechanism and cell stops growing permanently
- may be related to aging
99
Where is telomerase found?
- germline cells, stem cells, over 85% cancer cells
| - not found in somatic cells
100
What does telomerase do?
- reverse transcriptase
- uses its own RNA template complimentary to G rich strand of telomere, to extend 3' end of parental strand, length of telomeres is maintained
- key player in immortalization- can keep replicating
101
What is dangerous about cancer cells when it comes to normal checkpoints that lead to senescence?
these checkpoints fail which causes the cells to keep replicating their DNA even though the telomeres are dangerously short
102
What does the lack of functional telomeres lead to?
chromosomal instability, very few cells have telomerase that can be activated
103
Where did mitochondria evolve from? What does their genome contain? Where do they get other mitochondrial proteins?
- evolved from symbiotic bacteria in early eukaryotes
- contain 13 proteins that are transcribed and translated to function in mitochondria
- most other proteins are made in the nuclear genome, translated in cytosol, and are imported to mitochondria
104
What shape is mitochondrial DNA? How many copies of DNA per mitochondria?
- single, double helical circle, no histones
| - multiple copies of DNA per mitochondrion and multiple mitochondria per cell
105
T/F
All cells have the same amount of mitochondria.
False
every cell varies, number of mitochondria changes with changes in physiological conditions, some cells need more energy output such as muscle cells
106
Is replication in mitochondria the same as nuclear DNA replication? Is the genetic code the same?
- No, it is not limited to S phase, it resembles bacterial replication
- No, code is slightly different
107
Where does a person inherit mitochondrial DNA?
mother
108
Which is more economical, Mitochondrial DNA or Nuclear DNA?
mitochondria because nearly every base pair is used to make a functional product, nuclear genome has many repetitive sequences
109
Which has a higher mutation rate, mitochondrial DNA or nuclear DNA? Why?
What could these mutations lead to?
- Mitochondrial (5-10X higher) due to its close proximity to sites of generation of oxygen radicals, or could be due to less efficient repair mechanisms
- contributes to decreased efficiency of oxidative metabolism with increased age, and aging in general
110
What is the importance of junk DNA?
it correlates better to the complexity of an organism rather than number of genes
