DNA REPLICATION Flashcards
1
Q
The ____ of eukaryotic cells consists of DNA complexed with ___ in nucleosomes
A
chromatin
histones
2
Q
___ are relatively small, basic proteins with a high content of __ and ___
A
Histones
- arginine and lysine
3
Q
Prokaryotes do not have __
A
histones
4
Q
Eight histone molecules form an __, around which about__ base pairs of DNA are wound to form a __
A
octamer
140
nucleosome core
5
Q
The DNA that joins one nucleosome core to the next is complexed with___
A
histone H1
6
Q
- due to ___, 2 strands are complementary
the 2 strands of dna double helix - - - are ___, their chemical orientations are different - by convention, a base sequence is usually written with the ___ on the left
A
base pairing rules
antiparallel
5’ P terminal end
7
Q
models of dna replication
A
Conservative Replication
Semiconservative Replication
dispersive model
8
Q
DOUBLE HELICAL MODEL
Suggests that the strands can:
1) ___ and
2) act as ___ for the formation of a ___
A
1 separate
2 template
new complementary strand
9
Q
parental DNA strands remained together in 1 of the daughter cell the newly synthesized DNA strands went to the other daughter cell
A
Conservative Replication
10
Q
- each daughter cell received 1 parental DNA strand and 1 newly synthesized complementary
strand parental strand was the template
A
Semiconservative Replication
11
Q
In __, ____ and ___ showed that DNA replicates semi conservatively in the ____ expe.
A
1957
Matthew Stanley Meselson
Franklin Stahl
Meselson- Stahl
12
Q
DNA Polymerase
Direction
Function
Example enzymes
A
5’->3’
DNA synthesis
DNA Pol III (bacteria), DNA Pol delta (eukaryotes)
13
Q
Exonuclease (proofreading)
Direction
Function
Example enzymes
A
3’->5’
Error correction
DNA Pol I, III (bacteria), DNA Pol delta and epsilon (eukaryotes)
14
Q
Exonuclease (primer removal)
Direction
Function
Example enzymes
A
5’3-3’
Primer/ DNA Fragment removal
DNA Pol I (bacteria)
15
Q
Definition: The process of copying
DNA prior to cell division
A
DNA Replication
16
Q
Each new DNA molecule consists of one parentaland one newly synthesized strand.
A
Semi-Conservative Model
17
Q
*Cell Cycle Context: In eukaryotes,
DNA replication occurs during the ___of the cell cycle.
A
S- phase
17
Q
Basic Requirements for DNA Synthesis
1
2
3
4
A
Substrates
Template
Primer
Enzyme
18
Q
Substrates
- 4 deoxynucleotide triphosphates (dNTPs)
A
- deoxyadenosine triphosphate (dATP)
- deoxyguanosine triphosphate (dGTP)
- deoxycytidine triphosphate (dCTP)
- deoxythymidine triphosphate (dTTP)
19
Q
- directs the addition of a complementary nucleotide
- it acts in semiconservative replication, each of the parental DNA strands.
A
Template
20
Q
prepares the template strand for the addition of nucleotides
- new nucleotides are added to the 3’-end of the primer → new synthesis is said to occur in a 5’
→3’ direction
A
Primer
21
Q
Enzyme Requirements for DNA Synthesis
A
- DNA-dependent DNA polymerases
22
Q
Characteristics of Prokaryotic
in DNA Replication
*Genome Structure: ___, ___
*Origin of Replication:____
*Replication Speed: ___
*Replication Direction: ___
A
- Single, circular chromosome
- Typically a single origin known as OriC
- Approximately 1000 nucleotides per second
- Bidirectional from the origin
23
Q
Initiation in Prokaryotes
A
DnaA Protein
DnaB (Helicase)
Single-Strand Binding Proteins
(SSBs)
DNA Gyrase (Topoisomerase
II):
Primase (DnaG)
24
Binds to OriC, causing
localized unwinding of DNA.
DnaA Protein
25
Unwinds the DNA
helix ahead of the replication fork.
DnaB (Helicase)
26
Stabilize the unwound single-
stranded DNA.
Single-Strand Binding Proteins
(SSBs)
27
Alleviates supercoiling tension
ahead of the replication fork.
DNA Gyrase (Topoisomerase
II)
28
Synthesizes short
RNA primers to provide starting
points for DNA synthesis
Primase (DnaG)
29
Elongation in Prokaryotes
*Leading Strand Synthesis: DNA Polymerase III
*Lagging Strand Synthesis
* DNA Polymerase III
* DNA Polymerase I
* DNA Ligase
30
Continuous synthesis by DNA Polymerase III in the 5' to 3' direction.
Leading Strand Synthesis
31
Discontinuous
synthesis creating ___
Lagging Strand Synthesis
- Okazaki fragments
32
Extends RNA primers with DNA nucleotides
DNA Polymerase III
33
Removes RNA primers and replaces them with DNA
nucleotides.
DNA Polymerase I
34
Seals the nicks between
Okazaki fragments, forming a continuous DNA strand.
DNA Ligase
35
TERMINATION in Prokaryotes
*Tus Proteins
*Topoisomerase IV
36
Prokaryotes
*Termination Sites: Specific
sequences called ____ signal the
end of replication
Ter sites
37
Bind to Ter sites,
inhibiting helicase activity and
halting replication.
Tus Proteins
38
Decatenates interlinked daughter chromosomes, ensuring proper segregation.
Topoisomerase IV
39
Characteristics of Eukaryotic DNA Replication
*Genome Structure: ___
*Origins of Replication: __
*Replication Speed: Approximately ___
*Regulation: Tightly controlled by ___ to ensure accuracy.
- Multiple linear chromosomes
- Multiple origins along each chromosome
- 50 nucleotides per second
- cell cycle checkpoints
40
Initiation in Eukaryotes
* MCM Helicase
* Replication Protein A
(RPA)
* Topoisomerases I & II
* DNA Primase-Polymerase
41
In eukaryotes, it Binds to replication origins,
marking them for initiation.
* Origin Recognition Complex
(ORC)
42
Unwinds the DNA double helix at the replication fork.
MCM Helicase
43
Stabilizes single-stranded
DNA regions in Eukaryotes.
Replication Protein A
(RPA)
44
In eukaryotes, Relieve supercoiling tension ahead of the replication fork.
Topoisomerases I & II
45
Complex Synthesizes RNA primers
and initiates DNA synthesis.
DNA Primase-Polymerase
46
Primosome- a Combination Of
1
2
1. Prepriming complex
2. Primase
47
formed prior to RNA primer synthesis
–- group of several proteins
–- binds to the single strand of DNA → displacing some of the
____
–- displaces SSB in an ATP-dependent process
–- initiates ___formation by moving along the lagging strand in the 5’ → 3’ direction
Prepriming Complex
- single-stranded DNA-binding proteins
- Okazaki fragment
48
Elongation in Eukaryotes
*Primer Removal and Replacement: Removes RNA primers.
1
2
*Leading Strand Synthesis: Carried out by DNA Polymerase.
*Lagging Strand Synthesis: Carried out by DNA Polymerase, producing Okazaki fragments.
* FEN1 (Flap Endonuclease 1)
* DNA Polymerase
*DNA Ligase I
49
alter DNA supercoiling by making transient single strand breaks ___ or ___
TOPOISOMERASE
type 1 or double strand breaks
50
Activities of Type I DNA Topoisomerases (swivel)
1
2
- nuclease (strand-cutting)
- ligase (strand-resealing)
51
- relax only negatively supercoiled DNA (fewer helical turns
than the relaxed DNA) in __ by changing the linking number in increments of one
Type IA Topoisomerases
- E. coli
52
- relax negative and positive supercoils by a controlled
rotation mechanism in eukaryotic cells
Type IB Topoisomerases
53
Eukaryotic cells
Topoisomerase I and II
54
Prokaryotic cell
Topoisomerase IV
55
inhibit top 1 and cancer cells (eukaryote)
Irinotecan/ Topotecan
56
- bind tightly to the DNA double helix
- make transient breaks in both strands → allow a 2nd
stretch of DNA double-helix to pass through the break
→reseal transient break → relief of negative and positive supercoils (ATP-dependent)
-required in both eukaryotes and prokaryotes for the
separation of interlocked DNA molecules following
chromosomal replication
Type II DNA Topoisomerases
57
Inhibit Top 2 and cancer
cells (Eukaryote) and ____ in
(Prokaryotes)
Etoposide/Tenoposide
- Fluoroquinolones
58
TERMINATION in Eukaryotes
DNA polymerase
Telomerase
59
*End Replication Problem: Due to the inability of__ to fully replicate the _
DNA polymerases
- 3' ends of linear chromosomes
60
a ribonucleoprotein enzyme, extends telomeres using its RNA component as a template.
Telomerase
61
*Telomere Capping: __binds
to telomeres, protecting them from
degradation and preventing recognition as
DNA damage.
Shelterin Complex
62
are repetitive DNA sequences (__
in humans) at the ends of
eukaryotic chromosomes.
*They act as protective
caps, preventing the loss of
essential genetic information during DNA replication.
Telomeres
- TTAGGG
63
Checkpoints Ensuring Accurate Replication
*S-Phase Checkpoint
ATR & ATM Kinases:
*G2/M Checkpoint
p53 & Rb Proteins:
*Mismatch Repair System (MMR):
MLH1, MSH2, MSH6:
64
Ensures DNA damage is
repaired before replication continues.
*S-Phase Checkpoint:
65
Detect stalled replication
forks and activate repair pathways.
* ATR & ATM Kinases:
66
Prevents mitosis if replication is
incomplete.
*G2/M Checkpoint:
67
Halt cell cycle progression
if errors are detected.
p53 & Rb Proteins:
68
Detect and correct
base-pair mismatches after replication.
*Mismatch Repair System (MMR):
MLH1, MSH2, MSH6:
69
function of telomeres
Prevention of Genetic Loss
Chromosome Protection
Regulation of Cellular Lifespan
Cancer and Immortality
70
Due to the end-replication problem, DNA polymerase cannot fully replicate the 3' ends of linear chromosomes. Without __, this would lead to progressive
gene loss over time.
Prevention of Genetic Loss
- telomeres
71
1. Prevents chromosomes from fusing with each other, which could cause__
2. ___ binds to
telomeres and shields them from degradation.
Chromosome Protection
- genome instability
- The Shelterin complex (TRF1, TRF2, POT1, TIN2, RAP1, and TPP1)
72
1. ___ shortening acts as a biological clock for cellular aging (senescence).
2. When __ become critically short, cells undergo apoptosis or enter a
state of permanent cell cycle arrest (___).
Regulation of Cellular Lifespan
- Telomere
- telomeres
- replicative senescence
73
. Cancer cells reactivate __, allowing them to divide indefinitely.
2. ___of cancers show upregulated telomerase activity, making telomerase a key target for cancer therapies.
Cancer and Immortality
-telomerase
-85–90%
74
Cancer (Uncontrolled Cell Division)
*p53 Mutations:
*Defective DNA Polymerases:
75
* Prevents apoptosis in cells with
replication errors.
* Leads to accumulation of mutations
→ cancer development.
*p53 Mutations:
76
* Causes excessive mutations (e.g.,
Polymerase e mutations in colorectal cancer).
*Defective DNA Polymerases:
77
- *Defective RecQ Helicase (BLM
gene mutation) *Leads to increased
recombination and chromosomal
instability.
*Symptoms: __,__,__
Bloom Syndrome
- Short stature, cancer
predisposition, sun sensitivity
78
*Defective WRN Helicase, involved in telomere
maintenance.
*Causes __,__,__
Werner Syndrome
- premature aging, cardiovascular disease, and increased cancer risk.
79
80
*Defective Mismatch Repair (MMR) Proteins (MLH1,
MSH2, MSH6).
*Leads to microsatellite instability and increased risk of colorectal and endometrial cancers.
Lynch Syndrome (HNPCC - Hereditary Non- Polyposis Colorectal Cancer)
81
*Defective - Nucleotide Excision Repair (NER)
enzymes__,__,__
*Inability to repair UV-induced thymine dimers
→ extreme sensitivity to sunlight.
*Leads to early onset skin cancers.
Xeroderma Pigmentosum (XP)
- (XPA, XPC, XPV, etc.)
82
*Defect in ATR kinase
, which monitors
replication stress.
*Causes_,_,_
Seckel Syndrome
- growth retardation, microcephaly, and developmental delays.
83
*Defective DNA Interstrand Crosslink Repair.
*__,__,__
Fanconi Anemia
- Bone marrow failure, leukemia predisposition,
developmental abnormalities.
84
Prokaryotic DNA Replication: Simple, fast, single-origin, involves __,__,__
- DnaA, DnaB, Pol III.
85
Eukaryotic DNA Replication: Complex, multiple origins, regulated by
__,__,__,_
ORC, Pol a, Pol d, and e.
86
Critical for chromosome
stability.
* Telomerase Resolves End-Replication Problem:
87
Checkpoints Ensure Accuracy: __,__,_
ATR/ATM kinases, p53, CDKs regulate
replication.
88
Replication Defects Lead to Disease: _,_,_
Cancers, aging syndromes, and
genomic instability disorders.
89
Key Features of Mitochondrial DNA
Replication
*Circular, Double-Stranded DNA (~16.5 kb in humans).
*Multiple Origins of Replication ___
*Independent of Cell Cycle Regulation (Unlike nuclear DNA, which is___).
- Minimal Error Repair Mechanisms-> Higher mutation rates.
- (OriH and OriL)
- S- phase dependent
90
Overview of Transcription
*Template: DNA (sense and antisense strands;
RNA is transcribed from the antisense strand).
*Product: RNA (mRNA, tRNA, rRNA, and other
non-coding RNAs).
*Direction: RNA synthesis occurs in the 5' to 3'
direction, meaning nucleotides are added to the
3' end of the growing RNA chain.
*Enzyme: RNA polymerase catalyzes the
reaction.
*Regulation: Controlled by promoter sequences,
transcription factors, enhancers, and repressors.
91
*Product of Transcription
RNA (mRNA, tRNA, rRNA, and other
non-coding RNAs).
92
*Template: DNA (__ and __;
__ is transcribed from the antisense strand).
Transcription
- sense and antisense strands
- RNA
93
Transcription Direction: RNA synthesis occurs in __
direction, meaning __ are added to the
3' end of the growing RNA chain.
the 5' to 3'
- nucleotides
94
Transcription Enzyme:__ catalyzes the
reaction.
RNA polymerase
95
Transcription *Regulation: Controlled by__,__,__ and _
promoter sequences,
transcription factors, enhancers, and repressors.
96
*The process begins at a __, a specific DNA sequence that
RNA polymerase recognizes and binds to.
Initiation of transcription
- promoter
97
Prokaryotic RNA polymerase is a holoenzyme consisting of: – __ and __.
- Core enzyme
- Sigma factor
98
responsible for RNA synthesis
Core enzyme
99
recognizes promoter sequences and helps RNA polymerase bind correctly
Sigma factor
100
*Promoter regions contain:
* ___– Recognized by RNA polymerase for initial binding.
* ___ Important for
unwinding the DNA to allow transcription initiation.
-35 sequence (TTGACA)
- Pribnow box (-10 sequence, TATAAT)
101
102
*Once RNA polymerase binds to the promoter, it unwinds a small
section of DNA to form the __
*The first few nucleotides of RNA are synthesized, and once a
stable RNA-DNA hybrid is formed, the __is released,
allowing elongation to proceed.
transcription bubble(~17 base pairs)
- sigma factor
103
__ moves along the DNA, adding ___
complementary to the DNA
template strand.
*The enzyme does not require a
__ (unlike DNA polymerase).
*The RNA chain grows in the __direction.
- __ relieve
supercoiling ahead of the
transcription complex.
Elongation
- RNA polymerase
-ribonucleotides
- primer
- 5' to 3'
- Topoisomerases
104
There are two primary mechanisms of termination in prokaryotes:
a) Rho-independent termination (Intrinsic termination)
b) Rho-dependent termination
105
*Occurs when the RNA forms a hairpin loop followed by a
series of __
*The weak ___ between RNA and DNA cause the
RNA to detach, terminating transcription.
Rho-independent termination (Intrinsic termination)
- uracils (U-rich sequence)
- A-U bonds
106
*Involves the__, an ATP-dependent helicase.
*The __ on the RNA allows Rho to
bind and move toward RNA polymerase.
*When Rho catches up with RNA polymerase at a pause
site, it unwinds the __, releasing the __
Rho-dependent termination
- Rho protein,
- Rho binding site (Rut site)
- RNA-DNA hybrid
-RNA transcript
107
Eukaryotes have three major RNA
polymerases:
*RNA Polymerase I → Synthesizes rRNA (except 5S rRNA).
*RNA Polymerase II → Synthesizes mRNA and some snRNA (small nuclear RNA).
*RNA Polymerase III → Synthesizes tRNA, 5S rRNA, and other small RNAs.
108
Initiation
* Eukaryotic promoters are more complex than prokaryotic promoters.
* The core promoter includes:
- TATA box (~ -25 bp)
- Initiator sequence (Inr) (~ +1 position)
- Upstream elements
- General transcription factors (GTFs) (e.g., TFIID, TFIIA, TFIIB, TFIIF, TFIIE, TFIIH)
- TFIIH
109
– Recognized by TATA-binding protein (TBP),
part of the TFIID complex.
TATA box (~ -25 bp)
110
Overlaps the transcription
start site.
Initiator sequence (Inr) (~ +1 position) –
111
__ like enhancers and silencers modulate
transcription efficiency.
Upstream elements
112
assist RNA polymerase II in binding to the promoter and unwinding the
DNA.
General transcription factors (GTFs) (e.g., TFIID, TFIIA, TFIIB, TFIIF, TFIIE,
TFIIH)
113
has helicase activity, helping to separate DNA strands and initiate
transcription.
TFIIH
114
__ moves along the DNA, synthesizing RNA in the 5' to 3'
direction.
factors prevent premature termination.
* __ relieve supercoiling stress.
Elongation
* RNA polymerase II
- Topoisomerases
115
Unlike prokaryotes, eukaryotic termination mechanisms are less understood and
vary by polymerase type:
Termination
RNA Polymerase I
RNA Polymerase II
RNA Polymerase III
116
* Uses a termination factor similar to Rho-dependent termination.
RNA Polymerase I
117
* Terminates at a poly-U sequence similar to prokaryotic Rho-independent termination.
RNA Polymerase III
118
* Involves cleavage of the transcript and polyadenylation signals __
* The _ suggests that an exonuclease (e.g., Xrn2) degrades the remaining RNA,
causing polymerase dissociation.
RNA Polymerase II
- (AAUAAA)
- torpedo model
119
Post-Transcriptional Modifications (Eukaryotes Only)
Eukaryotic pre-mRNA undergoes extensive modifications before
becoming mature mRNA:
5' Capping
Splicing
3' Polyadenylation
120
*A _ cap is added to the 5' end of mRNA.
*Protects the RNA from degradation and facilitates translation
initiation.
5' Capping
- 7-methylguanosine
121
*Removes introns and joins exons.
*Conducted by the spliceosome, which consists of __(_,_,_,_,_)
Splicing
- snRNPs (U1,
U2, U4, U5, U6).
122
allows multiple proteins from one gene.
*Alternative splicing
123
*A __ is added to the 3' end.
*Increases RNA stability and facilitates export to the cytoplasm.
3' Polyadenylation
- poly(A) tail (~200 adenines)
124
Diseases Associated with Transcription
Dysregulation
*Cancer:
Mutations in transcription factors
*Neurodegenerative Diseases:
Huntington’s & Alzheimer’s Disease:
*Beta-Thalassemia:
*Lupus (SLE):
125
lead to uncontrolled cell growth.
* Mutations in transcription factors (e.g., p53, c- Myc)
126
Altered
gene expression.
Huntington’s & Alzheimer’s Disease:
127
* Mutation in
-globin gene affecting
transcription regulation.
*Beta-Thalassemia
128
* Autoantibodies against snRNPs affecting
splicing.
*Lupus (SLE):
129
The tRNA cloverleaf structure consists of five distinct regions:
1. The Acceptor Stem (3' End)
2. The D-arm (D-loop)
3. The Anticodon Arm (Anticodon Loop)
4. The Variable Arm
5. The TYC Arm (T-loop)
130
*Located at the 3' end of the tRNA.
*Has a CCA sequence (5'-CCA-3') where the amino acid is
covalently attached by aminoacyl-tRNA synthetase.
*The 5' end usually starts with a _.
The Acceptor Stem (3' End)
-G
131
*Contains _, a modified nucleotide.
*Plays a role in tRNA recognition by aminoacyl-tRNA
synthetase.
The D-arm (D-loop)
- dihydrouridine (D)
132
*Contains the anticodon triplet, which base-pairs with the
complementary mRNA codon during translation.
*Determines the specificity of tRNA for an amino acid.
The Anticodon Arm (Anticodon Loop)
133
*Size varies among tRNAs; some have a short
arm ___, while others have a long
arm __.
*Important for tRNA stability and function.
The Variable Arm
- (3-5 nucleotides)
- (13-21 nucleotides)
134
*Contains __,__ and _
*Important for ribosome recognition and binding
during translation.
The TYC Arm (T-loop)
ribothymidine (T), pseudouridine (Y)
and cytidine (C).
135
Tertiary Structure
*The 3D structure of tRNA is__ allowing it to fit into the ribosome’s __,__,__
L-shaped
A (aminoacyl), P (peptidyl), and
E (exit) sites.
136
Important Enzymes Involved in tRNA Processing and Function
1
2
3
4
1. RNA Polymerase III
2. RNase P (Ribonuclease P)
3. RNase Z
4. tRNA Nucleotidyltransferase
137
* Synthesizes tRNA transcripts in eukaryotes.
* The primary transcript undergoes several modifications before
becoming mature tRNA.
RNA Polymerase III
m
138
* A ribozyme (RNA-based enzyme) that cleaves the 5' leader
sequence of pre-tRNA.
RNase P (Ribonuclease P)
139
* Cleaves the 3' trailer sequence of pre-tRNA to help form the
functional 3' end.
RNase Z
140
* Adds the CCA sequence at the 3' end of tRNA, which is necessary
for amino acid attachment.
tRNA Nucleotidyltransferase
141
– Fixes specific DNA damage without breaking the DNA
backbone, such as __ for UV-induced thymine dimers.
Direct Repair
- photoreactivation
142
– Repairs small, non-helix-distorting base damage using __to remove damaged bases, followed by DNA
_ and _to fill and seal the gap.
Base Excision Repair (BER)
- glycosylases
- polymerase and ligase
143
Removes bulky DNA lesions, such as __ and __, by excising a short DNA
segment and filling the gap with new nucleotides.
Nucleotide Excision Repair (NER)
- pyrimidine dimers and chemical adducts
144
Corrects replication errors like base
mismatches and small insertions/deletions to maintain genomic integrity.
Mismatch Repair (MMR)
145
– Uses a sister chromatid as a template
for error-free repair.
* Homologous Recombination (HR)
146
Directly joins broken DNA ends,
which can introduce mutations but is crucial when a template is
unavailable.
Non-Homologous End Joining (NHEJ)
147
Double-Strand Break Repair (DSBR):
* Homologous Recombination (HR)
- Non-Homologous End Joining (NHEJ)
148
recog site for restrict enzyme
pallindromic sequence
149
- Main polymerase, responsible for mtDNA synthesis
DNA Polymerase y (Pol y)
150
- Unwinds mitochondrial DNA
Twinkle Helicase
151
- Stabilizes singel-stranded DNA
mtSSB (Mitochondrial Single-
Stranded Binding Protein)
152
- Provides RNA primers for DNA Pol y
PrimPol
153
- Regulates mtDNA replication and compaction
TFAM (Mitochondrial Transcription Factor A)
154
- Removes RNA primers
RNase H1
155
- Seals Okazaki fragments on the lagging strand
DNA Ligase III
156
- Resolves supercoiling during mtDNA replication
Topoisomerase 3a
