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BQ II > T.11 DNA REPLICATION > Flashcards

T.11 DNA REPLICATION Flashcards

1
Q

What is the general feature of DNA replication in most cells?

A

Most cells do not need to replicate DNA because they are not going to divide; however, transcription and translation occur continuously.

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2
Q

Why is DNA replication considered semiconservative?

A

Because the parental strand is conserved and used as a template to be copied, resulting in one old and one new strand after replication.

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3
Q

Where does DNA replication take place?

A

At the replication fork, which forms when double strands are separated, creating replication bubbles with two replication forks.

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4
Q

How does the progress of replication occur?

A

It is bidirectional due to the two forks formed being replicated simultaneously in opposite directions.

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5
Q

What is meant by DNA synthesis being semidiscontinuous?

A

New nucleotides are incorporated in the 5’ to 3’ direction; the leading strand progresses continuously, while the lagging strand forms Okazaki fragments.

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6
Q

How does DNA replication differ between prokaryotes and eukaryotes?

A

Prokaryotic DNA is circular with one origin of replication (monofocal), whereas eukaryotic linear DNA has multiple origins (multifocal) to allow faster replication.

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7
Q

What are the general properties of DNA polymerases?

A

All DNA polymerases have DNA polymerase and 3’→5’ exonuclease active sites, are processive enzymes, and require a template strand and a primer.

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8
Q

How do DNA polymerases ensure accuracy?

A

Their active centers are very specific, allowing only correct bases to fit, minimizing mistakes during DNA synthesis.

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9
Q

What substrates do DNA polymerases use, and how are nucleotides added?

A

They use deoxynucleotides (dNTPs) as substrates, adding them in the 5’→3’ direction; phosphates are stabilized by Mg2+ and added via nucleophilic addition.

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10
Q

What happens when a wrong base is incorporated during DNA synthesis?

A

Elongation is blocked; DNA polymerase slides back using its 3’→5’ exonuclease activity to remove the incorrect nucleotide, then resumes polymerization.

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11
Q

What is the role of DNA polymerase III in prokaryotes?

A

It contains three cores (two for the lagging strand and one for the leading strand), has a clamp loader to keep the complex together, and exhibits high processivity with a low mistake rate.

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12
Q

What are the roles of DNA polymerases ε and δ in eukaryotes?

A

DNA polymerase ε acts on the leading strand, while DNA polymerase δ acts on the lagging strand; the clamp loader in eukaryotes is known as RFC.

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13
Q

What is the function of the clamp in DNA replication?

A

It fixes the template strand and primer to the enzyme core to avoid mistakes; in prokaryotes, it’s a ring formed by two β subunits; in eukaryotes, it’s formed by three subunits referred to as PCNA.

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14
Q

How is the clamp loaded onto DNA?

A

The clamp binds to the clamp loader, consuming ATP; this binding causes a conformational change, opening the clamp subunit, allowing DNA to enter, promoting ATP hydrolysis, and releasing the clamp bound to DNA.

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15
Q

What is the function of helicases in DNA replication?

A

They are hexamers located at the replication fork ahead of DNA polymerase, unwinding DNA to obtain single-stranded DNA; eukaryotes require two helicases, one for each replication fork.

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16
Q

What is the role of topoisomerases during DNA replication?

A

They remove supercoiling ahead of the replication fork to prevent DNA tangling and ensure smooth replication.

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17
Q

How is single-stranded DNA protected during replication?

A

In prokaryotes, single-strand binding proteins (SSBs) protect DNA from nucleases; in eukaryotes, replication protein A (RPA) serves this protective function.

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18
Q

What synthesizes primers in eukaryotic DNA replication?

A

DNA polymerase α synthesizes primers in eukaryotes.

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19
Q

What synthesizes primers in prokaryotic DNA replication?

A

DNA primase synthesizes primers in prokaryotes.

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20
Q

What is the role of DNA polymerase I in prokaryotes?

A

It is involved in the maturation of Okazaki fragments by removing RNA primers and replacing them with DNA through nick translation; it has primer removal (exonuclease), DNA synthesis (polymerase), and proofreading (exonuclease) activities.

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21
Q

What is nick translation in DNA replication?

A

It’s the process where DNA polymerase I recognizes a nick (lack of phosphodiester bond between Okazaki fragments), binds to the primer, substitutes ribonucleotides with deoxyribonucleotides, and creates a new nick at the end of the substituted fragment.

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22
Q

What is the function of FEN1 in eukaryotic DNA replication?

A

FEN1 is an endonuclease that recognizes single-stranded DNA; it acts when DNA polymerase continues synthesis beyond the primer, creating a single-stranded flap, which FEN1 cuts to remove the primer.

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23
Q

How are Okazaki fragments ligated after primer removal?

A

Ligase performs three steps: adenylylation of DNA ligase, activation of the 5’ phosphate in the nick, and displacement of AMP to seal the nick, resulting in fragments bound by phosphodiester bonds.

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24
Q

What is the role of DnaA and ORC in DNA replication?

A

In prokaryotes, DnaA recognizes the origin of replication; in eukaryotes, the origin recognition complex (ORC) serves this function.

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25
How is the helicase loaded onto DNA during replication?
In prokaryotes, DnaC loads the helicase; in eukaryotes, Ctd1 and cdc6 perform this function.
26
What helicases are involved in DNA unwinding during replication?
In prokaryotes, DnaB helicase unwinds DNA; in eukaryotes, the Mcm2-7 complex serves as the helicase.
27
How is single-stranded DNA protected during replication?
In prokaryotes, single-strand binding proteins (SSBs) protect DNA; in eukaryotes, replication protein A (RPA) provides this protection.
28
Which DNA polymerases are involved in synthesis and proofreading?
In prokaryotes, DNA polymerase III performs synthesis and proofreading; in eukaryotes, DNA polymerases ε and δ carry out these functions.
29
What clamps are used to fix DNA to the active site during replication?
In prokaryotes, the β clamp is used; in eukaryotes, PCNA serves as the clamp.
30
What proteins load clamps onto DNA during replication?
In prokaryotes, the clamp loader loads the clamp; in eukaryotes, RFC (replication factor C) performs this task.
31
Which enzymes synthesize primers during replication?
In prokaryotes, DnaG (primase) synthesizes primers; in eukaryotes, DNA polymerase α carries out primer synthesis.
32
What enzymes substitute Okazaki fragment primers?
In prokaryotes, DNA polymerase I performs this substitution; in eukaryotes, DNA polymerase and exonuclease activities are involved.
33
What is the function of FEN1 in DNA replication?
FEN1 is an endonuclease that removes RNA primers during Okazaki fragment processing in eukaryotes.
34
How are Okazaki fragments ligated during replication?
Ligase seals the nicks between Okazaki fragments in both prokaryotes and eukaryotes.
35
What is the role of topoisomerase in DNA replication?
Topoisomerase cleaves and rejoins DNA strands to remove supercoiling during replication.
36
What proteins are involved in replication termination?
In prokaryotes, Tus protein terminates replication; in eukaryotes, telomerase performs reverse transcription and synthesizes telomeres.
37
What is the function of Dam methylase in DNA replication?
Dam methylase methylates 5’ (GATC) sequences at the origin of replication (oriC) in prokaryotes.
38
What is the goal of the initiation step in prokaryotic DNA replication?
To separate the double-stranded DNA into two individual strands at the origin of replication.
39
Who proposed the replication model in prokaryotes?
Jacob, Brenner, and Cuzyn.
40
What are the two components involved in initiation of prokaryotic DNA replication?
A replicator (a DNA sequence) and an initiator (proteins recognizing a DNA element on the replicator).
41
What is the structure of the replication origin in prokaryotes?
A tandem array mainly of adenine and thymine bases.
42
Why does the initiator protein prefer A=T base pairs?
Because A=T base pairs form weaker bonds than C≡G, making them easier to unwind.
43
What is the OriC sequence?
It contains binding sites for DnaA proteins, each formed by 9 base pair sequences.
44
What happens first during replication bubble formation?
The OriC segment supercoils just after the tandem array of three 13 bp sequences.
45
What is the role of DnaA in replication bubble formation?
DnaA molecules recognize the OriC segment, bind using ATP, and initiate bubble formation.
46
Which proteins assist in separating the strands after DnaA action?
Destabilizer proteins such as HU bind to the double helix and help divide it into two single strands.
47
What is the role of DnaC in DNA replication?
DnaC loads the helicase (DnaB) onto DNA.
48
What does DnaB do during bubble formation?
DnaB uncoils the double helix to form an open region, establishing two replication forks.
49
What drives the physical opening of the replication bubble?
Tension generated by DnaA acting on weaker A=T bonds.
50
What process is affected by the regulation of DNA synthesis in prokaryotes?
The initiation process.
51
Which enzyme is involved in regulation of DNA replication initiation?
Dam methylase.
52
When does replication initiation occur regarding methylation?
Only when both strands at the origin are methylated.
53
What is hemimethylation in DNA replication?
When the template strand is methylated and the newly synthesized strand is not.
54
What is the role of SeqA in replication regulation?
SeqA binds to the hemimethylated origin, preventing Dam methylase from binding and inhibiting re-initiation.
55
How is SeqA released to allow another round of replication?
ATP accumulates, leading to SeqA release and allowing Dam methylase to methylate the strand.
56
When can DnaA initiate replication after methylation?
Once the origin is fully methylated, DnaA can recognize and bind to initiate replication.
57
How does elongation occur at the replication fork in prokaryotes?
It proceeds bidirectionally, forming loops.
58
What is the difference between elongation on the leading and lagging strands?
Leading strand elongation is continuous, while lagging strand elongation requires Okazaki fragments.
59
What is the role of RNA primase in DNA replication?
It synthesizes RNA primers needed for DNA polymerase III to start DNA synthesis.
60
Where does DNA polymerase III bind during replication?
To the 3’ end of the RNA primer.
61
How does DNA polymerase III function on the lagging strand?
It synthesizes DNA up to the previous Okazaki fragment.
62
What happens after an Okazaki fragment is completed?
A clamp is released from DNA polymerase I to fix the primer and template strand.
63
What is the exonuclease activity of DNA polymerase I used for?
To remove the RNA primer and fill in the resulting gap with DNA.
64
What is the final step after DNA polymerase I action on Okazaki fragments?
Ligases seal the strands, forming two semiconservative double helices.
65
What proteins are involved in the maturation of Okazaki fragments?
Primases, clamps, and ligases.
66
Why are primases, clamps, and ligases continuously exchanged?
Because each Okazaki fragment requires its own primer, primase, and clamp loader.
67
Do synthesis and maturation of Okazaki fragments occur sequentially or concurrently?
They occur simultaneously (concurrently).
68
What defines the termination of DNA replication in prokaryotes?
Termination sequences (Ter) specific to each replication fork.
69
What is the role of Tus protein in replication termination?
Tus binds to Ter sequences and blocks the replication fork and helicase activity.
70
What are the two faces of the Tus protein and what do they do?
The permissive face allows replication to pass, while the non-permissive face blocks it.
71
In which direction does the Tus protein block replication?
When the fork moves toward the non-permissive face of Tus.
72
What phases make up the eukaryotic cell cycle?
G0, S, G2, and M.
73
What happens during the G0 phase?
Cellular content (excluding chromosomes) is duplicated.
74
What occurs in the S phase of the cell cycle?
DNA replication is rapidly completed; all 46 chromosomes are duplicated.
75
What is the function of the G2 phase?
The cell double-checks duplicated chromosomes for errors and repairs them.
76
What occurs during the M phase?
Mitosis and cytokinesis.
77
When is replication initiated in the eukaryotic cell cycle?
It starts in the S phase and must be completed by the end of S phase.
78
How often do chromosomes replicate during the cell cycle?
Only once per cycle.
79
What happens at the beginning of the G2 phase?
Genomic instability is induced to prepare for chromosome segmentation.
80
Why must duplicated DNA be separated from non-duplicated DNA in G2?
To ensure proper chromosome segregation.
81
How many origins of replication do eukaryotes have?
Multiple origins to allow complete chromosome duplication in limited time.
82
Why can no eukaryotic origin of replication be fired twice?
To prevent re-replication of chromosomes during a single cycle.
83
What controls origin selection in eukaryotic replication?
A variety of factors including sequence, structure, and chromatin context.
84
What is the difference between active and passive origins?
Active origins require activation to initiate replication; passive origins initiate when the replication fork reaches them.
85
What factors influence origin distribution and activation?
Fork rate and the presence of fork blocks.
86
How does a fast fork rate affect origin distribution?
It results in fewer replication origins.
87
How does a slow fork rate affect origin distribution?
It increases the number of replication origins.
88
What happens when fork blocks are present?
Dormant (passive) origins are fired to continue replication.
89
What does the eukaryotic initiator ORC bind to?
ORC binds to the origin of replication, whether active or passive.
90
What proteins are attracted by ORC at the origin?
Cdc6 and Cdt1.
91
What is the function of Cdc6 and Cdt1 at the replication fork?
They act as helicase loaders at the replication fork.
92
What happens to helicase loaders during S phase?
They are phosphorylated, ubiquitinated, and degraded.
93
What forms the licensing system at each replication fork?
The complex of helicase loaders and ORC recruits inactive helicases (Mcm2-7), forming the licensing system.
94
When does replication begin in relation to licensing?
Replication begins after licensing is complete and G1 ends.
95
What happens after replication is complete to prevent re-replication?
Helicase loaders are degraded so no more helicases can be loaded, ending the S phase.
96
Why can't new replication origins be formed during S phase?
Because licensing is blocked during S phase, so replication cannot continue if a fork is blocked.
97
How is replication ensured if forks are blocked during S phase?
All replication origins (active and passive) are licensed before S phase so dormant origins can be fired.
98
Why can replication only occur once per cycle?
Because origins cannot fire twice and licensing system formation is blocked during S phase.
99
Why is pre-RC assembly tightly regulated?
To ensure only one duplication wave per cell cycle.
100
What enzyme is key in regulating Pre-RC assembly and activation?
CDK (Cyclin-dependent kinase).
101
Why is CDK inactive during G1 phase?
Because there is no cyclin synthesis in G1, so CDK remains inactive.
102
What does inactive CDK during G1 allow?
Assembly of the Pre-RC.
103
What happens to CDK in the S phase?
Cyclins are synthesized, bind to CDK, and activate it.
104
What does active CDK do in S phase regarding Pre-RC?
It inhibits new Pre-RC assembly but activates existing Pre-RC.
105
List 3 functions of CDK during replication.
1. Inhibits licensing of new origins. 2. Phosphorylates replisomes (Cdc6, Cdt1). 3. Promotes recruitment of proteins to licensed origins.
106
Why do dormant origins exist in excess?
Because factors for replication are limited, and dormant origins can act passively when forks progress.
107
What happens to Pre-RC in dormant origins?
It is degraded, allowing passive replication.
108
How does eukaryotic replication initiation differ from prokaryotes?
Eukaryotes need Pre-RC assembly and activation, which is absent in prokaryotes.
109
What is similar between prokaryotic and eukaryotic elongation?
Elongation is nearly the same in both.
110
What is unique about eukaryotic replication termination?
It requires telomerase for Okazaki fragment maturation.
111
Why can't primase synthesize a primer on the last part of Okazaki fragment?
Because there's no room at the end of the chromosome, so DNA pol cannot continue synthesis.
112
What consequence occurs without synthesis of the last Okazaki fragment?
Chromosomes become shorter each replication cycle, risking gene loss.
113
How does telomerase prevent chromosome shortening?
It adds hexanucleotide repeats to the 3’ end of the template strand using reverse transcriptase activity.
114
What are telomeres?
Repeated sequences added by telomerase to protect chromosome ends and allow synthesis of final Okazaki fragment.
115
What happens after telomerase extends the 3’ end?
Primase and DNA pol synthesize the complementary strand.
116
What does the final Okazaki fragment contain?
Part original sequence and part telomerase-generated non-informative repeats.
117
Why is telomerase active in reproductive cells?
To maintain telomere length across generations and prevent extinction.
118
What is the telomerase length difference between sexes?
In women it has 61 nucleotides, in men just 28.
119
What happens to telomeres with each replication cycle?
Their length is reduced.

BQ II (17 decks)

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