Lec 03- DNA Replication, Repair, and Recombination 1 Flashcards
1
Q
What is the error rate following DNA replication?
A
1 mistake in every 10^9 base pairs
2
Q
How many base pairs are in the human genome?
A
3x10^9 base pairs
3
Q
How many nucleotides are changed every cell division?
A
3 nucleotides
4
Q
How are most DNA replication errors corrected?
A
- proofreading
- DNA repair
5
Q
How are DNA errors corrected post-replication?
A
by repair mechanisms
6
Q
Multicellular organisms need high ___________
A
fidelity replication
7
Q
What do germ cells have to have in order to maintain the species?
A
low mutation rates
8
Q
Why do somatic cells need low mutation rates?
A
to avoid uncontrolled proliferation/cancer
9
Q
How does DNA polymerase synthesize DNA?
A
by catalyzing the reaction:
(DNA)n residues + dNTP»_space; (DNA)n+1 residues + P2O7
10
Q
What does template directed DNA replication mean?
A
new chain is assembled in a preexisting DNA template that is complementary to the incoming bases
11
Q
What does DNA replication require?
A
dATP
dGTP
dCTP
dTTP
12
Q
What does DNA polymerase require?
A
Requires a primer with a free 3’OH to begin
13
Q
Both strands of the replication fork are ___________ replicated
A
simultaneously
14
Q
In what direction can DNA polymerase synthesize DNA?
A
5’»_space; 3’
15
Q
Which DNA strand is synthesized continuously?
A
leading strand
16
Q
Which DNA strand is synthesized in segments?
A
lagging strand
17
Q
What is the first step of proofreading?
A
- just before a new nucleotide is added
- enzyme must tighten its fingers around the active site
- easier if the correct base is in place
18
Q
When does exonucleolytic proofreading take place?
A
immediately after incorrect bases are added
19
Q
What type of terminus does DNA polymerase require?
A
a perfectly paired 3’ terminus
20
Q
What clips off unpaired residues at 3’ primer terminus?
A
3’»_space; 5’ exonuclease
21
Q
Why does 5’»_space; 3’ replication allow for efficient error correction?
A
because it conserves energy when correcting mistakes
22
Q
How is the lagging strand replicated?
A
through backstitching process
23
Q
What are the steps of the backstitching process if the lagging strand?
A
1- DNA primase synthesizes a 10 nt long RNA primer (to prime DNA synthesis)
2- RNA primer is erased by RNAseH and replaced with DNA
3- DNA ligase joins the ends
24
Q
Why can’t DNA initiate de novo synthesis in the lagging strand?
A
because it would increase the mutation rate
25
What does RNAseH do?
recognizes RNA/DNA hybrids
26
What does DNA helicase do?
unwinds DNA
27
What is DNA helicase?
a protein with 6 identical subunits that bind and hydrolyzes ATP
28
What happens when DNA helicase binds?
causes conformational change that propels it along the single stranded DNA
29
How fast can DNA helicase pry apart the helix?
1000 nucleotide pairs/second
30
What do single-stranded DNA binding proteins do?
- bind tightly and cooperatively to exposed single stranded DNA
- help stabilize unwound DNA
- prevent formation of hairpins
- DNA bases remain exposed
31
What does the sliding clamp do?
-keeps DNA polymerase on DNA when moving
32
When does the sliding clamp release?
when double stranded DNA is encountered
33
What does a clamp holder do?
- required for assembly
| - hydrolyzes ATP as it loads the clamp onto a primer-template junction
34
What happens to the clamp on the leading strand?
clamp remains associated to DNA polymerase for long stretches
35
What happens to the clamp on the lagging strand?
clamp loader stays close so it can assemble a new clamp at the start of each new Okazaki fragment
36
What is mismatch repair?
- removal of almost all errors that are missed by proofreading
- detects distortion caused by mispairing
37
How does mismatch repair know which strand is correct in E. Coli?
depends on methylation to distinguish new strand
38
How does mismatch repair know which strand is correct in humans?
depends on single strand breaks
39
Where are single strand breaks present in humans?
present on lagging strand before Okazaki fragments are ligated
40
What does MutS do?
binds to mismatch
41
What does MutL do?
- scans for the nick
| - triggers degradation of nicked strand
42
How many base pairs make one turn?
10 bp
43
What is DNA topoisomerase?
a reversible enzme
44
What does DNA topoisomerase do?
- breaks a phosphodiester bond to change superhelicity
| - relieves supercoiling
45
What does type 1 topoisomerase do?
catalyzes the reaction of supercoiled DNA
thermodynamically favorable process
46
How does type 1 topoisomerase work?
- creates transient single strand break in DNA
- allows DNA on either side of the nick to rotate freely relative to each other
- use other phosphodiester bond as a swivel point
47
Why doesn't DNA resealing require any energy after Type I topoisomerases?
- rapid
| - energy is stored in the phosphotyrosine linkage
48
What are Type II Topoisomerases?
-enzymes that make a transient double-stranaded break in the DNA
49
Where are Type II Topoisomerases activated?
at sites on chromosome where 2 double-stranded helices cross each other
50
How do Type II Topoisomerases use ATP?
1) Break 1 double-stranded helix reversibly to crease "gate"
2) Cause 2nd strand to pass through
3) Reseals break and dissociates
51
What are the 2 functions of Type II Topoisomerase?
- separate "decatenate" two interlocked DNA circles
| - prevent severe tangling problems that would arise during DNA replication
52
What are replication origins?
A-T rich regions where sequence attacks initiator proteins to pry open DNA
(similar process for PRO and EUK)
53
Why is initiation highly regulated for E. coli replication?
Because it is the only point of control for E. coli
54
Initiation of E. coli replication proceeds only when __________.
sufficient nutrients are present
55
What is the refractory period for the initiation of E. coli replication?
a delay in replication until the new strand is methylated
56
How long does it take for bacterial genomes to replicate using 2 replication forks?
40 minutes
57
How long does it take for EUK genomes to replicate using a single ORI?
800 hours for an average chromosome with a single ORI
| traveling 50 nt/sec
58
What proteins bind to specific sites in ORI to form a complex?
initiator proteins
59
What does the initiator protein complex attract?
- DNA helicase
| - helicase loader
60
What is placed around a single-stranded DNA that is exposed by the initiator complex?
helicase
61
What remains engaged until the helicase is properly loaded?
the helicase loader
62
Why does helicase unwind the DNA?
so the primes can make the RNA primer on the Leading strand
63
What do the remaining proteins do after helicase unwinds the DNA?
remaining proteins assemble to create 2 replication forks with complexes moving in opposite direction with respect to the ORI
64
During which phase does EUK DNA replication occur?
during DNA synthesis (S) phase
65
How long does EUK S-phase last?
8 hours
66
Why are chromosomes replicated?
to produce 2 complete copies
67
Until which phase are the chromosomes joined at centromeres?
until M phase
68
Replication is activated in ___________ consisting of 20-80 origins.
clusters/replication units
69
Depending on chromatin structure, different regions of each chromosome are replicated in a reproducible order during _______ phase.
S phase
70
With timing related to the packing of DNA in chromatin, what is late-replicating?
heterochromatin
71
X chromosomes of females:
Almost all of inactive X is condensed into heterochromatin and is replicated late in S-phase. The active homolog is __________ and replicates through ______ phase
- less condensed
| - S-phase
72
Which regions of a genome replicate first?
regions with less condensed chromatin
73
What are the 3 minimum requirements for a sequence to be ORI (yeast)?
- binding site for ORC (origin recognition complex)
- A-T rich stretch for easy unwinding
- binding site for proteins (Abf1) that help attract ORC
74
ORC interaction with ______ persists throughout the cell cycle.
ORI
75
What proteins bind to ORC to form a pre-replicative complex and regulate origin activity?
- helicase
| - helicase loading proteins (Cdc6 and Cdt1)
76
What 4 things does activated Cdks lead to in S-phase?
1) dissociation of helicase loading proteins
2) activation of helicase
3) unwinding of DNA
4) loading of DNA polymerase, etc..
77
Assembly of new ORC is prevented until ___________
next M-phase resets the cycle
78
When is the single chance for ORC to form in G1?
when Cdk activity is low
79
What is the second window for pre-replicative complex to be activated and disassembled in S-phase?
when CDk activity is high
80
Specific human sequences have been identified that can serve as ORIs. They are _______ nucleotide pairs in length.
1000
81
How can specific human sequences still function if they are moved to a different locus?
must be placed where chromatin is uncondensed
82
What does ORI function depend on?
distant sequences
83
ORI dependence on distant sequences also affects _______ and has a global effect of _________________.
- affects transcription
| - decondensing chromatin structure
84
What else is required for replication besides DNA replication?
synthesis and assembly of new proteins
85
EUK have ________ copies of genes for each histone
multiple
86
In what phase are histone proteins mainly synthesized?
S-phase
87
How many histone proteins are synthesized in S-phase?
amount made = highly regulated to meet requirements
88
What is needed for efficient replication?
chromatin remodeling proteins
89
What do chromatin remodeling proteins do?
destabilize DNA-histone interface
90
What does the histone octamer break into as the replication fork passes through chromatin?
- (1) H3-H4 tetramer
| - (2) H2A-H2B dimers
91
From where are the (2) H2A-H2B dimers released as the replication fork passes through chromatin and the histone octamer breaks?
released from the DNA
92
How is the H3-H4 tetramer distributed?
distributed randomly to daughter duplexes
93
What does freshly made H3-H4 do in the reassembly process?
fills in the spaces
94
What % of the H2A-H2B dimers are new and old?
50% new
| 50% old
95
What is required for the orderly reassembly of chromatin?
histone chaperones (chromatin assembly factors)
96
What directs the histone chaperones to the DNA?
PCNA (sliding clamp)
97
Some daughter nucleosomes contain only ________ histones or only _____ histones, but most are hybrids of old and new.
- parental histones
| - new histones
98
How are parental patterns of histone modification spread?
through reader-writer complexes
99
What may be responsible for some types of epigenetic inheritance?
Patterns of histone modification
100
What happens when there is an end replication problem on the lagging strand?
there is no place for the RNA primer
101
What kind of genome does bacteria have?
circular genome
102
_______ have telomeres
Eukaryotes
103
What is the special sequence that is at the end of each chromosome and repeated x1000?
GGGTTA
104
What enzyme replenishes the special sequence at the end of the chromosome?
telomerase
105
How does the telomerase replenish the end of the chromosome?
by elongating parental strand in the 5'-->3' direction using an RNA template on the enzyme
106
After extension of parental strand by telomerase, replication of lagging strand can be completed by ______________, using the extension as a template.
DNA polymerase
107
What mechanism & nuclease ensure the 3' end is longer, leaving a protruding single stranded end that loops back and tucks into the repeat?
telomerase replication mechanism using DNA pol and 5' nuclease
108
What are T-loops?
- structures that protect chromosome ends
| - distinguishes chromosome ends from broken ends that need to be repaired
109
Our somatic cells are born with ___________.
full complement of telomere repeats
110
What can retain full telomerase activity?
stem cells
111
Each chromosome end in a given cell contains ______________ depending on age.
variable number of telomere repeats
112
What causes loss of telomere repeats?
insufficient telomerase activity
113
What are the characteristics of daughter cells after many generations?
- have defective chromosomes
| - stop dividing
114
What is replicative senescence?
- the way the cell's lifetime is regulated to guard against cancer
- daughter cells will have defective chromosomes and will stop dividing after many generations
115
How many times do normal human fibroblasts divide before undergoing replicative senescence?
60 times
116
What happens to human fibroblasts after 60 divisions when telomerase is provided experimentally?
cells will continue dividing and not undergo replicative senescence
117
What is responsible for aging in animals?
replicative senescence
118
What is the result of transgenic mice that lack telomerase?
mice develop progressive defects in highly proliferative tissues
119
What are 2 of the consequences of progressive defects in highly proliferative tissues which are a result of a lack of telomerase in transgenic mice?
- premature aging
| - prone to cancer
120
What is dyskeratosis congenita?
a disease in humans in which they carry a mutant telomerase RNA gene
121
What does dyskeratosis congenita do to the telomeres?
develop prematurely shortened telomeres
122
How do humans with dyskeratosis congenita die?
progressive bone marrow failure
123
Why is it risky for an organism to control cell proliferation using replicative senescence?
- not all cells stop dividing
| - gives rise to variant cells that lead to cancer
