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MICRO 2 > Lecture 2: Replication > Flashcards

Lecture 2: Replication Flashcards

(57 cards)

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1
Q

the replication of the E. Coli chromosome is…

A

bi-directional

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2
Q

what was John Cairns able to demonstrate about the E. coli chromosome? using autoradiography and radioactive precursors

A

there is a single region, called oriC, where
replication is initiated and a single region, called ter, where replication
terminated

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3
Q

what does OriC feature?

A

~260 bp origin of chromosomal replication (oriC) features:
* AT rich regions (bonds easier to break to separate strands)
* Binding sites for replication initiation protein ATP-bound DnaA
* Consensus sequence TTWTNCACA; N=A,T,G or C; W=A or T
* Methylation sites GATC
* Binding sites for FIS protein (inhibition of replication initiation) and IHF protein (DNA
bending assistance)

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4
Q

______: Necessary process with required accuracy to successfully transmit the
genetic information upon cell division

A

DNA replication

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5
Q

what is the precursor of each new nucelotide in the DNA strand?

A

dNTP- triphosphate! two terminal phosphates are removed when inserted

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6
Q

DNA synthesis ALWAYS goes from…

A

5’ to 3’

The 5’ phosphate of the incoming nucleotide (i.e.
deoxynucleoside 5’ triphosphate) being attached to the 3’
hydroxyl (i.e. OH) of the previously added nucleotide

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7
Q

DNA synthesis is mediated by…

A

DNA Pol I (minor) and III (major)

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8
Q

T/F: DNA polymerase can initiate DNA synthesis on its own

A

false!! only adds to existing 3’ OH- needs an RNA primer synthesized by primase

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9
Q

To open up the strands, the activity of a _______ is required which is fueled by
the energy released by hydrolysis of ATP

A

helicase

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10
Q

helicase Separates the strands just in advance of a _________

A

replication fork

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11
Q

What happens during unwinding of a circular dsDNA molecule?
what relieves this?

A
  • Supercoils! → Relieved by a special topoisomerase – DNA gyrase
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12
Q

supercoils are inserted or removed in DNA by enzymes called
_______

A

topoisomerases

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13
Q

_______ results when the DNA is twisted about its
axis in the opposite sense from the right-handed double helix →
Is the form found in most cells

A

Negative supercoiling

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14
Q

_______ results when the DNA is twisted in the
same spiral direction

A

Positive supercoiling

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15
Q

_______ inserts
negative supercoils into DNA

A

DNA gyrase

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16
Q

______ – helps stabilize ssDNA template

A

SSB

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17
Q

leading strand synthesis goes…

A

towards fork joint

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18
Q

T/F: the leading strand is only primed once

A

true!! pretty much continuous because there’s always a free 3’ OH available

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19
Q

_______
follows replication fork

A

DNA polymerase III

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20
Q

where does the lagging strand go towards?

A

OriC

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21
Q

Lagging strand synthesis (5’ to 3’ direction) → DNA synthesis is
__________ due to lack of a free 3’-OH (located on the opposite end,
away from fork joint) so made in short segments

A

discontinuous

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22
Q

what are the short segments called on the lagging strand?

A

Okakazi fragments

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23
Q

T/F: the lagging strand is only primed once

Study These Flashcards
A

false!! multiple times, segements are joined later

24
Q

T/F: DNA polymerase can only extend from 3’-OH but cannot
catalyze a phosphodiester linkage with a 5’- PO4
2- of a pre-existing
nucleotide

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A

true!! we need ligase for that

25
The _______ is a large complex of replication proteins that come together to mediate DNA replication
replisome
26
DNA gyrase is a....
type II topoisomerase- makes 2 nicks in ssDNA to remove positive supercoils
27
The subcomplex ______ includes the helicase and primase → unwinds and primes the DNA
primosome
28
T/F: helicase and primase work seperately
false!! work together- that's why they're in a complex
29
the replisome polymerizes DNA in the...
5'-3' direction One core enzyme works on each template * One proceeds towards the Replication Fork – leading strand * One proceeds toward oriC – lagging strand
30
T/F: fork movement from OriC is bi-directional
true!!!
31
E. Coli replication is....
very fast and very precise!! It is very fast * Chromosome consists of ~ 4600 kbp * E. coli replicates DNA in 40 min * Overall replication rate is ~ 100 kb/min It is very precise * Error rate is ~ 1 mistake per 109 – 1010 bp * DNA polymerase I and III have proof reading activity
32
what are some of the mechanisms we use to reduce error rate of replication?
1. Incorporation of dNTP by DNA polymerases I and III * Complementary base-pairing rules 2. DNA polymerase I has 5’ to 3’ exonuclease activity to remove RNA primer * Even though RNA primer may have a mismatch, it is a temporary mistake and will be removed by DNA polymerase I * Important for lagging strand synthesis 3. Methyl-directed mismatch repair 4. 3’ to 5’ exonuclease activity by DNA polymerases I and III * Allows DNA polymerases to back up and remove mis-basepaired nucleotide * DNA polymerase III has DnaQ * DNA polymerase I has a component with dual function of polymerization and proofreading * Mismatch is sensed when distortion due to mis-basepairing appears in helix
33
when we write genes, how do we write them?
italicized, at least first letter lowercase
34
when we write proteins, how do we write them?
not italicized, uppercase
35
what is DnaQ of DNA polymerase III
proofreading subunit
36
T/F: initiation of replication must be tightly controlled
true!!
37
When chromosomal DNA is newly replicated, the daughter chromosomes will be _______ for a little while before being DAM’ed to achieve fully methylated status.
hemi-methylated
38
what is the first control mechanism of replication initiation?
competition for oriC binding Once replication is initiated, hemi-methylated oriC sequences are strongly bound by SeqA (sequestration protein A) which blocks DnaA binding sites (a.k.a. boxes) preventing re-firing of oriC by DnaA-ATP
39
what is the secind control mechanism of replication initiation?
repression of dnaA expression The dnaA gene is located close to oriC region * Expression of dnaA is autoregulated → DnaA-ATP binds to DnaA boxes within promoter region but only in fully-methylated state * Expression of dnaA gene is subject to SeqA control
40
The oriC and dnaA promoter region also contains GATC sites that is bound by SeqA only in _______ state blocking binding of DnaA-ATP
hemi-methylated
41
what is the third control mechanism of replication initiation?
reduction of DnaA-ATP levels Prevent binding of DnaA-ATP within oriC region by decreasing ratio of DnaA-ATP to DnaA-ADP * Controlled by ATPase HdaA which associates with DNA in the proximity of the replisome and specifically targets DnaA-ATP * Promotes hydrolysis of DnaA-ATP to DnaA-ADP in a replication- dependent manner
42
T/F: cellular level of DnaA-ATP oscillates during the cell cycle
true!! reachesmax amount when replication initiation is needed
43
Studies of chromosome replication in E. coli have shown that about ________ is required for genome replication with the generation time of 60 minutes in low nutrient conditions (but sufficient to meet needs of the bacterial cells)
40 minutes
44
T/F: In rich nutrient conditions, E. coli cells grow at twice the rate that its chromosome is replicating
true!! new replication round is initiated before previous round is completed
45
MukBEF and topoisomerase IV = ________
structural maintenance of chromosome (SMC) complex
46
Division apparatus of E. Coli cell = _______
divisome
47
In rod-shaped cells, formation of divisome begins with the attachment of ______ molecules in a ring precisely around the center of the cell → cell division plane
FtsZ
48
________ proteins help guide FtsZ to cell midpoint
MinC, MinD and MinE
49
______ forms spiral structure on the inner surface of cytoplasmic membrane along long axis of cell * Prevents FtsZ ring formation inhibiting cell division
MinCD
50
_____ moves from pole to pole pushing MinC and MinD aside
MinE
51
Center of cell will have ______ of Min proteins thereby allowing FtsZ formation
lowest concentration
52
Formation of FtsZ ring (~10,000 FtsZ proteins) attracts _______
FtsA and ZipA
53
_____ anchors FtsZ ring to cytoplasmic membrane thereby stabilizing it
ZipA
54
_______ also recruits FtsZ and other divisome proteins (FtsA, FtsI, FtsK) along those needed for peptidoglycan synthesis, and helps to connect ring to cytoplasmic membrane
ATPase FtsA
55
As the cell constricts, the ______ begins to depolymerize * GTPase FtsZ hydrolyzes GTP to yield energy to fuel polymerization and depolymerization of ring
FtsZ ring
56
translocation of chromosome by _____ at division septum
FtsK
57
_____ helps regulate decatenation of chromosomes through interaction with Topoisomerase IV topoisomerase IV also interacts with chromosome condensation protein ______ to remove catenanes before DNA is condensed
FtsK MukB

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