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Molecular Genetics > Exam I - Lecture 6-9 > Flashcards

Exam I - Lecture 6-9 Flashcards

1
Q

Meselsson & Stahl’s Experiments

A

The mode of replication

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2
Q

transfer of information

A

cell division

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3
Q

perpetuate

A

make (something) continue indefinitely; preserve(something valued) from oblivon or extinction

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4
Q

DNA replication

A

The duplication of the cellular genome in which the stored genomic information is handed down to the next generation

DNA structure carries information needed to perpetuate

Each parental strand: template for one daughter strand

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5
Q

semi-conservative

A

one new strand, one old

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6
Q

conservative replication

A

One original strand, one completely new

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7
Q

Mehelson and Stahl demonstrated that replication is _______

A

semi conservative

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8
Q

dispersive replication

A

Original strand would break into chunks, and from there replicate those new strands

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9
Q

Meselson and Stahl experiment

A
  • Used two isotopes of N to change the DNA density
  • Grew bacteria in media contianing only 15 N (heavy) or 14N (light nitrogen)
  • Extracted DNA from bacteria and used density gradient centrifugation to separate “heavy” DNA from “light”
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10
Q

After the 1st generation of the Meselson and Stahl experiment, only ____ could be eliminated, because it did not form a hybrid

A

conservative

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11
Q

After the 2nd generation of the Meselson and Stahl experiment, ___ could be eliminated

A

dispersive ( because there were no heavy 15N)

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12
Q

Replicon

A

a unit of genome in which DNA is replicated

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13
Q

each replicon, has an

A

origin of replication; a DNA sequence where replication initiates

Any DNA molecule that contains an origin can be replicated in the cell

Replication is controlled at the initiation stage

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14
Q

at a replication fork

A

the DNA of both new daughter strands is synthesized by a multi enzyme complex that contains DNA polymerase

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15
Q

replication fork

A

point at which replication is occurring

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16
Q

replication is initiated at ORIGINS and proceeds

A

BIDIRECTIONALLLY

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17
Q

bacterial chromosome: Theta Form replication

A

Origin - a sequence that can support replication of any DNA joined to it (replicon - DNA under a control of one origin

OriC - 245 bp

General feature: rich in A and T

E coli -> single origin, bidirectional replication (approx 30 proteins needed)

Rate: 1000 nucleotides per second

MOST of bacterial, viral and extrachromosomal eukaryotic genomes are circular

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18
Q

OriC

A

replication originin bacteria

245bp

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19
Q

**bidirectional replication

A

is the most common form of replication, but not completely universal

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20
Q

Eukaryotic chromosomes: multiple replicons

A

Each chromosome is composed of multiple replicons (40-100)

Many origins necessary because of slower replication and more DNA present (100 nucleotides per second)

ARS (autonomously replication sequence) elements from yeast. (Similar to OriC of E.coli

Any sequence containing ARS can be replicated within a yeast cell

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21
Q

The start of S phase (replication)

A

Activation of first replicon(s)

Not all replicons are activated at the same time

All of the DNA must be replicated ONLY ONCE prior to cell division

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22
Q

Replication is semi-discontinuous

A

only one daughter strand is synthesized continuously; the other is made as a series of discontinuous fragments

OKAZAKI fragments

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23
Q

What strand always contains the okazaki fragments

A

Lagging strand

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24
Q

Okazaki fragments

A

1-2 kb in bacteria

100 to 200 nucleotides in eukaryotes

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25
DNA polymerase requires:
A template strand A primer (to provide 3'-OH to add to new nucleotide)
26
DNA polymerase elongates in
5' to 3' direction always
27
proofreading
done by DNA polymerase in a 3'-5' exonuclease fashion
28
polymerase and nucleases activities
reside in different sites
29
removal of exonculeases (in e.coli)
increases mutation rates (mistakes) x 100
30
chemistry of polymerizsation
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 dNTPs 3) two phosphates cleaved off 4) a phosphodiester bond forms between two nucleotides 5) and phosphate ions are released
31
dNTP binding site
fingers
32
polymerase active site
palm
33
3-5 exonucleaise activity site
palm
34
Pol I - open form
dNTPs can bind to the finger domain
35
the accuracy of the polymerase functions at the level of
shape recognition
36
dNTP enter between
thumb and fingers
37
base pairing with template causes fingers to
close, positioning substrates in the catalytic site (in palm)
38
the conformational change of the finger domain after dNTPs have been bound
a conformational change brings dNTPs and primer into correct orientation with metal ions
39
DNA polymerase active site (palm) contains
two divalent metal ions (cofactors) that are required for catalysis Mg2+
40
mg2+ (or divalent atom)
deprotonates the primers 3'-OH group AND binds the incoming dNTP and facilitates departure of the pyrophosphate
41
the formation of ____ leads to the opening of the fingers domain and ___
phosphodiester bond and movement of template/primer by one basepair
42
proofreading by DNA polymerase
slow or no DNA synthesis "wrong" geometry of mismatched pair reduces its affinity for polymerase active site DNA slides down the exonuclease active site
43
removal of mismatched nucleotide(s)
removed by 3-5' exonuclease activity the slow incorporation yet rapid removal of a mispaired dNTP underlies the inherent accuracy of DNA polymerases. Accuracy is further enhanced by a vastly diminished rate of dNTP incorporation at a mismatched 3' terminus
44
error rate in DNA replication is less than
1 in 10^9 (a billion)
45
1. nucleotide selection by DNA polymerase
errors are 1 in 10^4-^5
46
proofreasing by DNA polymerase increases fidelity by
100 fold
47
mismatch repair system
increases fidelity another 100 -1000 fold
48
all DNA synthesis
5-3' direction all DNA polymerases link the alpha C-5 phosphate a new dNTP, to the 3' position of the nucleotide reside in the end of the chain
49
DNA synthesis is
semidiscontinious
50
lagging strand is opened
3-5'
51
SSB - single stranded binding proteins
unwind DNA< synthesize primers and keep strands apart
52
helicase function
slides 5-3 on the TEMPLATE for the lagging strand - it uses ATP to separate strands.
53
primase
initaiates on ssDNA containing a specific trimer (GTA in e coli); actvity increases when associated with a helicase
54
primase size
10-13nt
55
SSBs
stabilize ssDNA prior to replication cooperative binding ensures quick coverage of exposed template
56
topoisomerase
removes positive supercoils
57
repliosome
primase and helicase
58
Pol III holoenzyme
22 subunits 1,068 mass
59
DNA helicase (DnaB in prokaryotes)
slides on the template for the lagging strand (then in the 5-3') it uses ATP to separate strands 6 subunits in a ring shape to unzip the double helix
60
Topoisomerase (gyrase or II)
removes positive supercoils. untwists DNA by cutting one OR both strands of DNA to unwind it, then reaeals it. acts on duplex DNA ahead of the replication fork
61
primase - specialzed RNS polymerases
RNA primer in Ecoli come from DnaG primase; initiate each ot the thousands of okazaki fragments on the lagging strand; the leading strand is initiated by primase at a replication origin
62
In Ecoli - RNA primer is synthesized from
DnaG
63
E coli - DnaG
must be bound to the helicase for activity
64
DNA pol I
remove RNA primers at the end of each okazaki fragments and replaces with DNA. *RNaseH can also remove RNA primer, but not the last rNMP
65
ligase
seals the nick in the phospodiester bonds
66
SSB
protect the DNA from endonucleases; stimulates DNA polymerase activity by melting small DNA hairpin structures (i.e. separating base pairs ) in ssDNA
67
DNA pol III
exonuclease 3-5 YES exonuclease 5-3 NO
68
E coli, Pol III, holoenzyme
22 subunits
69
DNA sliding clamps greatly
increase speed and processivity of replication
70
clamp loader
assembles beta clamp onto DNA
71
Beta clamp is loaded onto DNA by clamp loader by using ATP
the beta clamp allows the DNA polymerase to have higher processivity, meaning it can add more dNTPs to the daughter DNA strand
72
clamp loader
five subunits ( gap between two of the five subunits) is where the beta clamp attaches to
73
DnaB helicase connect to the Pol III holoenzyme
increase helicase actibity
74
repliosome
POL III, DnaB, helicase, primase
75
leading strand
POL III - Beta clamp moves continiously with DnaB helicase
76
lagging strand
POL III - beta clamp repeadetely moves on and off the DNA to extend multiple primers
77
trombone model
looping DNA grows and shortens during lagging strand synthesis
78
finishing lagging stradn
okazaki fragments require removal of RNA primer and ligase mediates joining of DNA
79
Nick translation , activity by
DNA POL I 5-3"
80
primer removal
RNase activity 5-3' degrades both DNA and RNA
81
DNA synthesis
DNA pol 3-5' exonuclease for proof reading
82
replication fork in eukaryoes
30 - 50 nucleotides per second
83
CMG complex -
functional helicase CDC45, Mcm2-7, GINS 3-5' (opposite of ecoli - 5-3)
84
PCNA
proliferating cell nuclear antigen (DNA sliding clamp)
85
RFC
replication factor C ( clamp loader)
86
RPA
replication protein A ( equivalent to SSB in E.coli)
87
E Coli - 3 Pol III enzymes at the replication fork in Eukaryotes
pol alpha - primase -> extends each primer pol epsilon - synthesizes leading strand pol delta - synthesizes lagging strand
88
a specific sequence (replicator or origin) is bound by
initiator protein
89
initiator proteins
origin - CIS acting element Initiator - TRANS
90
cis acting DNA element
a short DNA sequence that acts as a binding site for a protein that has an affinity for that specific sequence
91
prokaryotic initiator protein
DnaA - 9 bp
92
the open complex
DnaA-ATP-OriC-HU
93
HU
small basic histone like protein
94
control of initiation: binding of DnaA at oriC
DNA methylation only methylated origins are functional
95
GATC sequence in oriC
N6 methylation 11 GATC within 245 bp in oriC
96
SeqA
binds hemimethylated DNA, preventing DnaA binding
97
DnaA- ADP
cannot destabilze A=T regions mainting the open complex, forming a closed complex
98
disenrangle the two daughter chromosomes
topoisomerase
99
MutS
recognizes mismatched base pair
100
MutS-MutL
scands bidirectionally
101
MutH
site specific endonuclease that cleaves unmethylated GATC sites
102
Mut S - L
recruits helicase II (UvrD). exonuclease degrades the newly replicated DNA

Molecular Genetics (9 decks)

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