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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
Q

DNA polymerase requires:

A

A template strand

A primer (to provide 3’-OH to add to new nucleotide)

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26
Q

DNA polymerase elongates in

A

5’ to 3’ direction always

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27
Q

proofreading

A

done by DNA polymerase in a 3’-5’ exonuclease fashion

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28
Q

polymerase and nucleases activities

A

reside in different sites

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29
Q

removal of exonculeases (in e.coli)

A

increases mutation rates (mistakes) x 100

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30
Q

chemistry of polymerizsation

A

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

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31
Q

dNTP binding site

A

fingers

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32
Q

polymerase active site

A

palm

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33
Q

3-5 exonucleaise activity site

A

palm

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34
Q

Pol I - open form

A

dNTPs can bind to the finger domain

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35
Q

the accuracy of the polymerase functions at the level of

A

shape recognition

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36
Q

dNTP enter between

A

thumb and fingers

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37
Q

base pairing with template causes fingers to

A

close, positioning substrates in the catalytic site (in palm)

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38
Q

the conformational change of the finger domain after dNTPs have been bound

A

a conformational change brings dNTPs and primer into correct orientation with metal ions

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39
Q

DNA polymerase active site (palm) contains

A

two divalent metal ions (cofactors) that are required for catalysis

Mg2+

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40
Q

mg2+ (or divalent atom)

A

deprotonates the primers 3’-OH group

AND

binds the incoming dNTP and facilitates departure of the pyrophosphate

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41
Q

the formation of ____ leads to the opening of the fingers domain and ___

A

phosphodiester bond

and movement of template/primer by one basepair

42
Q

proofreading by DNA polymerase

A

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
Q

removal of mismatched nucleotide(s)

A

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
Q

error rate in DNA replication is less than

A

1 in 10^9 (a billion)

45
Q
  1. nucleotide selection by DNA polymerase
A

errors are 1 in 10^4-^5

46
Q

proofreasing by DNA polymerase increases fidelity by

A

100 fold

47
Q

mismatch repair system

A

increases fidelity another 100 -1000 fold

48
Q

all DNA synthesis

A

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
Q

DNA synthesis is

A

semidiscontinious

50
Q

lagging strand is opened

A

3-5’

51
Q

SSB - single stranded binding proteins

A

unwind DNA< synthesize primers and keep strands apart

52
Q

helicase function

A

slides 5-3 on the TEMPLATE for the lagging strand - it uses ATP to separate strands.

53
Q

primase

A

initaiates on ssDNA containing a specific trimer (GTA in e coli); actvity increases when associated with a helicase

54
Q

primase size

A

10-13nt

55
Q

SSBs

A

stabilize ssDNA prior to replication

cooperative binding ensures quick coverage of exposed template

56
Q

topoisomerase

A

removes positive supercoils

57
Q

repliosome

A

primase and helicase

58
Q

Pol III holoenzyme

A

22 subunits
1,068 mass

59
Q

DNA helicase (DnaB in prokaryotes)

A

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
Q

Topoisomerase (gyrase or II)

A

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
Q

primase - specialzed RNS polymerases

A

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
Q

In Ecoli - RNA primer is synthesized from

A

DnaG

63
Q

E coli - DnaG

A

must be bound to the helicase for activity

64
Q

DNA pol I

A

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
Q

ligase

A

seals the nick in the phospodiester bonds

66
Q

SSB

A

protect the DNA from endonucleases;

stimulates DNA polymerase activity by melting small DNA hairpin structures (i.e. separating base pairs ) in ssDNA

67
Q

DNA pol III

A

exonuclease 3-5 YES
exonuclease 5-3 NO

68
Q

E coli, Pol III, holoenzyme

A

22 subunits

69
Q

DNA sliding clamps greatly

A

increase speed and processivity of replication

70
Q

clamp loader

A

assembles beta clamp onto DNA

71
Q

Beta clamp is loaded onto DNA by clamp loader by using ATP

A

the beta clamp allows the DNA polymerase to have higher processivity, meaning it can add more dNTPs to the daughter DNA strand

72
Q

clamp loader

A

five subunits ( gap between two of the five subunits) is where the beta clamp attaches to

73
Q

DnaB helicase connect to the Pol III holoenzyme

A

increase helicase actibity

74
Q

repliosome

A

POL III, DnaB, helicase, primase

75
Q

leading strand

A

POL III - Beta clamp moves continiously with DnaB helicase

76
Q

lagging strand

A

POL III - beta clamp repeadetely moves on and off the DNA to extend multiple primers

77
Q

trombone model

A

looping DNA grows and shortens during lagging strand synthesis

78
Q

finishing lagging stradn

A

okazaki fragments require removal of RNA primer and ligase mediates joining of DNA

79
Q

Nick translation , activity by

A

DNA POL I
5-3”

80
Q

primer removal

A

RNase activity
5-3’
degrades both DNA and RNA

81
Q

DNA synthesis

A

DNA pol
3-5’ exonuclease for proof reading

82
Q

replication fork in eukaryoes

A

30 - 50 nucleotides per second

83
Q

CMG complex -

A

functional helicase
CDC45, Mcm2-7, GINS

3-5’

(opposite of ecoli - 5-3)

84
Q

PCNA

A

proliferating cell nuclear antigen (DNA sliding clamp)

85
Q

RFC

A

replication factor C ( clamp loader)

86
Q

RPA

A

replication protein A ( equivalent to SSB in E.coli)

87
Q

E Coli - 3 Pol III enzymes at the replication fork

in Eukaryotes

A

pol alpha - primase -> extends each primer

pol epsilon - synthesizes leading strand

pol delta - synthesizes lagging strand

88
Q

a specific sequence (replicator or origin) is bound by

A

initiator protein

89
Q

initiator proteins

A

origin - CIS acting element

Initiator - TRANS

90
Q

cis acting DNA element

A

a short DNA sequence that acts as a binding site for a protein that has an affinity for that specific sequence

91
Q

prokaryotic initiator protein

A

DnaA - 9 bp

92
Q

the open complex

A

DnaA-ATP-OriC-HU

93
Q

HU

A

small basic histone like protein

94
Q

control of initiation: binding of DnaA at oriC

A

DNA methylation

only methylated origins are functional

95
Q

GATC sequence in oriC

A

N6 methylation

11 GATC within 245 bp in oriC

96
Q

SeqA

A

binds hemimethylated DNA, preventing DnaA binding

97
Q

DnaA- ADP

A

cannot destabilze A=T regions mainting the open complex, forming a closed complex

98
Q

disenrangle the two daughter chromosomes

A

topoisomerase

99
Q

MutS

A

recognizes mismatched base pair

100
Q

MutS-MutL

A

scands bidirectionally

101
Q

MutH

A

site specific endonuclease that cleaves unmethylated GATC sites

102
Q

Mut S - L

A

recruits helicase II (UvrD). exonuclease degrades the newly replicated DNA

Molecular Genetics (9 decks)

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