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Microbial Genetics > DNA Replication > Flashcards

DNA Replication Flashcards

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1
Q

DNA replication requirements

A
  1. energy supply to unwind helix
  2. SS-DNA will form intrastrand base pairs w/out intervention
  3. requires a number of enzymes
  4. development of proofreading safeguards
  5. geometric constraints - size (length) and circularity of DNA molecule
  6. not an unique mode of replication common to all
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2
Q

DNA replication prime role

A

duplicate base sequence of the parent DNA

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3
Q

Semi conservative vs. Conservative

A

S.C. first proposed by Watson and Crick (no enzyme action, could not prove their theory)
At the time: denaturation & strand separation thought to be impossible.

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4
Q

reasons denaturation/strand separation thought to be impossible:

A

a. Time for helix to unwind (large value) - wrong data
b. MW of DNA not halved by denaturation - wrong data
c. Length of DNA vs. length of cell - DNA to long (DNA 600x longer than cell in E. coli) to separate in short cell, result: conservative replication

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5
Q

Meselson-Stahl Experiment

A

determine conservative or semi-conservative replication
Method to distinguish between parental and daughter strands
proved semi-conservative replication

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6
Q

Meselson-Stahl Experiment 1

A

1 round of replication, then CsCl density centrifugation.

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7
Q

Meselson-Stahl Experiment 2

A

showed structure of first generation DNA

denatured, then CsCl density gradient - hybrid molecule with one strand heavy, one strand light

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8
Q

John Cairns in early 1960’s

A

Circular DNA
grew cells in media w/[3H] thymidine, isolated DNA w/o fragmentation, placed it on photographic film (3H decay exposed one silver grain) (3 months for end of exp.)
Indicated DNA replicated as circle

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9
Q

theta replication

A

DNA replicated as circle

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10
Q

Enzymology of DNA Replication

A
  1. high fidelity in copying base sequence
  2. physical separation of strands
  3. antiparallel backbone
  4. speed – 1000 nucleotides/second
  5. 20 known proteins are necessary
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11
Q

1957 - Arthur Kornberg discovered

A

DNA polymerase (Pol I)

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12
Q

Pol I required

A
  1. 4 DNA nucleotides (dNTP) with 5’-triphosphates

2. Template - SS-DNA to be copied

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13
Q

Pol III

A
  • actual enzyme for advancement of the replication fork

requires 5’-triphosphates + DNA template

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14
Q

Pol I Functions

3’–>5’ exonuclease activity

A

(running backwards) if error in DNA synthesis adds nucleotide to 3’OH that won’t H bond to template base, then must be removed before synthesis continues.

a. terminates polymerizing action -

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15
Q

Pol I Functions 3’–>5’

A

removes base *proofreading or editing function - post synthetic function

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16
Q

Pol I Functions 5’–>3’ exonuclease activity

A

a. nucleotides removed from 5’P end (also work on nicks if a 5’-P is present)
b. more than one can be removed, base paired to be removed
c. ribo- or deoxribo- sugar type
main function - remove ribonucleotide primers

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17
Q

nick translation

A

move nicks around the molecule - can start replication at a nick in DNA

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18
Q

strand displacement

A

at a nick
growing strand displaces the parental strand, mechanism of genetic recombination.
other Pol can do it, with aid of auxillary proteins

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19
Q

excision

A

repair system to repair damaged DNA

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20
Q

polymerizes

A

fill in short ss regions on DS-DNA

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21
Q

Pol I functions

A 
exonuclease activity
nick translation
strand displacement
excision
polymerizes
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22
Q

Polymerase III or Pol III (not as much is known)

A
  • complex enzyme
  • substrate more limited than Pol I
  • can’t unwind DNA helix
  • 3’-5’ exonuclease activity
  • main enzyme for synthesizing DNA
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23
Q

pol III holoenzyme

A

enzyme + 6 other proteins associated with it.

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24
Q

holoenzyme

A

several subunits, some activity when one or more are missing

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25
Q

core enzyme

A

mallest unit with activity (usually different activity than holoenzyme)

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26
Q

genes for 5 of the subunits

A

dnaE, dnaN, dnaQ, dnaZx

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27
Q

dnaE -

A

main polymerizing activity

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28
Q

other subunits:

A

a. catalytic efficiency
b. high processivity tendency to remain on single template rather than disassociate/reassociate (move along DNA strand)
c. 3’65’ exonuclease activity

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29
Q

3’ to 5’ exonuclease activity

A

editing function dnaQ (dnaE)

major editing function in DNA replication

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30
Q

DNA Ligase

A

joins a 3’-OH and a 5’ monophosphate group (5’-P) on adjacent base-paired nucleotides
can’t bridge a gap

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31
Q

Type I topoisomerase

A

– uncoils DNA helix, works ahead of the replication fork, attaches to one strand

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32
Q

Type II topoisomerase (gyrase)

A

converts positive supercoils from replication process to negative supercoils, ATP dependent reaction, attaches to both strands

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33
Q

Topoisomerases

A

(Five families/two main groups)

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34
Q

Requirements for Gyrase (Type II topoisomerase)

A
  1. bind 2 DNA segments may be distant
  2. hold free ends of cut DNA together
  3. pass free ends to other side of the molecule
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35
Q

Gyrase result:

A

Catenation

decatenation

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36
Q

Catenation

A

linking two circular DNA molecules to form a chain

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37
Q

Decatenation

A

reverse process, important in DNA synthesis, when a circle replicates sometimes 2 catenated circles result and must be separated

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38
Q

Source of Precursors(5’ triphosphate nucleotides)

A
  1. Salvage pathway

2. de novo synthesis/pathway

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39
Q

Salvage pathway

A

ree bases, nucleosides, nucleotides from degradation of nucleic acids or from growth media, built up to nucleoside monophosphates

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40
Q

de novo synthesis or pathway:

A

ribonucleotide (1 PO4) made from amino acids, CO2, NH3, phospho-ribosyl-phyrophosphate

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41
Q

Discontinous Replication

A

ragments in the Replication Fork since pol I and pol III add only to 3’OH group
choices

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42
Q

Discontinous Replication

possible explanantions

A

a. another polymerase (5’-P) end
b. two strands 5’–>3’ opposite ends of molecule
c. 5’–>3’ “discontinuous mode” - predicts newly made DNA consists of fragments

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43
Q

1968 Okazaki worked with E. coli and found:

A
  • found DNA fragments that attach to one another
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44
Q

A. Pulse-labeling experiment

A
  1. replicating (growing) cells
  2. add [3H] dTTP
  3. 30 seconds, DNA isolated
  4. sedimented in alkali (strand separation)
45
Q

A. Pulse-labeling experiment results:

A

labeled DNA sediments slowly/native DNA
(1000-2000 nucleotides length)
parental 20-50x larger

46
Q

Pulse Chase experiment

A
  1. growing bacteria
  2. [3H] dTTP for 30 sec
  3. replaced w/non radioactive dTTP
  4. grown for several minute
  5. sedimented in alkali (strand seperation)
47
Q

Pulse Chase experiment

results

A

edimented together-labeled fragments where joined (sedimented increased)

48
Q

Okazaki fragments

A

discontinuous model

49
Q

small fragments

A

large polymer as attached to synthesized DNA

50
Q

DNA fragment observation:

A

1/2 new DNA is fragments (yet all was fragments in Pulse Label experiment)
DNA 3’-OH end - not discontinuously synthesized
leading strand is continuous, fragmented later

51
Q

Fragment reason: Uracil Fragments

A

dTTP and dUTP present

pol III and pol I can’t tell the difference

52
Q

dUTPase

A

coverts dUTP to dUMP (not incorporated into DNA, not efficient) so some dUTP survives

53
Q

deamination

A

mechanism mutation - of cystosine to uracil GC–>AU–>AT

54
Q

Fragment reason;Cell repair

A

replace U with a T (works on any U)

55
Q

Reasons for DNA fragments

A
  • uracil fragments
  • cell repair
  • discontinuous replication
56
Q

Cell repair steps

A
  1. uracil N-glycosylase - removes uracil base, leaves deoxyribose in backbone
  2. AP (apurinic acid) endonuclease - frees one end of sugar
  3. Pol I - remove sugar + nucleotides fills gap w/correct nucleotides (excision-repair)
    reaction is slow so newly synthesized DNA appears fragmented.
57
Q

Discontinuous Replication

A

universal for bacteria, eukaryotes, phage, viruses
2. eukaryotes - 100-200 bases (higher amt dUTP)?
prokaryotes - 1000-2000 bases

58
Q

Initiation of DNA Synthesis

RNA Terminus of Precursor Fragments

A

no known DNA polymerase can initiate a DNA chain - must extend from 3’-OH end of primer.

59
Q

Initiation of DNA Synthesis

Need a _____ ______ that synthesizes primer ________

A

polymerizing enzyme, oligonucleotide (short)

60
Q

INitiation of DNA synthesis

Pol III then extends this

A
  1. leading strand, priming DS-DNA

2. lagging (precursor fragment), strand to be copied is already unwound - priming SS-DNA

61
Q

Enzymes (in bacteria)

A

RNA polymerase
primase
primosome

62
Q

RNA polymerase

A

same that makes mRNA + other RNAs

63
Q

primase

A

dnaG gene

64
Q

primosome

A

helicase and primase are often paired in bacteria

65
Q

Joining of Precursor Fragments

A
  1. ligase can’t seal ribose form triphosphate
  2. Pol I, nick translation removes RNA, replaces w/DNA
  3. growing 3’-OH reaches DNA base
  4. ligase closes the nick
    Second enzyme in E.coli - RNase H - riboendonuclease specific for RNA in RNA/DNA hyrid…also removes primers
66
Q

Initiation of Synthesis of the Leading Strand

A
  • unique base sequence
  • oriC
  • DnaA boxes or 9 mers
67
Q

unique base sequence

A

replication origin or ori - organism specific

68
Q

oriC

A

initation of replication in E. coli

69
Q

Pol I ___ unwind the helis- Pol III ___ unwind th e helix

A

can, can’t

70
Q

helicases

A

helix unwinding enzymes, (rep gene –> REP protein, helicase in E. coli)

71
Q

how the REP protein works:

A

hydrolyzes ATP –> unwind helix

2 ATP/bp broke

72
Q

lagging strand

A

large SS area left behind helicase

73
Q

leading strand

A

small SS area left behind the helicase

74
Q

SSB protein- binds to __ ___ and ___ ______

A

SS DNA, one another

75
Q

Pol III displaces ___ as it moves along

A

SSB

76
Q

some replication systems have a single protein that is both a _____ and ____ function

A

helicase, SSB

77
Q

Sliding Clamps - Processivity Clamps

A
  • proteins with no enzymic activity
78
Q

sliding clamps function

A

increase processivity of DNA polymerases

79
Q

sliding clamps form

A

a pseudohexameric ring shaped structure

-Ds-DNA in center

80
Q

sliding clamps also interact with these factors

A

a. DNA repair
b. recombination
c. cell cycle regulators
5. central nexus for coordination of proteins that process/join Okazaki fragments

81
Q

Clamp Loader

A
  1. supply ring opening and ring closing reactions
  2. usually a heteropentamer (1large/4 small subunits)
  3. in E. coli 3 different subunits make up the pentamer
82
Q

Summary of Events at the Replication Fork (6)

A
  1. enzymes unwind double helix
  2. proteins stabilize unwound parental DNA
  3. leading strand synthesized continuously by DNA polymerase
  4. lagging strand synthesized discontinuously. Primase, an RNA polymerase, synthesizes a short RNA which is extended by DNA polymerase
  5. DNA polymerase digest RNA primer adn replaces it with DNA
  6. DNA ligase joins discontinuous fragments of lagging strand
83
Q

Delays in Replication Fork:

A
  1. 3’–>5’ exonuclease editing

2. removal of uracil residues (uracil-N-glycosylase)

84
Q

Why is E. coli so complex when compared to phage/virus models?

A

larger the molecule/greater the error - need more replication proteins/each protein can minimize or correct an error.

85
Q

Two Methods of Initiation

A

DeNovo

Covalent

86
Q

DeNovo Inidiation

A

all DNA initiate within helix (even linear)
2 mechanisms:
1. DNA sequence-specific origin-binding protein
2. leading strand synthesis - RNA polymerase
replication “bubble” –> D-loop (displacement loop)
DS & SS loop
initially no gyrase (breathing because underwound)

87
Q

Covalent extension

A 
leading strand covalently attached to parental strand (rolling circle replication)
     several exceptions (DNA molecules)
88
Q

Rolling Circle Replication uses

A

phage replication, genetic transfer in bacteria

89
Q

Rolling Circle Replication

Concatemer

A
  1. intermediate in phage production, ex. lambda phage

2. linear molecule goes from host –> donor (conjugation?)

90
Q

Rolling Circle Replication

A
  1. polymerase III enzyme used
  2. primer unnecessary because 3’-OH available
  3. displacement
    called: sigma replication
91
Q

rolling circle displacement is a result of

A

helicase, ssb, pol III

92
Q

Looped Rolling Circle Replication (Plasmid transfer or conjugation)

A
  1. lag strand synthesis - SS progeny from DS parent (generates + strands) from template
  2. 1 copy - difference with rolling circle - displaced loop never longer than length of circle
93
Q

Bidirectional Replication

A
  • depends on which directions the helicases can go
    2 replication forks moving in opposite directions
    some unidirectional replication - phages and plasmids (stop signal)
94
Q

Denaturation Mapping

A

heating (melting) treat with formaldehyde

95
Q

bidirectional organisms

A

plasmids, phage, viruses, bacteria, eukaryotes

96
Q

Termination of Replication of a Circle

A

not well understood
unidirectional molecule - stops at the origin
2 types in bidirectional molecule

97
Q

2 types in bidirectional molecule

A
  1. defined termination sequence (plasmid-one end stops at a fixed pt.)
  2. 2 growing pts collide to terminate (E. coli phage)
    often results in catenane (pair of circles linked) - fixed by a gyrase
98
Q

Methylation of DNA

A

a. mismatch repair

b. Regulatory function (Restriction Endonucleases)

99
Q

Methylation of DNA method

A

a. methylase adds a methyl group to a base (cytosine and adenine)
b. gradient of methylation - least methylated DNA is closest to the fork on daughter strand - parent is uniformly methylated
c. back-up editing system
d. mismatch repair - recognizes a pair of non-H bonded bases, excises out polynucleotide sequence (removes one “bad” base)
e. remove from which strand (parent or daughter)? removes from under-methylated strand

100
Q

Archaea DNA Replication

A

Machinery (proteins) to perform DNA replication are more closely related to Eucarya. Archaea become a simple model to study complex eucaryal replication machinery.

101
Q

Eucarya genome

A

chromosomes contain linear DNA molecules

102
Q

Archaea/Bacteria genome

A

circular DNA molecules

103
Q

Many Archaea have ___ copies of the genome, allows _____ ______ since high temps can damage DNA

A

2, recombination repair

104
Q

Differences between Bacteria and Archaea:

DNA replication initiation sites

A

Bacteria - single site
Eucarya - multiple sites
Archaea - single or multiple sites, reason is slow replication rate in some extremophiles (6 kb/min vs 20kb/min)

105
Q

Differences between BActeria and ARchaea:

DnaA, MCM

A

Bacteria - DnaA binds and allows access for DnaB (helicase)

Archaea - MCM (minichromosome maintenance) replicative helicase opens DNA (similar to Eucarya)

106
Q

Differences between Bacteria and Archaea:

SSB

A

Bacteria - SSB homotetramer binds 65 nucleotides
Archaea - SSB-like to RPA-like (replication protein A)
Eucarya - RPA

107
Q

Differences between Bacteria and Archaea:

polymerases

A

Bacteria - Pol III (family C)
Eucarya - 3 types (family B)
Archaea - also family B, have unusual features to prevent mutations in hyperthermophiles

108
Q 
Differences between Bacteria and Archaea:
Sliding Clamps (processivity clamps)
A

Bacteria - B clamp
Eucarya - PC clamp
Archaea - PC clamp
Clamp loaders vary in Archaea

Microbial Genetics (4 decks)

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