Molecular Bio Unit 2

Front 1

Replication

Back 1

• new daughter strand is assembled on each parent strand
• Used complementary base pairing, and requires a series of enzymes
• Highly regulated process
• The new strand is synthesized in the 5’ to 3’ direction

Front 2

The incoming dNTP

Back 2

Needs a 5’ triphosphate because it provides the energy for rxn in the high energy phosphate bond when the diphosphate is released

Front 3

Template strand

Back 3

3’-5’

Front 4

Do old DNA strands pair with new or old DNA?

Back 4

Meselson and Stahl : semiconservative

Front 5

Meselson and Stahl : semiconservative

Back 5

o Grew bacteria in 15N heavy and E coli incorporated 15N into DNA
o 15N- labeled DNA can be distinguished from DNA containing 14N by density-gradient centrifugation

Front 6

DNA synthesis

Back 6

enzymatic rxn = identified using combination of genetics and biochemistry

Front 7

What proteins are required for DNA synthesis?

Back 7

dnaA, dnaB,dnaC, ssB, Topoisomerase, dnaG, Pol III, Pol II, Pol I, DNA ligase, Tus

Front 8

dnaA

Back 8

binds to 9-mer at Ori (A-T rich) and uses hydrolsis to melt DNA

Front 9

dnaB (Helicase)

Back 9

uses hydrolysis to unwind the db helix

Front 10

dnaC

Back 10

recruits dnaB to Ori and loads it on

Front 11

ssb

Back 11

prevents DNA strands from closing and reannealing

Front 12

Topoisomerase I

Back 12

relaxes supercoils by nicking and resealing one strand of DNA

Front 13

Topoisomerase II (DNA gyrase)

Back 13

relaxes supercoils by nicking and resealing both strands of DNA

Front 14

dnaG

Back 14

Primase - RNA Pol. contains the primosome which provides 3'OH for initiation
it is not the same RNA pol used in transcription

Front 15

How many primers are used for the leading strand and lagging strand?

Back 15

Leading: 1
Lagging: multiple

Front 16

Pol III

Back 16

extends 5'-3' from 3' OH
replicates DNA at a fast rate 500-750 bp
3'-5' exonuclease activity for proofreading
Holoenzyme: β subunit: sliding clamp = high processivity

Front 17

Pol I

Back 17

5'-3' exonuclease activity to remove primer and fill in gaps
3'-5' exonuclease activity for proofreading

Front 18

DNA Ligase

Back 18

uses ATP hydrolysis to form phosphodiester bond

Front 19

Tus (Contrahelicase)

Back 19

Binds to Ter site and inhibits helicase, so it blocks replication so the DNA Pol and c'somes dissociate

Front 20

Topoisomerase IV

Back 20

separates the concatenanes

Front 21

Components needed to synthesize DNA

Back 21

dNTPs
DNA template
Primer (free 3'OH)
DNA pol
Mg2+

Front 22

Kornberg: (1957)

Back 22

biochemical approach and identified the Pol I
cells > extracted > fractionate proteins + dNTPS = assay for DNA synthesis

Front 23

All DNA polymerases catalyze this reaction

Back 23

• (dNMP)n +dNTP → Mg2+ →(dNMP)n+1 +PPi
• All make DNA 3’-5’ (proofreading)
• All require dNTPS, template, primer with 3’= OH

Front 24

how do we know that Pol I is not the only DNA polymerase?

Back 24

column chromatography: two major peaks of DNA pol activity.
• Make pol I mutant = a third peak becomes visible = there are three Pol in E Coli

Front 25

Problems for DNA replication

Back 25

• DNA strands are annealed so must be unwound for replication to occur
• DNA pol requires a 3’ OH to initiate DNA synthesis
• DNA synthesis only occurs 5’-3’

Front 26

҉Cairn’s theta (θ) replication experiment (1963)

Back 26

• Grew e. coli in radioactive media, lyse the cells and expose to photographic emulsion
o Allowed visualization of the replication fork
o Could not distinguish between bidirectional and unidirectional
• Cairn’s theta (θ) replication
o There is only one initiation site in e coli

Front 27

how many replication/initiation sites are in E coli?

Back 27

One initiation site and 2 replication forks

Front 28

Gyurasits and Wake (1973)

Back 28

• Modified Cairns’ experiment. They grew Bacillus subtilis in weakly radioactive media (3H) briefly then shifted to very radioactive media
DNA is Bidirectional

Front 29

Replicon

Back 29

DNA under the control of one origin of replication

Front 30

how and where does replication begin and end?

Back 30

oriC: identification of the E. coli

Front 31

OriC Function

Back 31

1. Origin of replication
2. DNA partitioning

Front 32

DNA partitioning

Back 32

replication must be coordinated with cell cycle, and daughter strands must be partitioned into daughter cells

Front 33

what needs to happen first at a replication origin?

Back 33

@ A-T rich region because it is easier to break apart the bonds

Front 34

Structure of Ori Site

Back 34

9 mer and 13 mer because they are AT rich

Front 35

Initiation of the Replication bubble at oriC

Back 35

1. DNA strands must be separated to initiate replication.
2. dnaA = initiation factor that recognizes oriC and forms an open complex
3. dnaA uses ATP hydrolysis to melt the A-T regions
4. dnaB: Helicase furthers separation of the strands to allow DNA polymerase

Front 36

how do you prevent strain on the helix during replication?

Back 36

replication occurs very fast to prevent physical strand on the helix
o 500-750 nucleotides per second
o 10 bp per turn of the helix
• Topoisomerase (DNA gyrase) relieves the strain

Front 37

Okazaki’s experiment (1968)

Back 37

• T4 phage in E coli
•Pulse experiment
* Replication is semi-discontinuous
* use mutant Pol I no 5'-3' end

Front 38

Pulse experiment

Back 38

treated cells with short pulse of 3H thymidine. Isolated DNA, denature it, estimated size by ultracentrifugation

Front 39

why did they denature the DNA?

Back 39

they denatured it so they can look at the individual strands

Front 40

primosome

Back 40

a complex that moves with the replication fork, periodically igniting the synthesis of Okazaki fragments it provides the 3’OH group for initiation
contains dnaG

Front 41

how did they find out primer is RNA, not DNA?

Back 41

Okazaki showed that DNase could not destroy all small fragments

Front 42

What ensures that replication is precisely accurate?

Back 42

proofreading by Pol I, II, and III.

Front 43

Elongation: what has continuous synthesis and what has discontinuous synthesis?

Back 43

Continuous synthesis: Leading
Discontinuous synthesis: Lagging

Front 44

Primer Removal

Back 44

• Pol I digests the RNA primer using 5’-3’ exo activity and synthesizes a DNA strand to fill the gap left by the primer then DNA ligase joins DNA ends

Front 45

Different types of replication

Back 45

Eukaryotes: multiple Ori
Circular DNA: replicates rolling circle
Phages: some replicate: linear

Front 46

Telomere replication

Back 46

remove primer with Pol I 5'-3'
telomerase adds repeated DNA
formation of RNA primer (primase)
Dna synthesis from RNA primer
Removal of primer

Front 47

What happens without telomere

Back 47

c'somes get shorter and shorter after every replication

Front 48

Telomerase (protein + RNA)

Back 48

reverse transcriptase that carries internal RNA template for DNA synthesis

Front 49

Where is telomerase activity present?

Back 49

Germline cells (so progeny can have full length c'some)
activated cancer cells

Front 50

DNA Repair

Back 50

• DNA is the only molecule in the cell with a repair system to insure fidelity

Front 51

DNA damage

Back 51

errors during replication (inherent instability of DNA)
damage from environmental agents
change in structure

Front 52

Major enzymatic repair strategies:

Back 52

proofreading - replication
mismatch repair
excision repair
light repair (photoreaction)

Front 53

Q: How do organisms tells old strand from the new strand?

Back 53

• They add methyl group on the old strand and when the whole replication cycle is complete, they also methylate the new strand.

Front 54

Why is there post modification of DNA?

Back 54

• It serve as as a marker for the DNA
• It is used to distinguish self from non-self DNA
• Used to help guide DNA repair

Front 55

DNA Damage - Breakage of chemical bonds

Back 55

Thymine dimers
Pyrimindine dimers

Front 56

Thymine dimers

Back 56

DNA to be unable to replicate
Solution: Light activated repair

Front 57

Pyrimindine dimers

Back 57

distort the double helix by forming a “buldge”

Front 58

DNA damage - Loss of bases

Back 58

common with purines
occurs spontaneosly
secondary effect of other mutagens
strong acids will enhance purine loss

Front 59

DNA damage - Deanimination

Back 59

C > U
distortion in DNA helix
o Changed whole nucleotide resulting in whole protein change which can result in total loss

Front 60

DNA damage - chemical mutagens

Back 60

o Often work by bind to DNA (intercalating) btwn bases to change the structure
 Ie. Ethidium bromide
o Modify the chemical composition of a base so that base pairs pair incorrectly

Front 61

General strategy for post replication repair

Back 61

1. Proteins detect distortion in DNA helix
2. Generate gap in helix by removing damaged molecules using
a. Endonucleases
b. Exonucleases
c. Glyconucleases
3. DNA Pol (I) makes new DNA at the gap
4. DNA ligase completes the phosphodiseater backbone and require AT for energy

Front 62

Light repair (photoreactivation)

Back 62

o Not presents in mammals
o Reversal
o An enzyme, photolyase, uses light energy to break the bonds in the TT Dimer

Front 63

What does excision repair repair?

Back 63

defective nucleotide
deamination (Uracil DNA glycosylase)
nucleotide excision (thymine dimers) (UvrABC)

Front 64

Endonuclease must leave ?

Back 64

3’ OH and 5’P

Front 65

Coping with Damage

Back 65

Recombination repair
inducible repair: SOS pathway

Front 66

why is Pol I more involved in repair?

Back 66

it is slower
5'-3' and 3'-5'

Front 67

Recombination Repair

Back 67

recA uses ATP hydrolysis to catalyze strand exchange
gap is repaired using recombination with parental strand
dimer is removed with UvrABC
Pol I and Ligase

Front 68

Hutchinson-Gildord (progeria) syndrome

Back 68

• Premature aging and death
• Defect in gene (lamina) involved in uclear structure
• Caused by single base change
• Not inherited because de novo mutation (in early embryo or parent’s germ cells)
• Mutation is dominant