1
Q
DNA replication requirements
A
- energy supply to unwind helix
- SS-DNA will form intrastrand base pairs w/out intervention
- requires a number of enzymes
- development of proofreading safeguards
- geometric constraints - size (length) and circularity of DNA molecule
- not an unique mode of replication common to all
2
Q
DNA replication prime role
A
duplicate base sequence of the parent DNA
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.
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
5
Q
Meselson-Stahl Experiment
A
determine conservative or semi-conservative replication
Method to distinguish between parental and daughter strands
proved semi-conservative replication
6
Q
Meselson-Stahl Experiment 1
A
1 round of replication, then CsCl density centrifugation.
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
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
9
Q
theta replication
A
DNA replicated as circle
10
Q
Enzymology of DNA Replication
A
- high fidelity in copying base sequence
- physical separation of strands
- antiparallel backbone
- speed – 1000 nucleotides/second
- 20 known proteins are necessary
11
Q
1957 - Arthur Kornberg discovered
A
DNA polymerase (Pol I)
12
Q
Pol I required
A
- 4 DNA nucleotides (dNTP) with 5’-triphosphates
2. Template - SS-DNA to be copied
13
Q
Pol III
A
- actual enzyme for advancement of the replication fork
requires 5’-triphosphates + DNA template
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 -
15
Q
Pol I Functions 3’–>5’
A
removes base *proofreading or editing function - post synthetic function
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
17
Q
nick translation
A
move nicks around the molecule - can start replication at a nick in DNA
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
19
Q
excision
A
repair system to repair damaged DNA
20
Q
polymerizes
A
fill in short ss regions on DS-DNA
21
Q
Pol I functions
A
exonuclease activity nick translation strand displacement excision polymerizes
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
23
Q
pol III holoenzyme
A
enzyme + 6 other proteins associated with it.
24
Q
holoenzyme
A
several subunits, some activity when one or more are missing
25
core enzyme
mallest unit with activity (usually different activity than holoenzyme)
26
genes for 5 of the subunits
dnaE, dnaN, dnaQ, dnaZx
27
dnaE -
main polymerizing activity
28
other subunits:
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
29
3’ to 5' exonuclease activity
editing function dnaQ (dnaE)
| major editing function in DNA replication
30
DNA Ligase
joins a 3'-OH and a 5' monophosphate group (5'-P) on adjacent base-paired nucleotides
can't bridge a gap
31
Type I topoisomerase
– uncoils DNA helix, works ahead of the replication fork, attaches to one strand
32
Type II topoisomerase (gyrase)
converts positive supercoils from replication process to negative supercoils, ATP dependent reaction, attaches to both strands
33
Topoisomerases
(Five families/two main groups)
34
Requirements for Gyrase (Type II topoisomerase)
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
35
Gyrase result:
Catenation
| decatenation
36
Catenation
linking two circular DNA molecules to form a chain
37
Decatenation
reverse process, important in DNA synthesis, when a circle replicates sometimes 2 catenated circles result and must be separated
38
Source of Precursors(5' triphosphate nucleotides)
1. Salvage pathway
| 2. de novo synthesis/pathway
39
Salvage pathway
ree bases, nucleosides, nucleotides from degradation of nucleic acids or from growth media, built up to nucleoside monophosphates
40
de novo synthesis or pathway:
ribonucleotide (1 PO4) made from amino acids, CO2, NH3, phospho-ribosyl-phyrophosphate
41
Discontinous Replication
ragments in the Replication Fork since pol I and pol III add only to 3'OH group
choices
42
Discontinous Replication
| possible explanantions
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
43
1968 Okazaki worked with E. coli and found:
- found DNA fragments that attach to one another
44
A. Pulse-labeling experiment
1. replicating (growing) cells
2. add [3H] dTTP
3. 30 seconds, DNA isolated
4. sedimented in alkali (strand separation)
45
A. Pulse-labeling experiment results:
labeled DNA sediments slowly/native DNA
(1000-2000 nucleotides length)
parental 20-50x larger
46
Pulse Chase experiment
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
Pulse Chase experiment
| results
edimented together-labeled fragments where joined (sedimented increased)
48
Okazaki fragments
discontinuous model
49
small fragments
large polymer as attached to synthesized DNA
50
DNA fragment observation:
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
Fragment reason: Uracil Fragments
dTTP and dUTP present
| pol III and pol I can't tell the difference
52
dUTPase
coverts dUTP to dUMP (not incorporated into DNA, not efficient) so some dUTP survives
53
deamination
mechanism mutation - of cystosine to uracil GC-->AU-->AT
54
Fragment reason;Cell repair
replace U with a T (works on any U)
55
Reasons for DNA fragments
- uracil fragments
- cell repair
- discontinuous replication
56
Cell repair steps
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
Discontinuous Replication
universal for bacteria, eukaryotes, phage, viruses
2. eukaryotes - 100-200 bases (higher amt dUTP)?
prokaryotes - 1000-2000 bases
58
Initiation of DNA Synthesis
| RNA Terminus of Precursor Fragments
no known DNA polymerase can initiate a DNA chain - must extend from 3'-OH end of primer.
59
Initiation of DNA Synthesis
Need a _____ ______ that synthesizes primer ________
polymerizing enzyme, oligonucleotide (short)
60
INitiation of DNA synthesis
| Pol III then extends this
1. leading strand, priming DS-DNA
| 2. lagging (precursor fragment), strand to be copied is already unwound - priming SS-DNA
61
Enzymes (in bacteria)
RNA polymerase
primase
primosome
62
RNA polymerase
same that makes mRNA + other RNAs
63
primase
dnaG gene
64
primosome
helicase and primase are often paired in bacteria
65
Joining of Precursor Fragments
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
Initiation of Synthesis of the Leading Strand
- unique base sequence
- oriC
- DnaA boxes or 9 mers
67
unique base sequence
replication origin or ori - organism specific
68
oriC
initation of replication in E. coli
69
Pol I ___ unwind the helis- Pol III ___ unwind th e helix
can, can't
70
helicases
helix unwinding enzymes, (rep gene --> REP protein, helicase in E. coli)
71
how the REP protein works:
hydrolyzes ATP --> unwind helix
| 2 ATP/bp broke
72
lagging strand
large SS area left behind helicase
73
leading strand
small SS area left behind the helicase
74
SSB protein- binds to __ ___ and ___ ______
SS DNA, one another
75
Pol III displaces ___ as it moves along
SSB
76
some replication systems have a single protein that is both a _____ and ____ function
helicase, SSB
77
Sliding Clamps - Processivity Clamps
- proteins with no enzymic activity
78
sliding clamps function
increase processivity of DNA polymerases
79
sliding clamps form
a pseudohexameric ring shaped structure
| -Ds-DNA in center
80
sliding clamps also interact with these factors
a. DNA repair
b. recombination
c. cell cycle regulators
5. central nexus for coordination of proteins that process/join Okazaki fragments
81
Clamp Loader
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
Summary of Events at the Replication Fork (6)
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
Delays in Replication Fork:
1. 3'-->5' exonuclease editing
| 2. removal of uracil residues (uracil-N-glycosylase)
84
Why is E. coli so complex when compared to phage/virus models?
larger the molecule/greater the error - need more replication proteins/each protein can minimize or correct an error.
85
Two Methods of Initiation
DeNovo
| Covalent
86
DeNovo Inidiation
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
Covalent extension
```
leading strand covalently attached to parental strand (rolling circle replication)
several exceptions (DNA molecules)
```
88
Rolling Circle Replication uses
phage replication, genetic transfer in bacteria
89
Rolling Circle Replication
| Concatemer
1. intermediate in phage production, ex. lambda phage
| 2. linear molecule goes from host --> donor (conjugation?)
90
Rolling Circle Replication
1. polymerase III enzyme used
2. primer unnecessary because 3'-OH available
3. displacement
called: sigma replication
91
rolling circle displacement is a result of
helicase, ssb, pol III
92
Looped Rolling Circle Replication (Plasmid transfer or conjugation)
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
Bidirectional Replication
- depends on which directions the helicases can go
2 replication forks moving in opposite directions
some unidirectional replication - phages and plasmids (stop signal)
94
Denaturation Mapping
heating (melting) treat with formaldehyde
95
bidirectional organisms
plasmids, phage, viruses, bacteria, eukaryotes
96
Termination of Replication of a Circle
not well understood
unidirectional molecule - stops at the origin
2 types in bidirectional molecule
97
2 types in bidirectional molecule
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
Methylation of DNA
a. mismatch repair
| b. Regulatory function (Restriction Endonucleases)
99
Methylation of DNA method
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
Archaea DNA Replication
Machinery (proteins) to perform DNA replication are more closely related to Eucarya. Archaea become a simple model to study complex eucaryal replication machinery.
101
Eucarya genome
chromosomes contain linear DNA molecules
102
Archaea/Bacteria genome
circular DNA molecules
103
Many Archaea have ___ copies of the genome, allows _____ ______ since high temps can damage DNA
2, recombination repair
104
Differences between Bacteria and Archaea:
| DNA replication initiation sites
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
Differences between BActeria and ARchaea:
| DnaA, MCM
Bacteria - DnaA binds and allows access for DnaB (helicase)
| Archaea - MCM (minichromosome maintenance) replicative helicase opens DNA (similar to Eucarya)
106
Differences between Bacteria and Archaea:
| SSB
Bacteria - SSB homotetramer binds 65 nucleotides
Archaea - SSB-like to RPA-like (replication protein A)
Eucarya - RPA
107
Differences between Bacteria and Archaea:
| polymerases
Bacteria - Pol III (family C)
Eucarya - 3 types (family B)
Archaea - also family B, have unusual features to prevent mutations in hyperthermophiles
108
```
Differences between Bacteria and Archaea:
Sliding Clamps (processivity clamps)
```
Bacteria - B clamp
Eucarya - PC clamp
Archaea - PC clamp
Clamp loaders vary in Archaea
Microbial Genetics
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