ANDREWS UNIVERSITY
College of Arts and Sciences
Department of Chemistry and Biochemistry
BCHM 422 Biochemistry II
Chapter Twenty Four Lecture Guide
NAME ____________________________
Thought for the Chapter
“With man this is impossible, but with God all things are possible.” Matt 19:26
Outline
DNA Replication
Prokaryotic
Eukaryotic
Mutation
DNA Repair
Recombination
Lecture
DNA Replication (Generally Applicable Concepts)
The replication of duplex DNA is semiconservative. Half of the original is conserved.
Each parent single strand is used to make a complementary daughter strand.
Meselson and Stahl experiment
DNA polymerase actions
Used 15N to label new DNA
Template is 3’5’
Original parent hybrid remains
Replicated is 5’3’
Polymerase moves along SS DNA
NTP’s (CTP, ATP etc)ppp is attacked
by 3’ -OH
Hydrolysis of ppp powers reaction
The biological machine for replication
New bases must have Watson-Crick
is the DNA-directed DNA polymerase
hydrogen bonding
(DNA polymerase).
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Replication occurs at replication fork.
Duplex DNA cannot be copied
Duplex must be opened to allow DNA polymerase to operate.
“Opened” DNA duplex is called a theta structure ()
Once opened, replication can occur in one direction or both
This diagram shows that replication happens in both ways (bi-directional)
Replication is semidiscontinuous (Half of the duplication is discontinuous: 3’5’)
3’5’ SS DNA is continuously, without interruption, duplicated (leading strand)
5’3’ SS DNA is replicated in the 3’5’ direction which means (lagging strand):
a. the 3’5’ direction moves backwards to the direction of the opening replication fork
b. the DNA polymerase has to wait as newly exposed 3’5’ SS DNA is revealed in
lagging strand
c. the newly formed DS DNA is formed in short segments (Okazaki fragments)
1000-2000 in prokaryotes; 100-200 in eukaryotes
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d. Okazaki fragments are joined to form continuous DNA by DNA ligase.
DNA polymerase requires a 3’ OH to begin or continue replication
RNA “primers” temporarily attach to the DNA parent SS DNA to provide the –OH
Many primers are required for lagging strand, only one for leading strand
The RNA fragment is eventually replaced with DNA
Prokaryotic DNA Replication
Three prokaryotic DNA polymerases have been discovered. (Pol I, Pol II, Pol III)
They were named in order of discovery, not in order of importance.
Pol I was discovered first. (1957); Pol II and III were discovered later.
Pol I Has three activities on a single chain
1. DNA polymerase activity for DNA replication
2. 3’5’ exonuclease activity causes DNA replication
AND to act as a “proofreading” enzyme (primary role in the cell)
3. 5’-3’ exonuclease activity enables lagging strand synthesis by removing RNA primer
after the 3’5’ activity has created the Okazaki fragments.
The gap between Okazaki fragments is called a “nick” and is closed by DNA ligase.
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Pol II: Used in DNA repair; if lacking, not fatal; has polymerization activity.
Pol III
This is the bacteria’s primary DNA replicase protein.
Fatal if missing.
Is a holoenzyme with 10 subunits
Catalytic core consists of three subunits
- polymerase activity
- 3’-5’ exonuclease activity
- holE gene product
Also edits the polymerization reaction.
Can’t close nicks
Note the relative numbers of each Pol (much more Pol I in each cell)
Note the turnover numbers of each Pol (Pol III has highest turnover number)
Initiation of Replication in E. coli
Opening DNA duplex
Origin at oriC locus
DnaA protein accumulates at locus and “melts” DNA into single strands
DnaB protein (helicase) furthers unwinds DNA duplex in ATP dependent fashion
produces positive supercoils (makes further unwinding more difficult)
Chromosome is already negatively supercoiled (somewhat, but not enough).
Additional unwinding requires DNA gyrase to introduce more negative coils.
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SSB (single-strand binding protein) prevents reannealing.
SSB interferes with Pol III activity
SSB must be removed prior to replication
Creation of Primers for Initiation of Polymerization
Primosome is composed of a helicase (unwinds) and a primase (makes RNA primers)
Follows the 5’3’ DNA parent strand
Must reverse temporarily to make RNA primers for Okazaki fragments.
Required for production of Okazaki fragments
RNA primers can also be made by an RNA polymerase
Primosome is holoenzyme with multiple copies of 7 subunits.
Replication of Parental Duplex DNA in E. coli
The replisome is a multiprotein particle at the replication fork.
Two Pol III holoenzymes act at the replication fork, both operating simultaneously.
The SS DNA for the lagging strand loops around the two Pol IIIs.
The replisome creates continuous 3’5’ DNA duplex AND
Discontinuous 5’3’ lagging DNA duplex
RNA primers are replaced by the action of Pol I nick translation, nicks are sealed by DNA
ligase
Energy comes from hydrolysis of NAD+ or ATP
Termination of E. coli Replication
Occurs at a site directly opposite of oriC
Stopping replication at the termination site requires the Tus protein
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Tus protein blocks the action of DnaB helicase, stopping replication.
DNA proofreading
Cellular conditions also exist to encourage correct DNA replication.
1. dNTPs exist in equimolar amounts.
2. Polymerase reaction has good fidelity because
a. dNTP templates with DNA in noncatalytically environment
b. Protein conformation of polymerase changes during reaction.
3. Exonuclease activity is proofreader
4. Extensive repair mechanisms exist.
Eukaryotic DNA Replication
Remarkably similar to prokaryotic systems
Multiple versions of DNA polymerase exist
Initiation of Replication
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Telomeres and Telomerase
Thus each replication would have the DNA shortened by the length of the RNA primer. This
problem is overcome by the use of telomerases.
Telomerases
Telomers maintained by telomerases
Absence of telomerases
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Mutations
Errors occur despite proofreading activities.
Environmental factors can cause DNA errors
Chemical Mutations
Point mutations (one pair replaces another)
Insertion/deletion mutations
Point Mutations
5-bromouracil (5BU)
Nitrous acid (HNO2)
Alkylating agents
Cl
H2
C
H 3C
O
C
H2
N
H 2N
H 2C
CH2
Cl
C
H2
C
CH3
N
O
N
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Intercalating causes distortion of DNA strand. This in turn causes single insertions/deletions to
occur.
Ames Test- A test for mutagenicity.
DNA Repair
Nucleoside Excision and Repair (NER)
Pyrimidine dimers (covalently bound DNA units)
Prokaryote NER (nucleotide excision repair)
In E. coli, dimmers are repaired by protein products of three genes, uvrA, uvrB, uvrC
UrvABC endonuclease cleaves bonds on either side of the error
The SS DNA is removed
Pol I adds the correct DNA
Splice is completed with ligase
Acts on most distortions of DNA helix
Eukaryote NER
Removes 24-32 nt bases
Consists of up to 16 peptides
Defective NER is responsible for two diseases
XP (xeroderma pigmentosum)
Can’t repair UV damage skin
Autosomal, recessive
Extremely sensitive to light, lots of skin scars
Eye problems
Fatal skin cancers (2000 x the likelihood of dying than normal)
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Lots of unrelated physiological and neurological problems
CS (Cockayne syndrome)
Similar gene defects as XP (more gene defects)
Similar physical symptoms
Normal rate of skin cancer incidence
Glycosylases
Damaged DNA can be repaired by a group of enzymes called DNA glycosylases.
This set of enzymes can each cleave a specific DNA base from its sugar. This initiates repair
by the endonuclease activity of a DNA polymerase.
Recombination
Transposons-mobile genetic units
Two types
General recombination and site-specific recombination.
General recombination (Do not memorize this section)
Holliday junction forms a segment of heterologous DNA
This forms a crossover point
DNA is sealed with ligases.
Crossover point then moves in either direction
The four strands of DNA can be resolved into two duplexes in two ways:
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1. (jl, right) Cleavage of strands that did NOT cross over, then nick sealed, forms
recombinant DNA
2. (jl, left) Cleavage of strands that did cross over , then nick sealed, forms homologous SS
DNA strands.
RecA protein in E. coli promotes this kind of recombination. Similar proteins exist in
eukaryotes.
Site-specific recombination or transposition (Memorize this)
Transposons are common in both prokaryotic and eukaryotic genomes
Transposons code for the enzymes that insert it into the target DNA
General recombination has no specific DNA target
Three levels exist
1. IS Elements (Insertion sequences)
Bacterial chromosomes and plasmids
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E.coli have 8 copies of IS1, five of IS2
Elements codes for the protein responsible for its insertion
Sometimes has a regulatory gene included
IS inserts in a DNA region with direct repeated sequences
The size of the insertion (5-9 bp) is important, not the actual sequence
2. Complex transposons
Carries genes for antibiotic resistance in addition to their own genes
For example:
Tn3: inactivates ampicillin by carrying B-Lactamase
Region in between inverted repeats holds genes for three proteins
Transposase
A Regulatory Protein
Site-Specific transposon proteins
3. Composite transposons
A gene-containing region with IS modules on both ends
Will transpose any gene between the IS modules
Transposon mechanism
The transposon must be replicated at the new site.
1. Staggered cuts in target and insertion DNA is made
2. Each of the protruding SS DNA
3. The transposon is replicated (causes fusion of the two plasmids)
4. The fused plasmids are separated at internal resolution sites by a site-specific crossover
uses a resolvase enzyme
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