Chapter 25 DNA metabolism
25.0 Introduction
A. DNA metabolism includes:
Process that try to reproduce the information
replication (faithful reproduction) - which must be incredibly
accurate
Processes that try to preserve the current information
Repair and recombination
Processes to degrade DNA
Emphasis in this chapter is on the enzymes that perform these functions
Much of these discoveries were first found in E-coli
Figure 25-1 gives you a feel for how many enzymes we can potentially
study in even a simple organism like E coli
B. Terminology
look at 25-1 again
by convention bacterial genes named using 3 lowercase, italicized letters
letters generally reflect apparent function
if several genes affect same process, then add A, B, ...
A, B, reflect order of discovery, not position in a pathway
sometimes have already isolated the protein corresponding to a gene so
can refer to using either protein name or the gene name. Sometimes
haven’t isolated the protein yet, so continue to call by the gene name
to differentiate between the gene and the gene product
Remove the italics and capitalize the first letter of the abbreviation
dnaA is the gene, DnaA, is the protein produced by the
gene
Similar system used in eukaryotes, although not as systematically, so can
get confusing
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25.05 DNA Degradation
This book talks about some of the DNA degrading enzymes (page 1013) in the
section on replication. DNA degradation is a necessary part of several enzymes
in this section, so I have pulled this part out and put it here so we know what we
are talking about when we hit DNA degrading enzyme activities later in this
chapter.
A. DNA degraded by nucleases
Enzymes that degrade DNA called DNA nucleases or Dnases
Are specific for DNA not RNA
Two major classes
Exonucleases nibble in from end
May be 5' or 3' but not both
Endonucleases start somewhere in the middle
Endonuclease that attack specific sequences are called
restriction enzymes
A few endo and exo’s only work on single stranded DNA
Interestingly enough will see nuclease activity as a necessary and integral
part of many DNA synthesizing enzymes!
25.1 DNA Replication
A. DNA replication governed by a set of fundamental rules
I. DNA replication is semi-conservative
Each strand of DNA is used to make new DNA so new DNA
contains one old strand and one new strand
This was one hypothesis of Watson Crick (1953)
Proved 4 years later by Meselson and Stahl (1957)
Made heavy DNA using 15N
Could then see one heavy strand passed on to offspring
Figure 25-2
II. DNA replication begins at an origin and usually proceeds bidirectionally
Figure 25-3
done by placing radioactive DNA on a photographic plate
Could see extra loop of replicated DNA
By doing with a different DNA that had added denatured regions
Could observe that always used same origin and that was
bidirectional
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III. DNA Synthesis proceeds in 5'63' direction and is semi-discontinuous
(Semidiscontinuous -means continuous on one strand,
discontinuous on the other)
Not only bidirectional, but on both strands
And a bit amazing if you think of structure of NTP’s that can only
add to 3' end!
means are always attaching new nucleotide to free 3' of strand
go back to figure 8-7 to remind you what 3' and 5' means
Synthesis on 3' end makes sense - bringing in PPP-bases
phosphorylated on 5'end so take 2 P ‘s of the 5' end as you attach
and this gives you E and gets attached ONLY at the 3' end
Can’t get to work in any other orientation
If adding DNA in 5' 63' direction, then the template is being reading
3'65' direction
If synthesis only in 1 direction how do your get replication forks and
bubble growing on BOTH strands??
Figured out 1960's Okazaki
Figure 25-4
1 strand done continuously (called leading strand)
Other strand goes in small pieces (called lagging strand)
Short pieces of DNA on lagging strand called Okazaki
fragments
DNA degraded by nucleases - this section was moved to 25.05
B. DNA synthesized by DNA polymerases
1st polymerase isolated was by Kornberg in 1955 from E coli
called DNA polymerase I
(E coli contains at least 4 other polymerases)
Single polypeptide MW 103,000
Will see in a bit, is not ‘THE’ polymerase, simply first one discovered
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Mechanism is common to all polymerases
Figure 25-5
3' OH on 3' end of DNA does a nuclephilic attack on áP of an nTP
Releases PPi
Overall E should be about in equilib
Made one PO bond, broke one PO bond
Also get some E from base stacking of new base in DNA
But get major push (~19 kJ) from PPi 62Pi
Reaction requires a template DNA
That is obvious now, but when discovered that was the first time a
template had ever been used in biology
Remember this is isolated 1955, two years after Watson Crick
Model (1953), but 2 years before Messelson Stal (1957)
1955 would be first description of isolation, details we just looked at
would take years to come out!
Reaction requires a primer (a base already starting the new strand that
you can attach to. Need someplace to start can - only add to a preexisting stand)
3' end of primer called Primer Terminus
Primer may be DNA or RNA!
Will need to get a special enzyme to make primers (later)
Polymerase active site has two parts
Insertion site - where incoming nucleotide is positioned
Postinsertionsite - where nucleotide is located after polymerase
translocates (moves 1 bp)
After has added a base polymerase may either fall off, or may add
another base
Average number of bases added before falls off called processivity
May add a single base, fall off DNA then have to find it again, or
may stay attached to DNA was it adds thousands of bases. This
varies from enzyme to enzyme
C. Replication is very accurate
E coli 1 mistake in 109 or 1010 nucleotides
E coli chromosome 4.6x106 bp so makes a mistake once every 100010,000 replications
How do we achieve this accuracy?
5
Specificity not just in correct base pair, but in correct base pair geometry
and P-P position
See figure 25-6
Shows native base pairs and then several incorrect base pair that
can occur.
See how setting “box’ size and P position can rule out all incorrect
base pairs?
Incorrect base pairs will not fit in active site
Specificity of active site not perfect, should still get errors once every 104105
Most polymerase also have proofreading activity
Figure 25-7
A 3'-5' exonuclease that can remove incorrect bases
Usually if incorporate a bad base, the enzyme is slowed down
(inhibited) so next base is added slowly. This added time gives
exonuclease a chance to remove the bad base
Not simply reverse of forward reaction, since can’t get Ppi back
Can assay two polymerase and nuclease activities separately
Can have separate sites on the same enzyme
Have 2 binding events so complimentary each other
And multiply selectivity together
Say each binding is only selective to 1/100
1/100 X 1/100 = 1/10,000 so greatly increase selectivity with
a second binding event
Proofreading improves fidelity another 102-103
Accuracy of E coli replication higher still
Has a mismatch repair mechanism that is applied to DNA after it is
synthesized (will study later in chapter)
D. E coli has at least 5 polymerases
DNA polymerase I accounts for 90% of activity in E coli
But early evidence said wasn’t ‘the’ enzymes
1. About 100 x to slow to keep up with replication fork
measurements
2. low processivity (falls off often, probably why so slow)
3. Many other gene product known to be needed for replication
4. 1969 discovered an E coli strain with nonfunctional DNA pol I
6
that was viable
early 1970's discovered DNA pol II and DNA pol III (15-20 years later!)
Pol II is a repair enzyme
Pol III seems to be the principle replication enzyme
Properties compared table 25-1
Pol IV and V identified 1999, seem to be involved in DNA repair
Returning to Pol I
Thought to perform clean-up work in replication, recombination and
repair
Has a 5'63' exonuclease
In addition to 3'65' proof reading nuclease
Located on a separate domain
This activity allow it to remove or replace a segment of DNA
(or RNA it’s not fussy)
In a process called nick translation
Figure 25-8
Most polymerases don’t have this activity
Pol I minus 5'63' nuclease domain called large or Klenow Fragment
Can still polymerize and do proofreading
Pol III
Larger and more complex than pol I
10 different subunits (table 25-2)
á & å associate with è to form a core polymerase
á is polymerizing subunit
å is proofreading subunit
Can polymerize but limited processivity (falls off DNA
fast)
2 cores associate with clamp loading complex
Called ã complex
ô2ãää’
Add in ÷ and ø
And you have DNA polymerase III*
This has better processivity, but still not good enough
Now add 4â subunits that can encircle DNA
And form complete DNA Pol III
Can’t fall off so very good processivity
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E. DNA Replication requires many enzymes and protein factors
Besides the complicated DNA polymerase will need 20 more enzymes
and proteins
entire complex called DNA replicase system or replisome
Won’t go over all details here, just the salient points
To replicate DNA need way to separate strands (unwind from each other)
Need a helicase uses ATP energy to separate two strand of DNA
from each other in a short region
Once have separate strand they want to fold back together, so need
DNA-Binding Protein to stabilize separate strands
As you unwind, this puts in topological stress
Need topoisomerase to relieve this stress
Have already seen that DNA polymerases need a primer so
Primases synthesize short segments of RNA that polymerase then
extends
RNA primers need to be removed. This is where DNA Pol I is thought to
come in
But doesn’t seal the nick so need
DNA ligases to seal final gaps
All of the above must be coordinated and regulated
F. Replication of E coli chromosome proceeds in stages
initiation
elongation
termination
Different reactions and enzymes for each stage
I. Initiation
Origin of replication on DNA
Called oriC
245 bp of DNA with a sequence that is highly conserved among all
bacteria
Structure indicated in figure 25-10
Key features on DNA
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R sites
5 repeats of 9 bp
Binding site for key initiation protein DnaA
Region rich in AT pairs
Called DNA unwinding element (DUE)
I sites
Additional binding sites for DnaA
IHF (Integration host factor) binding site
FIS (factor for inversion stimulation) binding site
Last two used in certain recombination events - Will
study later in chapter)
Process involves at least 10 different proteins (table 25-3)
Open DNA at origin
Establish pre-priming complex
DnaA is key protein (figure 25-11)
Is a AAA+ ATPase family
AAA+ stands for “ATPase associated with diverse cellular
activities”
Typical AAA+ activity
form oligomers
hydrolyze ATP slowly
Slow hydrolysis is switch between two states
For DnaA
ATP bound for is active
Hydrolyzed,-ADP bound form is inactive
Eight DnaA proteins (all with ATP bound) assemble to form helical
complex in oriC (figure 25-11)
This binding event uses both R and I sites
DnaA binds to R site in both ATP and ADP forms
DnaA binds to I site only when ATP bound
Tight right hand wrap of DNA around structure
Make + supercoil
In turn opens up AT rich DUE region
Several other DNA binding proteins join in
HU (histone like protein binds non specifically
IHF and FIS at their specific sites
Also serve to bend DNA
DnaC protein (another AAA+ ATPase) loads DnaB onto separated
DNA strands
A hexamer of DnaC (with ATP bound)
Forms a tight complex with hexameric ring of DnaB
This opens up the hexameric DnaB ring
9
Now interacts with DnaA
2 rings of DnaB are loaded onto DNA in DUE region
1 ring on each strand of DNA
DnaC completes its slow hydrolysis of ATP
And this signals it to fall off complex
Loading of DnaB onto DNA is key event
DnaB is a helicase
Migrates along DNA in 5'63' direction
Unwinds DNA as it goes
Each DnaB complex Is the start of a replication fork
All other proteins in replication complex will be linked to DnaB
ô subunit of DNA pol III binds to DnaB
As strands are separated
Many molecules of SSB (Single strand binding protein) bind and
stabilize separated strands
DNA Gyrase (DNA topoisomerase II)
Relieves unwinding stress
Initiation is only phase of DNA replication that is regulated
Will only occur once each cell cycle
Regulation mech not entirely clear yet, but here is what we know
End of initiation occurs when DNA pol III is loaded on DNA
Hda, another AAA+ ATPase
With bound ATP, binds to â subunit of DNA pol III at
this time
Also binds to DnaA
Binding to DnaA make DnaA start its hydrolysis
of ATP, and this makes DnaA complex fall
apart
Binding of Fresh ATP 20-40 minutes later is
part of signal for next round of replication
Other part of signal comes from DNA methylation
Ecoli DNA methylated by Dam methylase
Methyl on N6 of A in sequence GATC
Chance of finding this sequence in 1 in 256 bp
But there are 11 GATC’s in 245 bp of ori
sequence
Since methyl group is added by Dam methylase, after
DNA is replicated, Newly synthesized DNA is
Hemimethylated, because only the old strand of DNA
has the methyl groups
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After initiation the hemimethylated oriC sequence is
bound by SeqA protein and sequestered in plasma
membrane (we don’t know how) After a time SeqA
falls off and it is released from membrane.
Now it must be methylated by Dam methylase before
DnaA will bind again
II. Elongation
All done on Pol III so lets look at the structural details of Pol III now
Figure 25-9, table 25-2
Assembled on site
Elongation process Figure 25-12
DNA unwound by helicases
Topological stress relieved by topoisoerases
Single strand DNA stabilized by SSB (single strand binding
protein)
Different enzymes for leading and lagging strands
Leading strand
DnaG Primase synthesizes 10-60 nucleotides of RNA on the
DNA template
Does this in conjunction with DnaB helicase that is on
Lagging strand!
Then DNA polymerase III takes over and start adding DNA
Proceeds down the replication fork as it open up the DNA
Lagging stand
DnaG Primase does its thing
DNA polymerase III takes over to make DNA
Extends until hits next primer
Seems pretty simple until realize that are doing BOTH AT ONCE IN
A SINGLE POLIII ENZYME COMPLEX
Accomplished by looping DNA as shown in figure 25-13
DNA helices unwinding DNA
Primase occasionally binds to helices and initiates a primer
on lagging strand
DnaG Primase dissociates and DNA/RNA â-clamp is loaded
onto DNA/RNA complex
When previous Okazaki fragment hits RNA of fragment
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before it
Its clamp is discarded from core
New clamp is added to core
Next fragment is polymerized
Clamp-loading complex consists of
ô2ãää’, and is another AAA+ ATPase
Binding of 3 ATP’s to complex opens up clamp so
DNA can get in
Hydrolysis of ATP to ADP seals DNA into clamp
Figure 25-14
Rapid process about 1000 bp added to each strand /second
After RNA clear complex DNA PolI binds, edits out the RNA
Then nick sealed by DNA ligase (25-15)
Summary of replisome proteins table 25-4
Ligase reaction shown figure 25-16
Enzyme activated by attaching AMP
Viruses and eukaryotes use ATP as source
Bacteria use NAD+ as a source
AMP transferred to 5'P of nick to reactivate that P
3'OH can attack to seal nick
AMP released
Now that you know the steps, watch an animation
http://www.youtube.com/watch?v=4jtmOZaIvS0&feature=related
III. Termination
Eventually 2 replicating forks meet
Not a random event
Figure 25-17
Meet at a sequence called Ter
Multiple copies of a 20 bp sequence
Ter sequence acts as binding site for protein Tus
(terminus utilization substance)
Ter-Tus complex will halt a replication fork from one
direction but not the other
Ordinarily replication forks stop when they meet, but this seems to
be a way to insure that both meet at the same place at the same
time
12
One fork halts when meets first complex
Other fork stops when it meets the stalled fork
DNA between complexes (a few hunderd bp) replicated
(mechanism unknown)
Get two DNA molecules but are twisted around each other
Called catenanes Figure 25-18
Separated by topoisomerase IV (a type II isomerase- ie breaks
both strand at once
Two molecules segregated into two daughter cells
G. Replication in Eukaryotic cells more complicated
Eukaryotic DNA lots larger
organized into chromatin
So will be different
But essential steps seem to be the same
Origins
AT rich sites
Vary from organism to organism
In Yeast called autonomously replicating sequences (ARS) or
replicators
150 bp several conserved sequences
400 replicators in 16 chromosomes in haploid yeast
~ 25/chromosome
~Origins spaced out about 30,000-300,000 bp apart
Does replicate bidirectionally
Regulation
Cyclins and cyclin dependent kinases (CDK’s)
Cyclins destroyed after mitosis
In absence of cyclins, pre-replicatvie complexs form on
initiation sites, but don’t do anything
In bacteria key initiation step was loading DnaB/DnaC
heterohexameric complex that was a helicase
Figure 25-19
Similar complex in Eukariotes with minichromosomal
maintenence proteins (MCM) proteins
MCM2-7) for hexameric helicase like DnaB
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Loaded on DNA with hexamer origin replication complex (ORC)
protein (equivalent to DnaC) also an AAA+ ATPase
Also needed are CDC6 and CDT1
Added controls - involve synthesis of cyclin CDK complexs that
bind to and phosphorylate several protein in the Pre-replicative
complex to activate them
Replication fork moves 1/20 the speed of bacterial
50 nucelotides/sec
If single origin would take 500 hours to replicate genome
(That’s why there are so many origins!)
Also several polymerases (á,â...)
Several linked to different functions
Replication of nuclear chromosomes involved polymerase á and ä
á similar in all eukaryotic cells
Has a primase and a polymerase
No 3'-5' exonuclease so no proofreading. Don’t think its ‘the’
polymerase
Thought to synthesize primers
Primers extended by ä
ä associated and stimulated by PCNA (proliferating cell
nuclear antigen)
PCNA heavily expressed in nuclei of replicating cells
3D structure similar to â portion of Ecoli Pol III
Make circular clamp of polymerase to stays on DNA
ä has 3'-5' exonuclease so can proofread
Seems to work on both leading and lagging strands
May be ‘the’ nuclease
å polymerase replaces ä in DNA repair
May act to remove primers like E coli DNA pol I
Protein to that binds single stranded DNA is called RPA
(replication protein A)
Clamp loader is called RFC (Replication Factor C)
Termination involved synthesis of special structures called telomeres at
end of chromosomes
Will look at details next chapter
(But nothing is said about termination within a chromosome)
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H. Viral DNA Polymerases provide targets for antiviral therapy
Many DNA viruses encode their own DNA polymerase, so if you can
specifically inhibit this enzyme, you have killed the virus
25.2 DNA Repair
if RNA or protein damaged, simply make a new copy
if DNA damaged have a problem
back in chapter 10 saw lots of ways DNA can be damaged
How do we repair this damage?
A. Mutations are linked to cancer
damage to DNA called a lesion
if lesion leads to a change in sequence and
Bad sequence passed on to next generation
now have a mutation
Mutations
Substitution of one base for another
Insertion of one or more new bases
Deletions of one or more bases
If affect nonessential DNA or has negligible effect - called silent
mutation
Occasionally will offer advantage - evolution begins
Often are deleterious - damaging
In mammals - strong correlation between accumluation of mutations and
cancer
Lead to Ames test
Add chemical to specialized bacterial strain
Watch for easily detected mutations to occur
Tie between bacterial mutations and cancer in humans?
90% of known carcinogens are mutagenic in Ames test
So strong correlation
B. All cells have multiple repair systems
have seen several different types of damage so several different repair
mechanisms
Repair mech can be extremely inefficient. Lots of ATP E is thrown away
yet want to be sure you have it right so need to do this
Repair mech relies on having two strand and assuming one is good
Figuring out the good one can be tricky
15
I. Mismatch repair
Cleanup synthesized DNA by a factor of 102 - 103
Assumes old strand is good and new strand is bad so need way to
recognize old strand
Done in E coli by tagging old strand with methyl groups
Mismatch repair involves at least 12 protein in e coli Table 25-5
Some for repair, some for strand identification
Start with Dam methylase
(DNA adenosine methylase)
It has already methylated the N6 of all A in the sequence
GATC on both strands
(Already saw this guy as part of control of initiation)
It takes a few seconds up to a few minutes before it gets
around to methylating the new strand
During this time can tell old from new
Do you need figure 25-21?
Mismatch near (within 1000 bp) a hemimethylated area
repaired using old strand as template Figure 25-22
(Mismatch repair >1000 bp more difficult so not discussed)
If both strands methylated no repair occurs
If neither strand methylated repair occurs but 50-50
chance of getting it right
MutL and MutS proteins hydrolyze ATP to form complex at
mismatched DNA (all except C-C mismatch)
Mut H bound to MutL/S complex and to a nearby GATC to
make a DNA loop
When Mut H finds a hemimethyated GATC
It cleaves the DNA on the unmethylated side
Now depends on if nick is 5' or 3' from mismatch
Figure 25-23
Mismatch on 5' side
Unwind and degrade DNA in 3'-5' direction until
gets to mismatch
Replace with new DNA
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Need DNA helicase II, SSB, exoI or exoX,
DNApol III, DNA ligase
Mismatch on 3' side
Same but use exoVII which can degrade either
5'-3' or 3'-5'
Mismatch repair costs lots of E
Will redo 1,000s of bases just to get 1 bad one
This means costs 1000 of ATP’s
Eukaryotic cells have similar protein to Mut L and Mut S
Error in these genes associated with cancer-susceptibility
(Box 25-1)
Some details given in text, but there is still much we do not know
Don’t even know how identify old and new strand
II. Base-Excision Repair
Class of enzymes that recognize common lesions
Let’s review lesion formed by spontaneous chemical reactions
(Chapter 8 pages 289-291)
Deamination (figure 8-30a)
C6U
5mC6T
A6Hypoxanthine
G6Xanthine
Depurination (figure 8-30b)
UV dimerization (figure 8-31)
DNA methylation (no figure)
Remove bad base by cutting base from sugar
Cleaving glycosidic linkage so called DNA Glycosylases
DNA has a apyrimidinic or apurinic site
Short called AP site
Each glycosylase specific for one type of lesion
Uracil glycosylase- removes C’s that deaminated to U’s
But will not remove U from RNA
Bacteria a 1 U glycosylase
Humans have 4! Indicates how important it is
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Another recognizes
hypoxanthine (adenine deamination)
3 methyl A
7 methyl G
Pyrimidine dimers
AP sites can also arise spontaneously
(Depurination)
Once AP site formed can’t simply attach a new base to the sugar
Need to replace the sugar and replace entire base
Need AP endonuclease cleave DNA
May be either 3' or 5'
Segment of DNA removed (not just the one bad sugar)
DNA replaced by DNA polymerase I and DNA ligase
Figure 25-24
III. Nucleotide-Excision Repair
The above lesions, methylations and demination, made minimal
distortions for the DNA helix so base excision was all that was need
for a first step
Lesions that cause larger distortion in DNA generally repaired by
removing entire region around a base and sugar in one step.
hence the name nucleotide excision repair
Used for repair of pyrimidine/cyclobutane dimers, 6-4 photo
products, and several other base adducts including
benzo[á]pyrene-guanine from by exposure to cigarette smoke
In e coli. nucleotide excision repair done by a multienzyme complex
called ABC exinuclease (figure 25-25)
Made up of UvrA (104,000) UvrB(78,000) and Uvr C(68,000)
And A2B unit scans DNA to find and bind to lesion
A then dissociates and B tightly bound
UvrC then bonds to B
UvrB then clips 5th P 3' of lesion
UvrC then clips 8th P 5'
Total of 12-13 depending on size fo lesion
UvrD (a helices) then removes the segment
DNA filled in with Pol I
Sealed with ligase
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In humans and other eukaryotes
Similar action
But requires 16 different polypeptides
None of the peptides has any sequence similarities to E coli.
enzyme
IV. Direct Repair
Some repairs can be made without removing base!
Direct photoreactivations of pyrimidine dimer
Done by DNA photolyase
Figure 25-26
Won’t go over mech, but in mammals required FAD and
another chromophore to help absorb light of the right E
Repair of O6-methylguanine
Common methylation site, highly mutagenic
Because G now wants to pair with T instead of C
Right margin page 1033
Repaired by O6 methyltransferase
Pulls methyl group from G and puts on an protein’s Cys SH
Not true enzyme because it suicides cannot regenerate
So used an entire protein to correct one mistake
Interestingly the dead enzyme is not simply discarded, but it
acts as a signal to activate the synthesis of its own gene and
a few other repair genes
1-methylA and 3-methylC
These amino groups sometimes methylated in single strand
DNA
Interferes with proper base pairing
In Ecoli oxidatively removed by AlkB protein
Figure 25-28
C. More extreme damage
double strand breaks, double strand cross-links, damage to single
stranded DNA during the replication or transcription process
All extremely harmful because there is no complementary strand to repair
from
1 method recombinational DNA repair
Go to the homologous chromosome for a copy
Will study more later in chapter
Note: this only works for diploid organisms ~ Eukariotes
19
Under special circumstances can be used in haploid bacteria
Have to catch during DNA replication but before cell division
Since can’t generally use this method In E coli had a second method
called error-prone translesion DNA synthesis (TLS)
Much less accurate, a state of desperation repair system
Turned on when cell getting heavy UV damage or in extreme
cellular distress
Part of the SOS response
Some SOS response protein already expressed at low levels
for DNA repair (UvrA & UvrB)
Under SOS,s level are boosted
Also start expressing other proteins (UmuC & UmuD)
UmuD cleaved to UmuD’
Makes complex with UmuC to make
DNA PolymeraseV
Much less finiky polymerase, can get around
many problems but error prone
Error can easily kill the cell
Only induced under extreme conditions
A few cells die
But some survive
Will talk in more detail on SOS response in chapter 28
Also another error prone polymerase, polymerase IV
Error prone Translesion polymerases like IV and V are found
in ALL organisms
Lack proofreading
Error rates 10-100x worse
Error rates as high a 1 in 1000!
In Humans are used for some specific repair mechs
And may only replace 1 or 2 bases at a time
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25.3 DNA recombination
Only works in diploid cells
rearrangement of genetic information within and among DNA molecules
three general classes
Homologous genetic recombination (general recombination)
Genetic exchanges between two DNA’s that share a large region of
nearly identical sequence, Actually sequence not important, just
overall similarity
Site specific recombination
Recombination occurs only at a specific sequence
DNA Transposition
Short segment of DNA that moves from one place to another
Functions and mechanisms are all different. Sometimes we don’t even
know the function
In general seems to be a repair mechanism, and, as such, is integrated in
to DNA metabolism
A. Bacterial Homologous Genetic Recombination - a repair mechanism
In bacteria used for DNA repair hence name recombinational DNA repair
used to reconstruct DNA around a replication fork that stalled due to DNA
damage
Also used in conjugation (mating) when DNA from a donor is integrated
into recipient cell -a relatively rare event
Figure 25-30
Replication fork hits a DNA nick or extensive DNA damage and has to
stop because can extend damaged strand
First degrades 5' end of short (damaged) strand
21
A recombinase binds to exposed 3' end of short strand to invade the long
(undamaged) double strand
When gets to complement in other strand have created a branched DNA
structure
This branch point can then move forward or backward in a process called
branch migration. These crossover structures are called Holliday
intermediates
Holliday structure then resolved with a special class of nuclease, and the
replication fork is reconstructed
Details
RecBCD complex - is both nuclease and helicase, works in step 1
clipping back the double stranded DNA to get some single strand stuff
figure 25-31
Binds at a double strand break
Unwinds and removes BOTH strands of DNA using ATP for E
RecB moves 3'65' on one strand
RecD moves 5'63' on other
Hits a chi sequence (GCTGGTGG)
Binds tightly to RecC
Then slows cutting 3' strand
Gets faster cutting 5' strand
There are about 1000 chi sequenced in E coli.
Centers of recombination
Sequences that promote recombination found in higher organisms
Recombinase is the Rec A protein
RecA active form is ordered helical filament of thousand of rec A
Figure 25-32
Starts coating the single strand DNA
Not simple process, since single stranded DNA is coated with SSB
The Rec BCD comples actually nucleates and starts the RecA
filament growing, and many other proteins are involved. In growth
and strand invasion.
Branch migration promoted by RuvAB Figure 25-33a
Cleaved by specialized nuclease called RuvC 25-33b
Nicks are sealed with ligase,
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Replication fork is restarted in a process called
Origin-independent restart of replication
Protein PriA, PriB, PriC, and DnaT act with DnaC to load DnaB
helicase onto reconstructed replication fork. Primase synthesizes
the primers and DNA polymerase reassembles on DnaB to restart
DNA synthesis
Complex of PriA,PriB, PriC DnaT DnaB and DnaC
called replication restart primosome
Restart also requires DNA pol II, but we don’t know why
B. Eukaryotic Recombination is required for proper Chromosome Segregation
during Meisos
Several roles for recomination in Eukaryotes
Occurs with highest frequency during meiosis
Meiosis
Diploid germ cell 6 haploid gametes
Figure 25-34
Diploid cell replicates DNA
Get 4 copies of 2 pairs of sister chromatids
Cell divides and two pairs of sister chromatids are separated
Cell divides again and each gamete cell gets a single copy of each
DNA
Now remember our cohesins that we saw in previous chapter that
provide physical links to guide chromosome segregation?
They aren’t around during the first meiotic division, so it is relatively
easy for the chromosomes to get tangled, and for recombination to
occur.
This is called crossing over, and genetic material is exchanged
between pairs of sister chromatids. Increases genetic diversity
Crossing over is largely random, but here are some ‘hot spots’
If assume total random then can use to calculate distance between
genes
Used to map genes
If 2 genes stay together often during crossing over then must
by physically close on the DNA
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If 2 gene often separated during crossing over, then must be
far apart on the DNA
Can identify 3 functions for homologous recombination in
Eukaryotes
1. Repair of several types of damage
2. Provides transient physical link between chromatids and
promotes orderly segregation in 1st meiotic division
3. Enhances genetic diversity
C. Recombination during Meiosis is initiated at double strand breaks
Possible mechanism figure 25-35a
See my diagram for product 2, its not obvious
4 main features
1. Homologous chromosomes closely aligned (physically touching)
2. Double strand break enlarged by exonucleases that nibble away
different parts on two strands
3. One strand invades homologous DNA, and in branch migration
Displaces one strand and is extended to migrate the branch point
4. end up with 2 interlinked DNA structures called a Holliday
structure that can be observed with an electron microscope
Very similar to bacterial process
This is called double-strand break repair model
Details vary from species to species
As shown in figure Holliday structure can be unlinked in two ways, both
are observed
1. Flanking DNA not recombined
2. Flanking DNA recombined
Since the two strands involved came from different parents, they may be
the same in overall sequence, but there can be differences in individual
bases, that leads to small changes in new genome
D. Site-specific Recombination - precise DNA rearrangements
just looked at recombination that can occur anywhere between two
homologous strands
Now examine a different process recombination at specific sequences
Occurs in all cells
May have different purposes in different cells
Regulation of expression of genes
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Promoting programed rearrangements during embryonic
development
Part of life cycle of some plasmids and viruses
Each recombination system consists of an enzyme called a recombinase
2 general types
Ser at active site
Tyr at active site
And a DNA segment it recognizes, the recombination site usually 20-200
bp
Also one or more auxiliary proteins for regulation
General pathway for Tyr type recombinase Figure 25-37
4 separate recombinases recognize 4 sites on DNA
(Book shows 2 sites on 2 different DNA’s, but can be 4 sites
on 1 DNA)
Protein associates as a tetramer bringing 4 sites into near contact
In each pair of recombinases, 1 recombinase cleaves one strand of
DNA and get covalently bond at the cleavage site though a
phospho-tyrosine
This linkage preserves energy of phosphate bond so can
regenerate DNA linkage without ATP
Protein now interacts with opposite in other pair to link strands in a
Holliday structure
Other half of pair now cleaves and binds and exchanges so get the
recombination
In Serine type recombinase both strands of each site are cut at the same
time and rejoined without going through Holliday structure
Can view recombinase as a site specific endonuclease and ligase.
Unlike many of protein-DNA binding sites, the sites recognized by
recombinases are NOT symmetric. Thus the recombinase binds in a
oriented manner and when sites on DNA pieces are aligned, the 2
combining sites are in the same orientation
This has some interesting consequences, in the overall recombined DNA
structure
If we have a single piece of DNA with the sequence of the two sites
inverted
when we go through the recombination event we simply invert the
intervening DNA
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(Figure 25-38 a)
However if we have a single piece of DNA with the sites in the
same orientation
the recombination event removes the intervening DNA and turns it
into a small circular loop!
(Figure 25-38b)
If the sites are on different DNA and either one or both of the DNAs is a
circular piece, then the recombination ends up inserting 1 DNA into the
other. We will explore this more in a minute.
Various recombinases tend to be specific for each of these different
pathways
Site specific recombination is also used in e. coli in one additional step
that sometimes has to be done after recombinational repair.
Look at Figure 25-39
Depending how you resolve the Holliday structure in recombination repair
you either get a normal chromosome, or you get a dimeric genome. The
dimeric chromosome cannot be segregated into two daughter cells, so it
becomes a trap for the cell.
To resolve this a specific recombination is performed by the XerCD
system using a mechanism like Figure 25-38b
E. Transposable genetic elements move from one location to another
Another use of recombination is in transposition - the movement of
transposable elements from one location to another
Transposons - segments of DNA found in all cells, that can hop from one
location to another
Terminology - hop from a ‘donor’ site to a ‘target’ site
New location usually random
If goes into a essential gene can kill
So very tightly regulated and not done too often
Transposon can be thought of as the simplest molecular parasite
Passively reproduced by host cell
If caries a good gene, can be a simple symbiosis
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2 classes of transposon in bacteria
Insertion sequences - simple transposons
Have the sequence required for transposition
And code for protein (transposases) that do the process
Complex transposons
Carry addition genes
For instance gene for antibiotic resistance thus making a
drug resistant bacteria
bacterial transposons have different structures, but here is usual scenario
DNA sequence has short repeated sequences that is binding site of
tranposase
these segments tend to be repeated in transposition process
Figure 25-40
2 processes Figure 25-41
1. Direct or simple
Cut at recognition sequences on both sides of transposon
(Leaves behinds a double strand cut for the Repair enzymes
to fix)
Transposase makes a staggered cut at a new location
Transposon inserts
DNA replicated to fill in gap
2. Replicative transposition
Replicate so leave copy behind at donor site
eukaryotic transposons same and different
some involved RNA intermediates
Will see next chapter
H. Immunoglobulin genes are assembled by recombination
an example of a programed developmental recombination events
Immunoglobulin your immune protein - binds antigens to fight infection
You are capable of expressing millions of different immunoglobulins
yet you only as about ~29,000 immunoglobulin genes!
Use recombination event to mix and match different immunoglobulin
genes together
May have evolved by early invasion of a tranposable element?
Look at immunoglobulin G (IgG)
First review protein structure Figure 5-21 page 176
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Now do gene structure
Figure 25-42 this is just kappa light gene
Protein is a dimer of 2 light and 2 heavy chains
Both chains have variable region, where sequences vary a
lot from one protein to the next. And a constant region,
where sequence is nearly identical from one to the next
2 different families of light chains, kappa and lambda
In picture
Have a single constant DNA
Lots of a short hypervariable DNA
And several longer variable region
Use recombination to mix and match
Use RNA splicing to get rid of unused DNA
Express protein
300 V segments (95 AA’s)
4 J segments (12 AA’s)
300x4 = 1,200 possible combos
But not nice clean recombination so 2.5 x more so
about 3000 combos
1 C genes
5000x1 = 5000 kappa light gene products
Combining VJ to C done by RNA splicing (Next
chapter) rather than recombination
Combining V to J done by recombination sites (RSSRecombination signal sequences) just after V and just
before J Figure 25-43
RAG1 and RAG2 (Recombination Activating Gene)
perform double strand breaks between RSS sites
then a second complex joins the DNA together
Genes for heavy chains undergo similar processes
Get about 5000 products for heavy chains
5000x3000 = 1.5x107 complete IgG’s
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Additionally high mutation rate in V sequences!
Each B lyphocyte cell will express only 1 IgG
Is this a left-over transposon?
Mech for double strand break by RAG1 &
RAG2 does resemble several steps in
transposition
Deleted DNA with RSS sites has structure like
a transposon
It the test tube RAG1 and RAG2 can inset this
DNA like a transposon into random places in
DNA