START 0918

1
DNA Packaging

• Progression of DNA compaction
• double stranded helix, no proteins 2 nm
  diameter
• histones (H2A, H2B, H3, H4) octamer generates
  nucleosomes
• Nucleosome histone octamer, 1.65 turns of DNA,
  approx 146 bp of DNA
• beads on string structure 11 nm diameter
• histone H1 generates solenoid structure 30 nm
  diameter fiber
• DNA eventually compacted to 700 nm diameter
• diameter of chromosome in mitosis
• condensins are 1 important protein complex in
  this compaction
• chromosomes most condensed during mitosis
  (10,000X compacted), less condensed and harder to
  identify during other stages of cell cycle

2
DNA Packaging

• chromatin DNA bound proteins
• heterochromatin DNA that is highly compacted
  relative to euchromatin, which generally refers
  to DNA that is translated into RNA
• no gene expression in heterochromatin, genes
  placed into region of heterochromatin (by
  experimentation or spontaneously) are silenced
  position effect
• position effect variagation the manifestation
  of position effects in the phenotype of an
  organism. Example mottled eye color in fruit
  flies
• three elements necessary to propagate a
  chromosome
• it must divide origin(s) of replication
• its ends must be properly replicated telomeres
• sister chromatids must be divided in mitosis
  centromeres

3
DNA Replication

• Ubiquitous Storage material for all genetic and
  heritable information
• Replication is essentially the reproduction of
  the polymer by careful addition of complementary
  monomers (remember Watson-Crick)
• Replication must occur with high fidelity, as
  most mistakes in coding sequence are not
  tolerated
• Replication is the point at which most errors in
  coding sequence arise -gt if not corrected they
  become mutations
• The new product is always the same chain length
  and MW try that in industrial polymer
  fabrication
• The observed error rate in replication in almost
  all organisms is approximately 1 per 109
  nucleotides per generation of a cell (our genome
  is 3.2 x 109)
• Using this rate, predictions of upper limit of
  gene number made.
• What is the biggest number of average-sized
  genes an organism can have at which it becomes
  impossible to inherit an unmutated copy of each
  gene
• Estimate approx 60,000 genes

4
Replication Occurs at S-Phase
Chromosome condensation
Replication DNA must be uncompacted to allow
replication
5
Predictions of Watson-Crick DNA Model

• Watson and Crick (and others), based on their
  structural model, predicted that DNA replication
  takes place by one strand TEMPLATING the
  production of a new strand (remember . . . it
  has not escaped our notice
• If true, this would be the first example of a
  biological template that guides the assembly of
  another macromolecule

6
Models for DNA Replication
parental
daughter
CONSERVATIVE new strands are bonded to each other
SEMI CONSERVATIVE (favored by
Watson-Crick) Each new strand bonded to older
parental strand
7
Deciphering between the two Models
Meselson and Stahl 1957 Bacteria grown on
heavy nitrogen (15N) for many
generations Replaced to light nitrogen (14N)
for 1 generation Light nitrogen second
generation At each stage, extract DNA and run on
CsCl density gradient DNA is ½ old and ½
new Hence, DNA replication is semi-conservative
8
Requirements for Replication in Semi-Conservative
Model

• Free template strand (strands must be separated
  somehow)
• Recognition of each nucleotide in template with a
  free complementary deoxyribonucleotide
  triphosphate
• Something to stitch the new nucleotides together

9
DNA Polymerase Synthesizes New DNA
1955 Stanford
10
New DNA is Synthesized by DNA Polymerase

• Catalyzes the addition of new nucleotides (5to
  3)
• Nucleophilic attack of 3OH of nascent strand
  sugar to 5 alpha phosphate of incoming
  deoxynucleotide triphosphate
• Requires a DNA template (DNA -gt DNA) and free
  3OH
• cannot synthesize de novo without a primer
  (because it needs a free 3OH)
• Energetically favorable (two phosphate bonds
  broken and coupled to reaction)
• Has a proofreading function (more on this later)
• But DNA Polymerase alone has low processivity
• After each nucleotide is added, can dissociate
  from template
• The number of nucleotides a polymerase can add
  before falling off
• Several different DNA polymerases exist

11
Energy for reaction carried on 5 triphosphate of
incoming free nucleotide
12
DNA Replication Occurs at Origins and is
Bi-directional
Replication forks
John Cairns 3H-Thymidine and E.
coli Autoradiography
13
Is One Origin Enough?

• Consider this problem
• Human genome size 3 X 109 bp
• Polymerization rate 25-50 nucleotides/sec
• (1 micron/min)
• Average mammalian chromosome 5cm long
• To replicate 1 chromosome from 1 rep fork would
  take over a month
• S phase, we know, only takes 6-8 hours to
  complete
• Solution multiple origins of replication (about
  40,000 observed in normal dividing human cells)
• Hmm .. . with this many origins, replication
  would take about an hour, so why up to eight
  hours?

14
DNA SynthesisBoth Strands Simultaneously?
Would appear to require both 5 to 3 and 3 to
5 synthesis, BUT only 5 to 3 occurs So how to
do both at once?
15
Replication Forks are Asymmetric
Discontinuous synthesis in 5 to 3 Nucleotides
added in opposite direction of new chain growth
Okazaki Fragments ca 100-200 nt in length
Joined by ligase
16
DNA Polymerase Proofreads

• If left simply to correct base pairing
  combinations, the error rate would be much higher
  than is observed (that is, the affinity for 1
  nucleotide for its correct match would still
  result in higher error rate than observed)
• Incorrect base pairs can occur
• Added mechanisms in DNA Polymerase
• Correct nucleotide has higher affinity for enzyme
  than incorrect
• Incorrectly bound nucleotides more likely to
  dissociate as polymerase moves on recognized by
  polymerase
• Self correction with 3 to 5 exonuclease
  activity (aka. Mismatch repair)
• chews back incorrect base pairs immediately
  behind
• Leaves free 3 OH on last correct base pair
• Continues forward adding nucleotides

17
looks like T
18
Why 3 to 5 polymerization doesnt work the
editing mechanism
The reaction site has no available energy source,
only 5 monophosphate
19
Polymerase is not enough

• How does it access a single strand of DNA when
  DNA is bonded to complementary strand and
  compacted into chromatin (histones too)?
• When pulled-apart, ssDNA will self-anneal or try
  to re-anneal with complementary strand
• DNA is twisted, so pulling apart will cause kinks
• New nucleotides must be polymerized into a new
  strand at very high accuracy
• There is a lot of DNA, the job has to be done on
  a reasonable time scale (roughly 6-8 hours)
• Many other proteins at work

20
Starting Events in DNA Replication

• Initiator Proteins bind at origins of replication
• Helicase binds and begins to separate strands

Insert pic from MBC p 260
21
Helicase in Action
Hexamer forms ring
Hydrolysis of ATP required to pull apart DNA Can
unwind at up to 1000 nucleotides per second
22
Single Stranded Binding Proteins (SSBs)aka
Helix Destabilizing Proteins
23
DNA Synthesis by DNA Pol Requires an RNA Primer
DNA Primase Remember, DNA polymerase requires a
free 3OH on nascent strand to begin Adds short
segments of RNA to DNA template (about 10
nucleotides long) Leaves open 3OH for DNA Pol.
To subsequently add nucleotides to Are eventually
replaced with DNA
24
Lagging Strand Fragments Must be Sealed Together
RNA/DNA hybrid RNA degraded by RNAse H and new
DNA added by DNA pol and ligated to strand with
ligase
25
Clamps Enhance Polymerase Processivity
PCNA
26
Synthesis along both Strands involves a Protein
Complex
27
DNA Synthesis more like it really works
Complex involves 2 polymerases bound together and
moving in same direction For this to work, the
lagging strand is looped around and spooled
through
28
Preventing Tangles During Unwinding Enter
Topoisomerase
Every 10 bp unwound is 1 turn of helix helicase
unwinds at 1000 bp/sec or 100 turns per second
29
TOPOISOMERASE I
30
other considerations during replication

• what happened to all those histones? How does
  replication proceed through nucleosomes?
• not understood BUT the replication complex
  appears to be able to polymerize through
  nucleosomes WITHOUT causing dissociation of the
  histones
• The new strand quickly has histones added behind
  the replication fork
• how do you replicate the end of a chromosome -
  telomeres
• linear chromosome at 3 end creates a problem
  for the lagging strand synthesis. These ends are
  composed of repeat sequences and are noncoding
  heterochromatin
• every round you would lose some nucleotides
  (evidence is about 50-100) off the end
  eventually with enough cycles you would lose
  coding sequence
• problem corrected by telomerase

31
Telomerase adds nucleotides to the 3 end of DNA
of template strand using a built in RNA strand as
a template
32
Telomere Shortening and Cell Lifespan

• best data in fibroblasts of the skin
• These cells have finite number of divisions
  before they senesce (stop dividing but not dead)
  called replicative cell senescence to
  distinguish it from other mechanisms of
  senescence
• Divisions related to age of source (young
  derived vs. old)
• fibroblasts have little or no telomerase
  activity
• believed that once coding sequence becomes
  abridged cells programmed somehow to die or stop
  dividing permanently
• perhaps a safety mechanism for cancer
• addition of telomerase can make fibroblasts
  immortal

33
Chromosome Compaction and Replication Interplay

• Heterochromatin replicates later than euchromatin
• housekeeping genes replicated early in all
  cells
• Cell-type-specific genes replicated early only in
  those cells which express the gene
• condensed heterochromatin regions do not
  replicate slower but they replicate later during
  S phase
• Because replication at all regions is not
  synchronous S phase takes longer than 1 hour,
  up to 8 hours

34
Summary of the DNA Replication Players

• Initiation Proteins bind to Origins of
  Replication
• Open portion of DNA
• Helicase pulls apart the two strands
• SSBs bind to single stranded DNA
• An assortment of proteins assemble the
  replication complex
• DNA Primase makes RNA Primers
• DNA Polymerase functions as a dimer
• Sliding clamp proteins attach to DNA polymerase
  and enhance processivity
• Topoisomerases relieve rotational tension on DNA
• DNA ligase joins fragments along lagging strand
• Telomerase lengthens ends of chromosomes

35
DNA REPAIR MECHANISMS
36
The Fidelity of DNA Replication

• After replication, on average, only 1 error per
  109 bases, or 3 per human genome!
• Only 1/1000 accidental base changes ultimately
  results in permanent mutation
• Less than expected by fidelity of Watson-Crick
  base pairing alone
• Proofreading mechanisms are in place

37
Errors in DNA

• Factors that cause DNA damage and errors
• UV light
• Chemicals that modify or destroy bases
• Spontaneous alterations tautomerization
• DNA replication errors
• Free radicals
• Random thermal collisions with solvent!
• 5000 purines lost per day per human cell due to
  disruption of N-glycosyl linkage to deoxyribose
  (most common form of spontaneous change)
• Uncorrected Errors lead to mutations

38
Mutations

• Permanent change to nucleotide sequence
• Can be single base sub, deletion, addition of
  many bases
• Often deleterious
• Silent mutations
• Undetected
• Not in coding sequence
• Do not change amino acid
• Amino acid change is tolerated

39
Mutational Load

• Observed mutation rate places upper limits on the
  amount of coding information/gene numbers we can
  have
• Above this, probability of inheriting working
  copies of every gene is too low
• Estimate is approx. 60,000 genes

40
MOST COMMON FORMS OF DNA DAMAGE
Red spontaneous oxidative damage Blue
hydrolytic attack Green uncontrolled methylation
41
Most common spontaneous forms of serious DNA
damage
5000 times/day
100 times/day
42
Pyrimidine dimers formed by UV light
If not corrected before replication, can lead to
deletion or substitution
43
Modification to Mutation
C-G becomes U-A, later becomes T-A (because U not
normally found in DNA)
44
Heavy Investment in DNA Repair Machinery

• In DNA and Yeast several coding capacity is
  in repair genes
• Mutations in repair genes have serious
  consequences

10-15 human colorectal tumors display massive
instability of microsatellites short repeating
sequences scattered throughout genome
45
Expansion of Repeat Sequences

• Repeats create difficulty for DNA Polymerase
• Replicative slipping
• Corrected by repair enzymes
• But mutations in repair enzymes can affect this
  process
• Expansion of repeats can cause severe pathologies
• Colon tumors, fragile X syndrome (neurological),
  myotonic dystrophy, Huntingtons disease, ataxia
  type I

6 CTG repeats
8 CTG repeats
.CTG CTG CTG CTG CTG CTG .GAC GAC GAC
GAC GAC
.CTG CTG CTG CTG CTG CTG .GAC GAC GAC
new strand temporarily dissociates and reanneals
off register
46
Multiple DNA Repair Systems at Work

• Despite all the damage, fewer than 1 in 1000 base
  pair changes results in mutation
• Can be upregulated in response to damage
• Generally rely on one good copy to template the
  repair of the bad copy

47
Whos the Good Copy?

• one strand is used to template the repair of the
  other
• If done randomly, correct base only inserted 50
  of time
• E. colis solution methylate your DNA (dam
  methylase at GATC sequence)
• The key - methylation of newly synthesized DNA is
  latent

48
Base Excision Repair
Deaminated C is Uracil, which looks like a
thymidine and thus pairs with T when copied
Identifies small substitutions or alterations in
bases Uses variety of enzymes collectively
called DNA glycosylases each recognizes a
specific altered base and removes it Followed
by AP endonuclease, which recognizes nucleotides
with no base (depurinated damage starts
here) Then filled in by DNA pol and sealed with
ligase
Scan along DNA
49
Nucleotide Excision Repair
Removes large lesions Large complex of enzymes
that recognize gross distortions Cuts flanking
distorted region, peeled away by helicase Repair
from here is the same as for others
50
DNA Damage Delays Progression Through Cell Cycle

• Checkpoints in cell cycle ensure proper
  completion of steps
• If not, progression is delayed, repair enzymes
  upregulated, and problem corrected