MCB 110 Study Guide

 Part 1: DNA structure, energetics, restriction enzymes

o Chemical level
 Sugar phosphate - deoxyribose or ribose (differ by hydroxyl group which
can create instability)
 Nitrogenous base
 2 purines - adenine and guanine ‘Pure as Silver AG’
 3 Pyrimidines - ‘cut the py’ cytosine, uracil, guanine
o pyrimidines are single ring (like pi)
 Actually MANY more bases produced through modification
 Phosphodiester linkages join the backbone
 Glycosidic linkages bind the ribose sugars to nitrogenous bases
 Both bonds have spontaneous reactivity and can undergo hydrolysis easily
o Watson Crick Model
 Linear Chains DNA strands have polarity running from 3’-5’
 DNA strands bind together into dsDNA
 Base pairing
 Creates consistent diameter for the structure : 1 Purine + 1
Pyrimidine ~ 11 angstroms
 Hydrogen bonding creates only certain favorable matchups - G
bonds with C and A bonds with U/T
 Strands run anti-parallel
 The angular pairing of the bases creates a major and minor groove
 Even though most of the information is actually packed inside the
DNA strand, reading the pattern of hydrophobic groups in the
major groove allows specific binding of enzymes without strand
unzipping
 The minor groove contains nonspecific pattern of hydrophobic
groups that allows for indiscriminate binding
o Energetics
 Base stacking pi interactions
 Hydrogen bonding
 Helical twist is due to repulsion of the negative phosphate backbone -
produces a horizontal displacement between negative charges; this twist
involves a tradeoff in energy between reduced base stacking and negative
charge repulsions
 DNA melting/denaturation : pairing, annealing, hybdridizing
 Difference AT and GC - greater percentage of GC produces a higher melting
temperature, i.e AT regions are less stable strands
o Melting temperature: 50% ssDNA 50% dsDNA
o In general a mized sequence Tm is dependent
o B and A forms of DNA
 Both are right handed helices
Type B A
Molecule dsDNA dsRNA + dsRNA + DNA

Width 2nm 2.6nm

Helical turn /10.5 base pair
Vertical Packing 0.34 nm rise / base
Functionality Less stable, ideal for DNA
unzipping; large accessible
major groove for recognition
 Restriction Enzymes
/11 base pair
0.24 nm rise / base
Tightly packed together, more stable, better for rigid
RNA structures, major groove is tucked inside
 o Cut DNA at specific palindromic repeat sequences - palindromic
sequence is equal to its reverse compliment
o GAATTC
o CTTAAG
o Different types of overhangs: 5’, 3’, blunt
o Example: EcoRI which binds to major and minor groove sites
 Methylases
o Methylate the H of the nitrogenous bases
o Hemimethylated or fully methylated
o Methylation of DNA prevents regonition by a cell’s own restriction
enzymes - this allows restriction enzymes to function as a sort of
immune system and attack foreign unmethylated DNA
o DAM methylase creates 6 methyl adenosine
 DNA Packing
o Packing requirement
 10 cm long strand packed into 10um wide cell - compaction
factor of 10^4
o Levels of packing
 Double strand of DNA
 Nucleosome array - coiling around histone proteins
 30 nm fiber of nucleosome arrays
 Euchromatin - loops of the 30 nm fiber ; usually the highest
level in a non replicating cell
 Heterochromatin - genes are silenced, packed structure not
known
 Chromosome
o Nuceleosomes
 DNA double strand wraps around the 8 subunit histone protein
 Histone tails contain post transcription epigenetic
modifications that control the expression of genes wrapped
around the histone
 Modification of histone determines higher level structures
 DNA Topology
o DNA topology is generally described by linking number which is the
sum of twist (Tw) and writhe (Wr)
o DNA twist is simply the number of 360 degree turns in the helix (for
DNA A there are 10.5 bp per 360 degree turn)
o DNA writhe is the number of overlaps of the double strand with itself,
can be positive or negative
o DNA Lk # cannot be changed in a closed loop
o Example. Imagine a 20 Twist loop with no supercoils; if two twists are
removed the DNA can assume a circular chromosome if some unpaired
sections or it can assume a completely paired structure with 2 -
supercoils
o Negative supercoils are related to the energetics of unwinding the
double strand - the unfavorable energy of strand separation can be
balanced by relief of negative supercoild tension
 Topoisomerases change the linking number of DNA structures
o No ligase needed, reversible binding
o Type 1
 No ATP required
 Changes Lk by 1
 Makes a knick in one strand; a single tyrosine hydroxyl binds
to the phosphate breaking one of the linkages on one strand
o Type 2
  Requires ATP
 Changes Lk by -2
 + supercoil becomes a - supercoil
 2 tyrosine hydroxyls break the phsophodiester linkages on
both chains making a 2nm gap through which it passes the
other strand ‘strand passage’
 Enzyme has complex coordination of steps involving breakage
of strand 1 and trapping of strand 2

Part 2 DNA synthesis

 DNA polymerase
o Discovered by Arthur Kornberg
 An in vitro assay in which components of cell were fractionated and tested
for ability to incorporate radioactive nucleic acids into a chain
o 4 requirements for synthesis
1. magnesium ions (needed for protein active site catalysis)
2. 3’ RNA primer with 3’ hydroxyl
3. triphosphate nucleotide - deoxyribonucleotides triphosphate
dNTP (triphosphate bond cleavage drives the reaction forward
energetically)
4. template strand
o synthesis only 5’ to 3’ direction which is consistent with the fact that the new bond
is being formed through condensation between 3’ hydroxyl and phosphate 5’
o Pol 1 - found in E.coli
1. Made of 3 subunits - a 5 - 3 polymerase and a 5’ - 3’ exo nuclease and a 3’ -
5’ exonuclease (exonucleases cleave individual nuceolotides at the ends
where as endonucleases cleave anywhere)
2. Uses the 5-3 exo to cut out sections of DNA after a nick which are then
replaced by the 5-3 pol ‘ nick translation’
3. As the pol lays down nucleotides in the 5-3 direction, the 3-5 exo checks
and proofreads and is capable of removing the last nucleotide layed down -
it removes the non template strand converting it into dNMPs
deoxynucleotide monophosphates;
4. for this editing step to occur, pol activity in the 5-3 direction must stop; thus
the strand actually switches location between the two active sites. Incorrect
nucleotide addition means the strands don’t bind as strongly which favors
switching to the editing site
o Fidelity = frequency of incorrect nucleotide addition; high fidelity replication means
lower polymerization rate
o Pol 3 - found in E.Coli, required for a genome replication (cell only typically has 2 - 3
molecules to copy entire genome)
Pol I Pol III
Subunits ( types) 1 3 and accessory
3’ - 5’ exonuclease Yes Yes
(proofreading)
5’ - 3’ exo Yes No
rate 16 - 20 nuc / sec 250 - 1000
Processivity 3 - 200 500,000

o Eukaryotic DNA polymerases

 Alpha - primer synthesis
 Delta (has 3 - 5 exo) genome replication (mostly lagging strand)
 Epsilon (has 3 - 5 exo) genome replication (mostly leading strand)
o Summary of Polymerase basics
  Several different types of polymerases with specialized functions
 Bacteria have two type Pol I (nick translation with 5 - 3 exo) and Pol III
(much more processive, genome replication)
 Eukaryotes have Pol a (primer) Pol delta (lagging strand synthesis) and
Pol epsilon (leading strand synthesis)
 4 main requirements ; 1) Mg ions 2) RNA primer with 3’ end 3) template
strands 4) dNTP (deoxynucleotide triphosphate)
o PCR: 2^N strands for each cycle
o Gel electrophoresis: agragrose vs. acrylamide, visualization agents; ethidium
bromide or acridine orange
 Replication Factors
o Primer
 RNA polymerase begins the chain without a primer
 Only need Mg ions and NTP
 RNA primer is 4 - 10 nucleotides in length
 E.coli - uses a primase DNAG to create DNA-RNA ds duplex with a -form
geometry
 So in eukaryotes primase, Pol alpha extends the RNA primer with DNA
before pol delta/epsilon takes over - an extra DNA primer
 Primase and Pol alpha (not processive_ form complex
 Primer removal
 in E.Coli by Pol I using the 5-3 exo
 in eukaryotes Pol delta or Pol epsilon displaces the RNA primer and
the RNA primer is cleaved by a endonuclease FEN 1 (flap
endonuclease)
o Ligase
 Joins backbones at nicks
 Requires a ATP and releases ADP
 The enzyme binds AMP adenosine monophosphate to the backbone after it
creates a complex with AMP with lysine amine group
o Lagging and Leading strand
 All polymerases add nucleotides in the 5 - 3’ direction only
 Thus one strand is made up of okazaki fragments - as the form opens a
short fragment is added eading away from the form
o Sliding Clamp
 6 lobed rings
 Beta (E.Coli) dimer
 PCNA (eukaryotes) trimer
 Helps bind polymerase to increase processivity by association with the C-
terminal face
o Clamp loader
 Y complex in E.Coli, RFC in eukaryotes
 5 subunits
 requires ATP activiates binding to clamp; clamp + clamp loader binds to
ss/ds DNA primer junction at a 5’ overhand; the clamp interaction with
DNA allows it to close which promotes loader release
o DNA Helicase
 DNA helicases actually bind to ssDNA and move along the strand with
either 5-3 or 3-5 directionality, when it encounters dsDNA it hydrolyzes
ATP and unzips the strand
 E.Coli use DNAB a hexameric protein that moves in 5-3 direciton
 DNAB is 5 - 3 (on lagging strand) and MCM is 3 - 5
o ssDNA binding proteins
 ‘E. coli use SSB
 SSB homotetramer binds 70 nucleotide long sequences
  Bind cooperatively thus if a SSB is already present keeps binding mo
 re
 Eukaryotes use RPA (replication protein A) RPA heterotrimer binds 40
nucleotides
o Topo
 TopoII (DNA gyrase)
 Removes positive supercoils, changes linking number by 2
 TOPO IV
 Seperates intertwined chromosomes (seperatre rings but locked
together)
 Decatenation
Prokaryotes Eukaryotes
Primer (‘green’ - DNAG) DNAG Primase + Pol alpha
Primer removal 5-3 exo Pol 1 FEN 1
Ligase Ligase Ligase
Clamp Loader (load weights at Gamma (Y) complex RFC
RSF and YMCA)
Sliding clamp (slides on Beta PCNA (Proliferating cell nuclear
playgrounds are PC but not antigen)
applicable )
Polymerase Pol III Pol epsilon and delta
Helicase DNAB MCM2-7 complex
ssDNA Binding protein SSB RPA (RFA)
TOPisomerase TOP II, IV Multiple

 Replication Initial and Termination: Lecture 6 - 7

o Ori C - replication origin in E.Coli
 Found by using restriction enzymes to slice up the bacterial genome into a
bunch of small plasmids with ampicillin restitance gene, putting into E.coli,
then seeing whats the similar code sequence between E.coli that have amp
resistance
 A DNA sequence that has 13 bp AT rich repeats followed by 9 bp repeats
o Steps to loading of helicases ( which have trouble starting on a normal DNA strand
because the are no free ssDNA ends)
 DNAA - binds cooperatively to 9bp repeats of ori, induces conformational
changes from ATP binding that creates + supercoils which help unwind
strand IN AT RICH SEQUENCE REGION (weaker)
 DNAC - chaperone for DNA B with affinity for DNAA
 DNAB - helicase
 * DNAA has binding for B and C because it needs to load two DNAB
molecules going in opposite directions, so the directionality of binding is
carefully controlled
 Helimethylated origins are resistant to initiation which is a clever way of
preventing DNAA from rebinding immediately to the copied DNA origins
o Eukaryotes don’t have a single replication origin
 ARS - autonomous replicating sequences
 ORC - Origin recognition complex to bind MCM2-7
 Only activated in some part of the cell cycle G1 S (replication) G2 M
(mitosis)
 Replication Termination: Lecture 7
o Tus proteins bind to TER sites
o Tus proteins have polarity, they stop DNAB depending on direction of travel, if they
hit the permissive face they dislodge the stuff itself
 Telomeres
 o End replication problem !! - inability to fill gap left by the last RNA primer on the
lagging strand
o TTAGGG telomeric repeats
o Problem in that to recreate the overhang at the end (by 5-3 exonuclease activity) the
telomere is repeatedly shortened
o So new DNA is added by telomerase using a RNA template and then Pol alpha +
primase extends the other part (telomerase is a reverse transcriptase)
o Telomerase is in high levels in stem cells
 Mutations Lecture 8 -9
o Specialized germ cells are passed on, these don’t undergo the same degree of
replicative cycles as somatic cells preventing too much mutation from occurring
o Sequence polymorphism - functionally neutral
o Single Nucleotide Polymorphism !!??
o Types of mutations
 Replication errors
 Double strand break
 Abasic side (base hydrolysis)
 Deamination ( methylated cytosine -- > thymine, cytosine -- > uracil )
 Dimerization to form double stranded DNA - UV light induces pyrimidine
dimerization between thymine molecules for example
o DNA Repair
o 1) Direct Repair - very specific, single base repair
 specific enzymes for specific individual base damage; enzymes degrade
after use but its worth it
 example: methyl transferase removes the methyl group from incorrectly
methylated guanine which would otherwise pair with thyme
o 2) Base Excision Repair - appropriate for deamination, repairs a single base missing
or altered
 glycosylase generates abasic site by recognizing damage
 endonuclease and phosphodiesterase removes sugar phosphate from abasic
site
 DNA polymerase adds correct base it and ligase seals it up
o 3) (oligo) Nucelotide Excision Repair - takes out an entire surrounding strand, good
for repairing dimerization (from UV light)
 Uvr A / B recognize bulky lesions from distorted geometry
 They make cuts at both 3’ and 5’ ends
 Helicase displaces the cut strand
 Pol I and ligase repairs and reseals
 Discovered that NER is very important for UV/radiation protection from
skin cancers
 RAD genes in yeast
 XP proteins in humans
o 4) Mismatch repair - both bases okay but paired combination not
 MutS recognizes error, MutH recognizes strand, MutL links the two together
 MusS - tries to bend the strand to test for bad pairing, bad pairing more
easily distorted
 MutH exploits transient DNA methylation to detect the recopied strand as
that lacking methylation, makes a nick at GATC
 Helicase UVrD and exonucleoases then remove nucleotides back to the
mismatch and Polymerases resynthesis the strand
 Eukaryotes: don’t have MutH (or Dam methylase)
 Errors in MR linked to colon cancer
o 5) SOS response
 Psorelin - intercalcates at AT/TA, causing extreme damage which can result
in double stranded break
  Rec A activates Pol V which is highly error prone and just adds random
nucleotides with a preference for A
 If the bacteria is undergoing major DNA damage then there’s a chance
that these mutations may throw up something useful. It’s a sort of insta-
evolution for times of stress.
o Alternatives for dsDNA breaks
 Non Homologous End Joining (NHEJ) - mostly humans
 Repaired DNA suffers from deletion of nucleotides
 Homologous End Joining (HEJ) - all organisms ; restores site by copying
from second chromosome
 HR - Homologous Recombination
 homologous chromosomes is a set of one
maternal chromosome and one paternal chromosome that
pair up with each other inside a cell during mitosis
 HR
 Step 1 create ssDNA
o (from double stranded break) - use a 5- 3 exonuclease to create
a 3’ overhang
 Step 2
o Find the homologous regions
o Rec A in E.coli, Rad 51 in humans
o Rec A bind to the damaged DNA strand, creating a protein
polymer filament via cooperative interactions; the geometry is
such that a double strand can be threaded around the single
strand and sampled for homology at which point it can break
from its double strand to pair with the new strand due to
favorable Rec A interaction, ATP is only required for Rec A
binding
 Step 3
o Fourway holliday junction - four way ds DNA junction
o Held in place by RuvA
o Homolgous cross over area
o Extend the exchange of strands beyond the initial homology
‘branch migration’
 Ruv A + Ruv B ( a helicase)
 Step 4
o Ruv C - a endonuclease
o Cleaves both the Watson or both the Crick strands
o Depending on the type of cleaving either get recombinant ends
or whole strands basically switch over
 Because we paired two homologous chromosomes which might not be
completely identical there can be now be mismatch repair which will
fix some sort of mismatch - this can result in a loss of hetezygocity
(LOH)
 Inappropriate HR
 Sometimes 2 near sequences on a chromosome can have very similar
sequences: they can be direct repeats or inverted repeats
 Direct repeat  - - -  or inverted repeats  ---- 
 Inappropriate homologous recombination results in inversion (inverted
repeats) or deletion of in between region (direct repeats)
 Innapropriate HR is responsible for color blindness
 Inducing double stranded breaks is a strategy for genome engineering: example:
homing endonucleases (HE) can be used to delete particular sequences and then
filled in by something else
o Site Specific Recombination (ssr)
  Site specific has 100%probability of recombinant ends vs. 50% for homologous
repair
 Important in the lytic cycle - allows the lambda DNA of bacteriophages to be
inserted into the host genome in the prophage pathway
 Site specific recombination provides the means to switch between
prophage and lytic pathways
 Uses a tyrosine recombinase
o Covalent binding similar to the topoisomerases - therefore a
reversible reaction
o 1) cleaving of strands by covelent linkage with tyrosine 2)
binding of strands to other strand 3) isomerization 4) cleaving
5) rejoining
o A very short region of strand exchange which is specific to the
recombinase
 Salmonella uses site specific recombination to evade the host immune
system
o Transposable Elements
 DNA transposon
 Have direct repeats at each end and inverted repeats as well
 The IS region contains the inverted repeats and all the important coding
stuff in the center of the protein
Non - replication transposition (cut and paste)
 The transposable element is removed by a
transposase from ints original location - an OVERHANG cut
is made and the transposon pasted in between, the overhang
then gets filled in to become the short direct repeat region -
this stays on even after the transposable element leaves
creating a permanent mutation

Replicative Transposon

 Mobile DNA elements like transposons often carry useful genes

 Retrotransposition requires reverse transcriptase
o DNA viral -- > DNA in cell  RNA
 DNA rearrangements is intentionally sloppy to provide adaption
o Example adaptive imunity
o Binding sites on antibodies
o Made by B cells which each create 1 particular / unique antibody
o Light chain and heavy chain - each B cell makes 1 type
o A transposable elemnt in the genome between a V and J element is
removed to create the light chain gene
 RAG recombinase
 Bind to specific sequences of repeats surrounding the
V, D, J elements
 Heavy chain V - D - J
 Light chain V - J
 The excised circle is not maintained
 Sequence diversity abundant
o The
Origin
 OriC was common sequence among different plasmids (generated by using
res enzymes to slice of bacterial genome) that expressed antibiotic resistance
when a amp resistance gene was added to the plasmid
 Ori C has two regions of bp repeats; 1) AT rich 13 bp repeats and 9 bp
repeats
 DNAA undergoes cooperative binding via ATP to 9bp repeats induces
conformational supercoil formation that favors strand separation in the AT
13 bp repeat region
 DNAC acts as a chaperone for helicase DNAB; the binding directionality is
important; in bacteria helicase unzips in a direction: thus C and B both have
affinity for A
o DNAC and DNAB are hexameric and require ATP
o DNAB proceeds in a 5 - 3 prime direction
 The origin on the newly replicated strand is now helimethylated (DAM
methylase forms methylated adenine) but this binds competitltey to other
factors besides DNAA (which binds best to fully methylated)
Single stranded binding proteins bind cooperatively to straighten and prevent
recombination of the strands with themselves - RPA / SSB
The next step is clamp loading
A clamp loader, y-complex or RFC complex becomes activated with 3 units of ATP
enabling to bind to the glamp (beta or PCNA) that then undergoes a conformational
change that opens it up up the clamp - this enables the clamp to bind to a ds/ss
junction where ATP hydrolysis results in the clamp closing and release of the loader;
the C - terminus of the clamo has a high affinity for polymerase

Primer synthesis
Primers are RNA ar first; only requires Mg, template, and NTP
DNAG and primase/pol alpha lays down the RNA primer
The rna primer is removes by a flap endonuclease FEN1 in eukaryotes after strand
displacement by copied strand and removes by 5 - 3 exonuclease of pol I in e .coli

The final steps are for ligases to join together any strands that aren’t fully paired
DNA ligase acts by first using a lysine amine group to bind to ATP which becomes an
AMP; the AMP leaves the enzyme and becomes bound to the phosphate of a nick, the
hydroxyl attacks and the AMP leaves createing a new phosphodiester linkage

Termination of of replication in E.coli

Occurs at Ter sites where polar Tus proteins bind which depending on directionality
of intereaction with pol will displace it

Topoisomerases are also essential

Top I
Top 2
Both bind covalently via a tyrosine residue hydroxyl
I requires NO atp ii REQUIRES ATP, I only drives toward Lk0 no supercoils, higher
twist
Words by nick, rest of DNA freely rotate
Type two works by two nicks which split it apart carefully coordinated strand
passage

Pathway Substrates How it words Proteins

Direct Repair Incorrect Specialized enzymes Mythyl transferase
methylation (degrade) to correct particular
problems
Base Excision Repair Depurination, Base removed (or already Glycosilase
(BER) Deamination abasic) , Phosphodiesterase
AP (apurinic) endo removes AP Endonuclease
evything else Pol and ligase
Pol I and beta
Nucleotide Excision Dimerization 1) Uvr A scans and activate B UvrA / B (ultra violet
Repair (NER) (UV light) which has helicase activity resistant )
Bulky ‘lesions’ 2) Uvr C is recruited and cuts UvrC exo
either side of lesion (12 in pro Helicase
29 in euck) Pol and ligase
3) Uvr D cuts the entire thing XP in humans
out and pol takes over
Mismatch Repair Don’t match 1) MutS attempts bending MutS
recruits H / L (ATP) MutL and MutH
2) MutH endonuclease looks Helicase UvrD
for hemimethylation GATC
nearby and knicks
3) MutL connects Mut S and H
4) helicase removes not
methylated strand, degrades
strand by 5-3 or 3-5 exo

SOS Intercalciation RecA activates Pol V

NHEJ Non Double strand
homolougous end Break
joining
Homologous Double strand 1) first we have detection of a Rec A
Recombination break double stranded break - RuvA/B
exonucleases create a RuvC
overhang
2) Rec A or Rad 51 (+ BRCA2
to increase affinity over RPA)
bind into polymer that can
sample homologus strands for
similar sequences
3) holiday junction with RuvA
‘branch migration’
4) Ruv C resolves the complex