Notes

Exam III

Summary
● Nucleotides and Nucleic Acids
● Genes and Chromosomes
● DNA Replication, Repair, and Recombination
● DNA Structure, Hybridization, Topology
○ https://www.youtube.com/watch?v=o_-6JXLYS-k
○ DNA structure contributes to function
● Gene Regulation in Prokaryotes
● Gene Regulation in Eukaryotes
● Genetic Engineering
● Epigenetics

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Nucleotides and Nucleic Acids
Functions

● Nucleotide
○ Coenzymes
○ Regulatory molecules

Nucleotide v nucleoside

Pyrimidine v purine

● Aromatic = absorb UV light (only NMP where N is A,G,U,dT,C)

Tautomeric equilibrium (favored forms, source of error)

Furanose in RNA v DNA

B-N-Glycosidic bond

● Syn v anti (B-DNA)

● Restriction bond 4
● Pyrimidines are always anti

Phosphate groups in DNA v RNA synthesis

● Phosphatase enzyme & hard bomb to detonate

MEMORIZE deoxyribonucleotides and ribonucleotides

● 2 minor bases of DNA

● 4 minor nucleosides of RNA
● 2 methylation systems of E. coli
○ Defense & distinguish nascent from parent
● Methylation *
○ CpGs repress gene activity (methyl transferases)
○ Accidental deamination of methylated C to T
○ DNA methylases?

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 ○ ¾ CpGs lost from vertebrate genome except in promoters of house-keeping
genes *
● Epigenetics*
○ changing the activity of different genes, not by changing DNA sequence but by
manipulating the DNA ie methylation
● Deamination v Depurination
● Anapurinic residue
● Why we have T in DNA and U in RNA
● Polynucleotides (sugar-phosphate backbone)
○ DNA v RNA stability
○ Read from 5→ 3
● Enzymes read DNA like braille (consistent bp geometry)
● Stability of DNA comes from hydrophobic nature of bases (pi-stacking)
● Intercalation of dyes in DNA
○ Aromatic dye added to DNA
○ in UV light, will release energy by photon (fluorescence)
○ Will not release energy by thermal collision because in DNA less freedom of
motion stacked in DNA base pairs
● Base flipping
○ One base sticks out of the double helix
○ Impt for DNA repair and DNA methylation
○ Enzymes scan DNA by base flipping in homologous recombination
○ Transcription flips out bases - the RNA polymerase enzyme has
holes
● Photolyase repairs pyrimidine dimers
○ Source of skin cancer
○ In E Coli?*
● Major v minor grooves
○ Major - gives you the sequence
○ Minor - just tells you its a valid base pair (can identify mismatch)
○ Both grooves provide information to proteins

● A, B, Z form of DNA

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 ○ B form is MOST COMMON
○ Base pairs stack together closely excluding water
○ 10.5 base pairs per turn
○ Right handed
● Denaturation
○ High T
○ Change in pH
○ Disruptions to H bonds and base-stacking interactions
○ Complete denature is two step process
○ ™ (melting temperature) when DNA is half denatured
● G:C content - more H bonds
● DNA length - more H bonds, base pairs
● Ionic strength - strengthens H bonds, stabilizes neg charged backbone
● pH of solution - *
● Hypochromic & Hyperchromic effect
● Hybridization of DNA
● Southern Blotting
● PCR - cloning DNA, in vitro (recombinant DNA, application of Hybridization)
https://www.youtube.com/watch?v=iQsu3Kz9NYo

https://www.youtube.com/watch?v=uKeMiAZ8Zu4

1. DNA Taq polymerase (Thermus aquaticus) heat stable

2. 4 dNTPs (monomers/nucleotides)
3. MgCl2 = Mg 2+ cofactor of DNA Polymerase
4. 2 primers (forward and reverse primer) because DNA polymerase cannot initiate
a new strand on its own can only extend 3’ into a preexisting strand
5. Buffer - Tris HCl, pH 8.3
6. Template DNA (what we are going to copy)
○ STEPS:
● Denature DNA using high T
● Primer attach at binding sites
● Anneal primers to template
● 3’ OH will be extended by DNA polymerase
● 2 copies with long tails all single strand
● PCR CHAIN TERMINUS
○ Has no 3’ OH at the sugar
○ Dideoxyadenosine 5-triphosphate vs normal DATP
○ Can’t add another nucleotide, stops chain

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Genes and Chromosomes
● Central Dogma
● DNA is SO much larger than the cell, and it fits in
nucleus!
● Chromosome Anatomy
○ Exon and intron - unique to eukaryotes
○ Centromere
○ Chromatin
○ Telomere - protect ends from degradation AGING
○ Telomerase
○ Hayflick limit - cell can’t divide anymore
○ DNA is found in other places than nucleus
● Mitochondria (mtDNA)
● Chloroplast
➢ Endosymbiosis - suggests that these organelles must have once been
bacteria that was eaten by predatory eukaryote
➢ Circular, double stranded DNA
● Supercoiling
● What causes it? Due to unwinding of DNA (tension)
● Why impt for cellular functioning? Must relieve this tension otherwise it will stop
the enzyme
● Does not change the number of turns but compresses them to a shorter segment
● MOST ORGANISMS HAVE (-) SUPERCOILS
1. Compacts DNA****
2. Easier to unwind
➢ Thermophilic archaeans exception - need to prevent DNA
from denaturing → +supercoil
https://www.youtube.com/watch?v=HyP0cEbqKTc
● Topology - how elastic materials properties stay fixed under deformations
○ Defined as those that can be altered only by breaking 1 or 2 strands (when DNA
ends are fixed)
○ https://www.youtube.com/watch?v=5hwaDamU-jo
○ Underwinding (-2) LK down, more bp/turn
● Easier to separate
● Cells maintain this state!

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 ● When we want to replicate DNA, transcribe it
○ Relaxed - circle LK=200
○ Overwinding (+2) LK up, less bp/turn
● Harder to separate
● When we replicate DNA in S phase, trying to pull sister chromatids apart
(knots and tangles)
○ Twist - in the strands
○ Writhe - the number of times the whole thing folds over itself
○ Linking number = twist + writhe
● Constant UNLESS you cleave a strand
● Topoisomers
○ Type I - for twists, no ATP needed, changes LK by 1
○ Type II - for writhes, ATP needed, changes LK by 2
● Topoisomers in Electrophoresis
○ Shape - different, more compact faster
○ Size - same mass
○ Charge - constant charge to mass ratio
● Topoisomerase IV - separates daughter chromosomes in bacteria DNA replication
● DNA is organized into Chromatin
○ Higher order folding and compaction of DNA when non-dividing
○ Histone proteins - nonspecific bindings (at minor groove)
1. Are positively charged (Lys, Arg)
2. Need to be escorted by histone chaperones in order to form
octamers; otherwise would stick to DNA randomly
3. If only methylated - gene silenced
4. If any acetylated - gene is expressed
➢ STEPS:
a. 2A and 2B dimerize
b. H3 and H4 dimerize
c. H3H4 and H3H4 form into tetramers
d. Tetramer binds to DNA
e. Two 2A2B dimers can join now
● Nucleosomes - beads on a string
1. Compacts DNA x7
2. Regulates access to DNA
➢ When wraps DNA, reduces linking number = neg
supercoil
➢ Nucleosome is made of 8 histones (octamer)
a. Histone tails keep DNA from falling off

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 ➢ The perfect binding site is an alternating GC rich/AT rich regions
b. A-T minor groove touches histone
c. Part of DNA away from nucleosome (C-G in minor groove) will bend DNA
toward major groove, allowing for
tighter bind*

● Nucleosomes at replication fork

○ CAF-1 histone chaperones associates with the sliding clamp (PCNA)
○ Nucleosomes go to both strands - Allows chemical markings to be passed on to
daughter cell so that the histone modifications (gene silencing, gene expression)
are preserved/retained
● Level of Compaction
○ Nucleosome compacts DNA about 7x
○ Needs to be 10,000x to fit into eukaryotic nucleus
○ Histone H1 compacts by 40x more
● Causes tighter wrapping (brings nucleosomes together)
● Near DNAs exit from the nucleosome
● At the center of DNA (chromatin fiber?*)
○ Loops of DNA attached to a protein scaffold (daisy chain)
● SMC Proteins (eukaryotic DNA)
○ Cohesins - hold sister chromatids together until separated
○ Condensins - help compact DNA chromatid into higher order chromosome
○ Two coils of SMC proteins wrap around each other and make ATP binding sites
● Organization of Bacterial Chromosome
○ Loops with anchors

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DNA BASICS

● Gene: a portion of DNA that codes for something specific

● Chargaff’s Ratios - AT and CG are found in equal amounts to e/o
● 1% of DNA codes for proteins
● https://www.youtube.com/watch?v=ofjyw7ARP1c MITOSIS G, S1, S2 stages of DNA
replication
● https://www.youtube.com/watch?v=mcEV3m9SG9M BASICS chrom
● DNA is packaged: chromatin → chromosomes only when about to divide

DNA Replication, Repair, Recombination

The Central Dogma of Biology DNA → RNA → PROTEIN

3 Fundamental Rules of Replication

1. Replication is semiconservative
○ produces two copies that each contained
one of the original strands
2. Replication begins at an origin and proceeds
bidirectionally
3. Synthesis of new DNA occurs in the 5 → 3 direction AND is semi
discontinuous (at LAGGING STRAND)

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DNA REPLICATION (SYNTHESIS) making another copy of DNA

● Enzyme - DNA polymerase (like palm with thumb and finger)

○ LIMITED - can only add nucleotides if there are already nucleotides
there; cannot start any sequence from scratch; can only continue
sequences
○ Proofreads with exonuclease site
○ Addition of nucleotides requires energy, which comes from the nucleotides
themselves (cleaving phosphate)

● Primer template junction (PTJ)

○ dNTP comes in
○ Phosphate end (𝛂) binds to 3’ OH
at the sugar
○ Release of (PPi)
○ PRIMER IS IMPT PRIMER GROWS
■ DNA cannot extend itself?
■ Nucleotides will attach to
primer, using DNA as a guide template

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 ○ Pyrophosphatase enzyme cleaves to 2 phosphates (EXPLOSIVE)

● Leading Strand v Lagging Strand

○ Leading : the one being replicated TOWARD the fork
○ Lagging : replicated AWAY from the fork (going in reverse is slower)
■ Okazaki fragments - result of DNA PLY only
synthesize from 5 → 3 tail end

● Base pairing is required

○ Puts the 𝛂 phosphate in the perfect position for nu attack by 3’OH of the primer
○ If mismatched, P will not be in the correct position

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 ○ Mismatches will float away since without base pairing there will be no H bonding
and no sugar-P backbone formed (because rxn is slow?*)
● DNA polymerase Active Site
○ Incorrect base pairs will not fit nicely into the active site
○ Palm will recognize any sequence of DNA *
○ Palm and minor groove is W&C bp specific*
● DNA Polymerase Players
○ 2 Aspartate in active site (-)
○ 2 Mg 2+ cofactors
○ Cofactor shields neg charges on phosphate and primer so that O
can attack P
○ Phosphatase cleavage has a high -ΔG IRREVERSIBLE
○ Adding dNTPs/nucleotides/monomers in the 5 → 3 direction of
primer
○ RNA POLYMERASE DOES NOT REQUIRE PRIMER
○ Primers target gene (amplicon)

● DNA Polymerase Mechanism

○ Hydrogen bond

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 ■ Nucleotide pair bound by Hydrogen bonds (perfectly placed)
○ Close active site
■ O helix (part in fingers) closes around a bound nucleotide (dNTP) to
prevent water from hydrolysis of dNTP (big bomb, lots of energy released)
■ O helix has positive charges (LYS, ARG, TYR)
■ dNTP has neg charges from oxygens around Ps
■ Two Mg 2+ cofactors are also present
○ TYR forms pi-pi bonds with base
○ LYS-ARG binds triphosphate
○ Repulsion opens active site
■ Release of pyrophosphate releases O helix (+) away from Mg (+)
○ Push down 1 bp
■ 3’ OH of the new nucleotide is elevated out of the
high affinity site
■ Does not require any energy since palm has non-
specific interactions?**
● DNA Replication is rapid, complete, accurate
○ E coli - 1 mistake in every1010 bp (in every 1000
generations)
■ Proofreading
■ DNA repair mechanisms
○ Humans - every 3x109 (in every 3 cell divisions) most of our defects are non-
coding, neutral mutations :)
● Tautomerism Causes Mispairings
○ Because C can turn into imino (prefers amino)
○ Because G can turn into enol (prefers keto)
■ ⅕of the time G will be enol form and pair with T (totally
legit bp Genol-T)
■ But then Genol - T will turn into Gketo - T which is
wrong! (Gketo - C correct)
■ DNA polymerase will stop synthesis because frayed
apart
■ Exonuclease enzyme site steps in to fix immediately

● Enzymes that cut DNA

○ Endonuclease - cut WITHIN the polymer (staggered cut =
sticky ends)

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 ○ Exonuclease - remove nucleotides from the ENDS ( 3 → 5 )
■ Synthesis is irreversible after PPi is cleaved
■ Therefore this is not the reverse of synthesis
■ It is a separate enzyme from DNA polymerase but it is part of the same
PEPTIDE (exonuclease is a specific site on the palm - base of palm)

● Exonuclease Mechanism
○ Mismatch → frays ends → stalls DNA polymerase → moves to active
site
○ Wrong nucleotide cut off plus extra
○ Single strand DNA will reanneal correctly to template
○ Shift until 3’OH is in the correct spot for continuing synthesis (out of base, toward
fingers)
● There are 5 DNA Polymerase Enzymes
○ DNA Polymerase I

■ Exonuclease off the ends 3 → 5 like normal but also 5 → 3

off the start
■ Low processivity
■ Repairs okazaki fragments! (5 → 3)

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 ○ DNA Polymerase II
■ Proofreading exonuclease (DNA repair)
■ Low processivity
○ DNA Polymerase III Core - main enzyme of replication
but low processivity
■ DNA Polymerase III Holoenzyme - very high processivity
■ Both have exonuclease activity (mismatch repair?*)
○ DNA Polymerase IV & V
■ No exonuclease
■ Very low processivity
■ lesion/bypass repair*?

DNA Polymerase III HOLOENZYME

Made up of many enzymes:

● 3 β sliding clamp - increases processivity (coach)

● 3 DNA Polymerase III core
○ main replicating enzyme in E Coli
○ Made of ⍺ ε 𝛂 (3 subunits)
● 3 Tau
○ holds DNA Poly III core to the clamp loader
○ Connected to helicase?*
● Clamp loader
○ holds Tau
○ Made of 4 different subunits, machinery
● Primase - enzyme made of RNA, which can add nucleotides together to make primer
● Helicase - unzips DNA

Replisome

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The 20 enzymes/proteins required for E Coli DNA replication

includes :

● Helicases - unzip
● Topoisomerases - relieve stress
● DNA binding proteins - keep strands separate
● Primases - make primers of RNA
● DNA ligases - seal nicks on lagging strand (okazaki fragments)

Helicase E Coli

Six subunits, each subunit has a hairpin that functions like paddle along
body of DNA PULLS THROUGH HOLE:

ATP binding, hydrolysis and release of ADP + Pi

● ATP binds → paddle reaches up to grab a base

● ATP → ADP + Pi paddle shifts down with base
● ADP + Pi is released and paddle lets go of DNA
● https://www.youtube.com/watch?v=bePPQpoVUpM HELICASE ANIMATION

Transcription DNA → mRNA https://www.youtube.com/watch?v=dKubyIRiN84

Translation RNA → string of amino acids

● RNA travels to outer part of cell

● Components of a molecular machine come together around RNA to form a ribosome

https://www.youtube.com/watch?v=kmrUzDYAmEI

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DNA SUMMARY
)

Replication (in both directions)

● DNA Polymerase III holoenzyme

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 ● Exonuclease is for proofreading AND removes primer
● DNA has an origin specifically for replication, a
location on DNA rich with A-T bp
○ Humans have most # of origin sites, faster
DNA replication!!!!
● ➲⤽Helicase is first one to origin (separates H bonds)
● Single strand binding proteins coat strands to keep
from reannealing
● DNA Polymerase III needs a 3’OH end to hook on to
to add a nucleotide
● Primase makes an RNA primer that provides this 3’ tail
for DNA POLY III to extend
● Topoisomerases keep DNA tension free so helicase
can keep going
● Beta clamps keep DNA POLY III from floating off when
it falls off to get to new site on lagging strand
● DNA Polymerase I fills in gaps after primer is removed
(BY ____)*
● DNA ligase seals nicks between the primase
replacements and the nucleotides set by DNA POLY III
● https://www.youtube.com/watch?v=TNKWgcFPHqw
DNA REPLICATION
●

Transcription - RNA can escape the nucleus

Translation - mRNA attaches to tRNA which carries amino acids

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Proteins Required to Initiate Replication
● Dna A - recognize origin, bind to R and I site to begin unzipping & replication
● Dna B - HELICASE that unwinds DNA, loaded at origin
● Dna C - escorts Dna B (bound so that Dna B does not unzip prematurely)
● Primase/Dna G - adds first primer at origin
● SSB (single stranded binding protein)
● DNA gyrase (topoisomerase II) - unwind + supercoils downstream
● Dam methylase - helps to discriminate new strand from parent strand

Bacteria Origin Sites - Initiating Replication

● Dna A is part of a AAA + ATPase family

○ Turns off (stops causing supercoil) when - ADP (hydrolyzed)
○ Activated when - ATP
○ Binds to R site whether ATP bound or ADP bound?***
○ ***can bind to I sites when ATP or ADP bound, less picky site
● DUE region : A-T rich, easy to unwind DNA here
● 5 R sites form the binding site for initiator protein Dna A
○ When Dna A is bound to these sites, causes helix to twist, supercoil
○ DUE region is denatured to release tension caused by Dna A activity
○ Load helicase Dna B with Dna C Hallmark of initiation
○ Dna C has an affinity for Dna A (loaded)
■ Member of AAA+ ATPase family
■ When ATP bound, binds to Dna B
■ When ADP bound, releases Dna B (free to unzip)
○ PRIMING INACTIVATES C (hydrolyzes its ATP)
○ Primase makes primer in opposite direction of helicase and frees helicase
■ If primer’s 3’ end points toward the fork, it is leading strand
○ Helicase is bound to Tau/ DNA Polymerase II Holoenzyme binds onto Dna B
at Tau initiation is complete
○ Hda protein will make Dna A hydrolyze its ATP and disassemble at origin
(leaves?*)

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 1. DnaA binds to R and I sites
2. Dna A causes supercoiling
3. DUE region unwinds to relieve supercoiling
4. Dna C is attracted by Dna A, brings along Dna B
5. Dna A and Dna C interact, which opens Dna B and loads it to single strand

6. Two Dna B face opposite

directions (toward forks)

7. Primase makes primer in opp dir

to Dna B
8. Priming makes Dna C hydrolyze and release Dna B

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9. Dna B free to start unzipping, but
a. everything must attach to Dna B first via Tau
b. Dna A must be hydrolyzed by Hda protein
c. THESE TWO THINGS COMPLETE INITIATION
10. Ready to replicate! (attaches to helicase and keeps going until primer?)

11. Sliding clamp loader attached to three DNA pol III

recognizes the PTJ
a. binds to primer and puts on a sliding clamp (circle)
b. Now clamp around PTJ can be recognized by DNA pol III core.

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12. Elongation begins
a. ONE DNA Poly III adds dNTPs to 3’ OH end in 5 → 3
direction
b. Both of these strands are leading!
c. The other two DNA Polys are waiting for primers on lagging strands

13. Primase (DnaG) attracted/associates with DnaB helicase

a. Makes primer in direction opposite to DnaB move

14. When primase released, sliding clamp binds

DNA Poly III to PTJ
a. Lagging strand synthesis elongates
primer at 3’ end moving away from
replication fork
b. RATE LIMITING STEP

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 15. Once Helicase has cleared 1 kb of DNA,
primase will make another primer and engage third DNA Poly III
a. The second will be engaged again

Cooperativity among Enzymes

1. Tau stimulates DNA Helicase activity

2. Primase stimulated by Helicase
3. Sliding 𝛂 clamps stimulate processivity of DNA Poly III until done

𝛂 clamp

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 ● ATP - clamp loader opens B clamp
● Attaches to DNA and primer at 5 end
● Primer on DNA interacts with active site on clamp loader
that causes ATP hydrolysis
● ADP - clamp loader falls off, leaving behind B clamp

Replication Fork - TROMBONE MODEL (Bacteria)

Okazaki Fragment Repair

● Want to remove primer and replace

with DNA
● Leaves gap between 3’ OH
● RNase H degrades the primers, all but the last nucleotide (cannot remove the one
attached to the last dNMP because it lacks the 2’ OH required by the enzyme)
● Need exonuclease to remove the last rNMP and some dNMP
● Left with the perfect PTJ Primer Template Junction
● DNA Polymerase I (in bacteria) fills in gap in DNA (has normal 5 → 3
AND also 3 → 5)
● Ligase enzyme seals by linking 5’ Phosphate to 3’ OH (can only seal adjacent nucleotides)
○ Needs AMP (from ATP or NAD+) to activate ligase (phosphorylate)

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○ Phosphate will activate the phosphate in the nick
○ OH in nick will attack activated phosphate, releasing the AMP and sealing nick

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DNA Repair (POST synthesis)

Mismatch Repair & Methylation

● Mis-incorporation of bases by DNA Pol

generates mismatches ~1 out of 10 5 bp.
Proofreading by the 3’ → 5’ exonuclease
of DNA Pol reduces the error rate to ~1
in 10 7 bp.
● Mismatch repair further reduces the error rate to 1 in 10 10bp.
● E. coli uses HEMI methylation to distinguish parental strand from the nascent strand

Methylated -Directed Mismatch Repair (Early Steps)

● MutS scans DNA for mismatch

● MutSand MutLrecruit MutH (exonuclease)
● Both strands of DNA are threaded through this protein complex, until MutH
locates a methylated A in the sequence 5’-GATC-3’.
● MutH, acting as an endonuclease, cleaves the non-methylated strand on the 5’
side of the G.

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Mut S + ADP scans
double strand
following replication
randomly

When Mut S finds a mismatch, it ADP is changed for ATP and grabs DNA tighter marking site

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It recognizes mismatches through the hole in Mut S (only bent DNA fits through this channel.
Mismatches or indels will bend DNA, W-C base pairs will be more rigid)

Mut S and Mut L recruit Mut H an exonuclease

The whole 3 member complex will thread the DNA until it finds closest hemimethylated site,
recognizes GATC

Mut H will cut the

unmethylated strand (at this
point
the nascent strand) on the 5’
side of the GATC

When mismatch is UPSTREAM from cut (on the 5’ side of cleavage

site), unmethylated strand degraded 3’ up toward 5’ from cleavage
site to mismatch. Enzymes used include :

● helicase II
● SSB
● exonuclease I or exonuclease X (degrades ssDNA 3’ → 5’)
● exonuclease VII (degrades ss in either direction).

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When the mismatch is DOWNSTREAM from the cut (on the 3’ side of the cut),
unmethylated nascent strand is degraded from 5 → 3. Enzymes
used :

● helicase II
● SSB
● RecJ nuclease (derades single strand DNA 5 → 3)
● exonuclease VII (degrades ss in either direction).

ECOLI MISMATCH REPAIR EXONUCLEASE ENZYMES

Will keep going until finds another bend, then look for methylated site nearby

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32
Nick - Directed Nascent Strand Detection (more DNA to repair vs CH3
versn)

1. Recognize mismatch (Mut S homolog)

a. Recruits a Mut L homolog (with endonuclease activity)
i. ATP bound and inactive
ii. Cuts only one strand of double stranded DNA, dictated by binding to
sliding clamp ring

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 1. MutL binds to MutS
a. Hydrolyzed to ADP becomes active endonuclease
b. Cuts the one strand with the nick
i. Sliding clamp has two faces -
only one face has a DNA
Polymerase III binding site
ii. DNA polymerase will be filling in
gap so it is impt to note the
direction it will move in
iii. MutL needs to bind to this same
site on sliding clamp
iv. Orientation of the sliding clamp tells MutL which DNA strand to cut!!!

The cut made by Mut L needs to be on the opposite side of the

mismatch to allow for its excision by exonuclease (Exo 1) in 3→ 5 5 → 3

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Exonuclease 1 will excise the orange region

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DNA Poly III holoenzyme in bacteria

Base - Excision Repair

1. Glycosylase enzyme removes the damaged base

only (not the nucleotide).
a. This leaves an AP site.
b. Note: Most common glycosylase is uracil
glycosylase.
2. An AP endonuclease cuts DNA 5’ to AP site.
a. This leaves a 3’-OH behind.
3. A 5’ → 3’ exonuclease removes the AP
phosphodiester backbone plus 1 -3
additional bases.
4. DNA polymerase and DNA ligase replace and
seal the gap.

RNA Transcription (HR part A)

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Initiation of RNA POL HOLOENZYME (in e coli)

RNA Repair ( P editing, Hydrolytic editing-exonuclease)

Two Types of Termination (rho dep/indep, operons)

Holiday Junction

Study topics for Exam III :

1. Keto-enol tautomerization and mis-incorporation by DNA polymerase
(ready to sketch structures).

2. Anti- and syn-conformations of nucleotides (ready to sketch structures).

3. Rarity of CpG islands in vertebrates.
● Why?

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4. Evolution of DNA
● Significance of deamination of C
5. Characteristics of DNA polymerase that enable PCR.
● TAQ polymerase (heat resistant)
● Needs primer
● 3 → 5 DNA Poly
6. DNA recognition by DNA polymerase that enables the active site to
catalyze 16 different reactions.
● MINOR GROOVE - bp look the same to DNA Poly
● RNA POLY Combinatorics
● There are only a very few transcription factors
● We must use them in unique combinations to code for a specific
7. Factors that influence the melting temperature of DNA.
8. Benefits of maintaining negatively supercoiled DNA.
● Easier to unwind
● histones/nucleosomes generate neg supercoils in order to
compact DNA

9. How are negative supercoils generated in pro- and eukaryotes.

● Prokaryotes - gyrase intentionally makes (-) supercoils
● Eukaryotes - don’t have gyrase, use nucleosomes made of
histones to make (-) supercoils

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10. Pyrophosphorylytic editing in transcription

11. Events at DNA polymerase’s active site involving the O-helix and
positioning of the incoming dNTP.

12. interpretation of gel of topoisomers.

● Supercoiled travels faster
● Shape > size > charge

13. reactions that require ATP

14. Exonuclease activity in various DNA polymerases.

★ Exo 1 3 → 5
★ RecJ 5 → 3
★ Exo 7 BOTH
15. DNA processivity
● Ranges - all low except the holoenzyme III
● B sliding clamp will keep it on DNA until finished

16. Cooperativity among proteins at the replication fork.

**** Reactions that use ATP

17. Loading helicase onto DNA in prokaryotes and eukaryotes.

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*MORE POINTS OF REGULATION*
● DNA Polymerase III Holoenzyme in REPLICATION
● ORC (DnaA initiator) loads onto dsDNA
● Dna C → Cdt-1 + Cdc6 (helicase loaders)
● Cdt-1 bound to Mcm2-7 (helicase)
● Mcm2-7 triggers ATP hydrolysis in Cdc6
● Cdc6 opens 2Mcms (2 helicases and wraps around dsDNA)
● Cdc6 and Cdt 1 released
● ORC-ATP → ORC-ADP helicase is released, the whole
process can start over again
● Helicase is inactive

1. Happens in G1 phase of cell cycle before replication

2. Loads around dis DNA
3. Helicase is ALL inactive
4. Marks all potential origins
a. every ORIGIN has a helicase not all will be used

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 ● DBFREH ( transcription)

18. DNA repair (after synthesis, proofreading is immediately during);

especially DNA mismatch repair.
● Base-excision repair - mutated base
● 2 Mismatch repair - base mismatch
○ methylation directed (prok.)
○ nick-directed (euk.)
● Nucleotide-excision repair (NER)
○ - large f**k ups in the DNA
○ (used to remove pyrimidine dimers formed by UV radiation as well
as nucleotides modified by bulky chemical adducts)
○ Four of the complementation groups, or genes, encode proteins that
play major rules in NER; they are uvrA, uvrB, uvrC and uvrD.

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 ● Recombinational DNA Repair - double strand breaks

19. Protein actions involved in homologous recombination, e.g.,

RecA and RecBCD. CHAPTER 25 DNARepHR-Part B
● Rev A

20. Comparisons of DNA polymerase III and RNA polymerase

from E. coli.
● DNA polymerase III is a hand RNA polymerase is a claw
● RNA polymerase has no helicase, no sliding clamp
● RNA polymerase requires zinc in addition to Mg 2+ or it
wont work
● RNA polymerase has a high error rate because lacks
exonuclease site
● RNA polymerase binds to promoter regions on DNA
● RNA polymerase has 5 main subunits (⍺ 1, ⍺ 2, β, β’, ω)
plus a certain 𝛂
○ Sigma subunits - help enzyme find promoter for specific gene
■ Each class of RNA polymerase holoenzymes has a
different 𝛂
○ ⍺ - bind to upstream promoter UP and acts as hinge
○ β - main catalytic subunit
○ β’ - for DNA binding
○ Sigma 1, 2, 3,4 are part of RNA Poly Core

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21. Structure of bacterial RNA polymerase.
● downstream DNAchannel - where dsDNA enters the polymerase)
● template channel - ssDNA template strand in the active site of the
enzyme
● non-template channel
○ (ssDNA non-template strand) when dsDNA is in the open
configuration
○ Delivers nucleotide monomers
● RNA exit channel
● RNA POLYMERASE HOLOENZYME = core (RNA P) + sigma factor

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 ○ Core - synthesis of RNA
○ Sigma - identifies the DNA sequence to be transcribed to RNA
by binding to a promoter region
● Sigma blocking exit
● Sigma 2 - drives a 3 and 4 unwinding of DNA at origin
○ binds to ssDNA backbone and makes based flip out (causes
unwinding!!) COMMITTED STEP when in open complex form
● 𝛂 70 - guides RNA to promoter region for housekeeping genes
● 𝛂 32 - guides RNA to region for promoting heat shock genes

22. CAUSES of abortive initiations of transcription

● 𝛂4 and 𝛂3 are blocking exit channel
● _____
● UNSTICK from promoter
● Scrunching of RNA causes escape promoter region? sigma to leave?
● Looks like promoter region becomes reannealed as result of
scrunching and that allows RNA P to be free to move along

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23. CTD of RNA Pol II, its phosphorylation and interactions

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 ● C-terminal domain
● Largest subunit of the core RNA POLY II
● has OH from R groups
● Phosphorylate serine 5 or 2
● Unphosphorylated - binds mediator (more surface for proteins bind)
○ Mediator attracts transcription factors bound to enhancer
sequences (far away) HELPS FIND PROMOTER
● Phosphorylated - release mediator, promotor release (transcription
factor TFIIH phosphorylates at Serine 5)
● P-TEFb phosphorylates Serine 2 on CTD → now start
elongation!!!
○ Causing NELF to leave
24. Energy that drives peptide bond
formation.
25. Details of peptide bond formation.
(tRNA?)

26. Recognition of sites on pre-mRNA by

snRNPs of spliceosome.
● U1 →
● U6 →
● BBP & U2 are mutually exclusive

27. Elaboration of eukaryotic machinery in terms of additional protein and

RNA components.

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 1. Instead of core sigmas 1,2,3,4, has “general transcription factors”
which bind to core promoter regions (not regulated) minimal sequence
required for transcription
2. Also has proximal promoter regions - REGULATE transcription
a. Unique transcription factors TF are the ones that regulate and
express a particular gene, these only bind at proximal promoter
regions
3. Enhancer - express enhancer RNA eRNA which helps bring in more
regulatory elements to the site, by having this long range portion
where other structures can bind to and then be brought to promoter
site when loop on itself

Initiation of Transcription in Eukaryotes?

Only 4 steps in bacteria

Uses RNA POLY II and proteins (6+) in Eukaryotes

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