DNA Topology

- B form DNA conformation:

 B-Form conformation is the most relaxed and preferred conformation of
DNA at physiological conditions.

Relaxed DNA contains 10.5 Base

Pairs Per Turn
Overwound DNA contains <10.5
Base Pairs Per Turn (+ Right)
Underwound DNA contains >10.5
Base Pairs Per Turn (- Left)

- Mathematical Supercoiling (Lk = Tw + Wr)

- Topologically Equivalent = No cuts or Re-ligation = Lk is Constant (Find Tw using
Lk and Wr)
o Linking Number (Lk): The number of helical turns in a closed dsDNA
 Count the Number of peaks in the closed circle.
 Example: Lk in 2100bp B-Form  2100/10.5 = 200
o 10.5 since each turn (peak) is 10.5 BP apart.
 Lk = # Peaks
o Twist (Tw): Number of Helical Turns
 Tw = BP/BP per Turns – Separations
 Unwind = Tw -Δ , Rewind = Tw +Δ
o Writhe (Wr): Number of Supercoiling
 A single dsDNA in a circle with no supercoiling as Wr = 0
 How to determine Wr:
- Why does DNA in front of replication fork form positive supercoils?
o Chromosome arm prevents the rotation of an arm.
o To prevent knots forming before the replication fork that would prevent helicase
from unwinding.
o If Tw goes down, Wr must increase to maintain the same Lk in the closed system.
- Topoisomerases (Removes Supercoils) (Lecture 9 Slide 7 Mechanism)
o Type I (Breaks only one strand)
 Unbroken strand is passed through break and Lk changes by -1
 Tyr OH acts as a nucleophile that attacks the sugar phosphate backbone
and forms a 5’-phosphotyrosy intermediate
 Creates opening that the strand can be passed through.
 3’ OH attacks the intermediate and reforms the phosphodiester bond.
 Camptothecin prevents regeneration and the buildup of 5’-
phosphotyrosy intermediate and the blocking of the replication
fork.
o Causes dsDNA breaks due to many ssDNA regions that are
unstable.
o Causes apoptosis due to mis replication.
o Type II (Breaks both strands)
 Changes Lk by -2 ATP required.
 Cuts both strands and reverses the loop (-1 to +1) and therefore changes
Lk by 2.
 Cuts dsDNA and brings over.
 Forms same 5’-phosphotyrosy intermediate only on both strands
 All topoisomerases relax DNA
 DNA gyrase (Type II Bacteria) induces negative supercoils
 o Underwinding in the circular genome preemptively
unwinds DNA and promotes process.
o Histones are the human equivalent.

DNA Repair
- Studying DNA Topology

- Transition and Transversion Mutations

- MMR
o MutS slides along DNA, stalls at mismatch.
 Mismatch bulge is detected by MutS (Sensor)
o MutS recruits MutL (Linker)
o Old DNA is methylated at both sides
 New DNA is only methylated on one side.
o MutH binds the hemi-methylated GATC
 MutS-MutL forms a loop
 MutH recruits an endonuclease and cleaves the unmethylated GATC
o UvrD unwinds towards the mismatch site and degrades via exonuclease
 SSB binds to the new ssDNA
 Pol III remakes the DNA  sealed by ligase.
 Dam methylated by DAM methylase
- Mutations and Cancer (Development of Cancer requires the accumulation of mutations
on multiple genes.
 o DNA Repeat expansion error

o CAG repeats can cause the insertion of extra residues and are responsible for
disease such as Huntington’s
- Repair Pathways

- Cytosine deamination

o Thymine is used because if uracil is detected, it knows that a cytosine

deamination occurred.
 o Uracil DNA glycosylase detects uracil by pushing base into pocket.
o If uracil, will make cut on the glycosidic bond
- Base Excision Repair (BER)  Does not bias a strand
o An aparine site (sugar without base) is formed.
o AP endonuclease cleaves abasic site and creates nick,
o DNA Pol, FEN1 and Ligase then remove the segment. Ligase seals the nick

- Depurination (Loss of AG)

o Anything could be inserted in the OH spot and result in decreased fidelity.

o Can also be repaired by the base excision repair pathway.
- Guanine Oxidation
o Reactive oxygen species formed by the mitochondria result in the oxidation of the
8’ guanine. Results in a G  T mutation.
o Repaired by OGGI
- 8-OxoGuanine Glycosylase/Lyase (OGGI)
o Lyase cuts the 5’ and the 1’ to remove the 8-guanine
o Fed into the BER
- Guanine alkylation by ethylmethanesulfonate (EMS)
o Alkylation of the Guanine 6’ O group.
o Results in resonation between isoforms that allow both C and T to base pair with
it.
o CpG islands, SAM, and Nitrites can result in the endogenous alkylation of the 6’
O
- AlkylGuanine DNA alkylTransferase (AGT)
o Cys will transfer the methyl group to the AGT and then yote itself.
o Will result in the reversal of the 0alkylation
- Thymine Dimers
 o When 2 thymine are next to each other under UV light, will form thymine dimers
o Linkage will result in the halting of DNA repair
- Bulky Alkylation Products
o Addition of these products will be oxidized by cytochrome P450 and form a
reactive cyclic group.
o Guanine will then bind to the reactive cyclic group and block DNA replication.
- Nucleotide Excision Repair and (Xeroderma pigmentosum)
o UvrA and UvrB travel along the DNA
 When thymine dimer is encountered, will stall at dimer.
o UvrC makes cut on both sides of the thymine dimer strand.
o UvrD (3’  5’ helicase) will come in and remove the damaged region containing
the dimer.
o DNA Pol I will come in and fill in the gap. And Ligase will connect to original
strand
- Ion Torrent Sequencing
o DNA chopped up and fed onto beads.
o Beads Placed on well
o Flooded with nucleotides
o When base paring occurs, H+ released  Changes pH of well
o Ion sensitive layer measures change in pH and converts to voltage
 Base is detected via voltage change.
o Repeated every 15 seconds with a different nucleotide.

Transcription RNA Synthesis

- RNA polymerases use DNA to make more DNA
o Bind to DNA and use it as a template.
o Requires dsDNA and are template dependent
o No primer needed  rNTP needed
o Reads template in 3’  5’ direction
 Synthesizes in 5’  3’
- Involves the interaction between the RNAP and promoters
o Regulators help regulate the transcription of RNA
- Bacteria Transcription
o RNAP contains 2 parts
 2A + 1B + 1B’  Core Enzyme
 Carries out transcription
 2A + 1B + 1B’ + 1O  Holoenzyme \
 Tells the enzyme where to carry out transcription
 Sigma recognizes promoter sequences on DNA
 Promoter gives RNAP directionality
 Beta’ Binds to DNA
  B Binds NTP and interacts with the sigma
 Alpha is used for assembly and activation of regulation proteins
o Scans DNA for promoters  Binds to Promoter  Initiates Transcription 
Elongates RNA  Terminates Transcription  Repressive to regulatory proteins

- Transcription Initiation

o Sigma Recognizes the -35 and -10 regions.  Leads RNAP in closed complex
(still dsDNA)
o RNAP opens DNA (at -10) to for the open complex
o Template strand makes RNA from strand
 Abortive transcription occurs when RNA starts transcription and then
terminates by releasing transcribed RNA early
o Scrunched Complex
 Binds to promoter with high affinity at -35 and -10 regions
Makes it harder to move away  pulls the DNA into the active sites and
scrunches it up as it moves down instead of moving forward (stuck to -35)
 Energetically unfavorable and causes abortive transcripts
o Sigma Domains (s2 + s4)

 Location of the promoter-10 and -35 is asymmetrical upstream

 Can only bind in one direction as a result.

a  a2  a2B  a2BB’ = core enzyme

- Assembly Pathway regulation
o Different sigma factors can bind to the same core
 Will direct the same RNAP to a different promotor and transcribe a
different gene.
 REGULATION OF THE SIGMA BINDING CAN DETERMINE THE
GENE TRANSCRIBED
- Promotor properties
o 2 Consensus (similar sequences across many promoters) sequences
 -35 TTGACA
 -10 TATAAT
 o Consensus sequences appear at the highest frequency between all promoters but
not 100% of the time.
o Pribnow box ideal for unwinding.
o Spacer is not important and not conserved (does not matter what it is)
 If twists increase or -10 and -35 move apart  sigma would have to twist
to bind
o If RNA goes beyond a certain length  enters exit channel  Changes B flap
 Alters interaction between Core enzyme and sigma  weakens the -35 
Allows RNAP to escape -35 hold.
- Mutations in Promoter
o Mutations in the -10 and -35 region can increase or decrease the transcription of
RNA  And by extension the expression of a gene.
o Environments can shift the RNA transcribed by shifting sigma factors.
- How does RNA open the duplex?
o T-7/A-11 fit into cavities in the sigma  results in the opening of the RNAP