Lec 15 - Genes & Chromosomes

Question 1

Genome structure

introns, exons (human genome makeup); centromere, telomere

Answer 1

• exons (expressed) vs introns (transcribed, not translated)
  -bacterial genes uninterrupted; eukaryotic genes split by many introns
• human genome: 1/3 introns + exons, 2/3 simple or complex repeats
• centromere: DNA sequence attachment point for proteins that link chromosome to mitotic spindle during cell division
• telomere: sequences at ends of eukaryotic chromosomes that help stabilize chromosome

Question 2

linking number (Lk)

Lk definition, Lk vs Lk0

Answer 2

linking number (Lk)
- # of times 2nd strand pierces “surface”
- can’t link/unlink without breaking at least one; Lk stable/fixed if DNA intact (no break)
- R handed = + (neg Lk not found in DNA)
- if break in either strand, Lk undefined; DNA adopts Lk + 1 every 10.5 bp

• in relaxed molecule (no supercoiling), Lk designated Lk0:
  Lk0 = #bp/bp per turn (equal to Lk if unstrained)
• DNA underwound if Lk less than Lk0

Question 3

linking number (Lk), twist (Tw), writhe (Wr)

connection, signs (confusing)

Answer 3

• coiling leads to twist (Tw); supercoiling leads to writhe (Wr)
• relaxed DNA has writhe 0, (when Wr=0, Lk rep by Lk0)
  Lk = Tw + Wr
• For Tw R-handed is + // L-handed is -
• For Wr R-handed is - // L-handed is +

Question 4

superhelical density (σ)

Answer 4

• expresses change in Lk in quantity independent of DNA length
  σ = ΔLk / Lk0
• ratio of # turns removed to Lk0
• ΔLk = Lk (have) - Lk0 (want)
• for most cellular DNAs, σ = -0.05 to -0.07; our genomic DNA is underwound and in a negative supercoiled state

“negative superhelical density is underwinding negative supercoiling”

Question 5

most cellular DNA is underwound

impact? why?

Answer 5

• observed as underwound/negatively supercoiled when isolated from cells (can see via gel electrophoresis; highly supercoiled moves further)
• “primed” for strand separation; allows access to DNA strands
• negative supercoiling fundamental for DNA packaging in chromatin

Question 6

transcription requires melting DNA (strand separation)

impact on Tw and Wr

Answer 6

• slight negative supercoil: DNA 5-7% underwound to favor strand separation
• favors melting DNA; neg supercoil realeased (no change to Lk): Tw decreases, Wr increases (less -)

“permanent negative supercoil helping transcription start”

Question 7

Topoisomerases

general function?

Answer 7

• enzymes that change DNA topology by cleaving and re-ligating DNA
• 2 types

changes Lk (bond breaking)

Question 8

type I topoisomerases

general mech? affect on Lk? nuchleophile? nucleophile for resealing?

Answer 8

• transiently nick DNA (break 1 strand)
• move unbroken strand through nick
• Lk up or down by 1 (at a time)

mechanism:
- topoisomerase creates nick via nuc attack via tyrosine hydroxyl, leaving protein cov linked to DNA (ensuring nick is reversible)
- topoisomerase guaranteed to remain at site because physically bound to DNA
- reversible rxn with nuc attack via **3’ - OH **reseals the backbone

Question 9

type II topoisomerases

general mechanism?

more complicated

Answer 9

• breaks both strands
• change linking number +/- 2

mechanism (general)
- both strands of 1 double helix broken and 2nd double helix passed through break & resealed
- unlike type I, this requires energy (they hydrolyze ATP)

removes supercoiling –> affects Wr

Question 10

Intercalating dyes can alter twist/writhe

Answer 10

• fluorescent + aromatic intercalating agent, ethidium bromide (EtBr), slips b/w bases (it is flat)
• alters structure: less twist (stretches & decreases)

Question 11

interchangeable shapes of supercoiling

2 types? info abt bacterial DNA?

Answer 11

plectonemic (bacterial)
- wrapping around self; right-handed plectonemic = negative supercoil
- supercoiling from gyrase (type II isomerase)
- “circular but still supercoiled… coils on itself”
- anchoring at base constrains topology of each loop separately (no consistent boundaries)
- 10,000 bp looped domains

solenoidal (eukaryotic)
- wrapping around something else; left-handed solenoidal = negative supercoil
- (interchangeable states)

***DNA is in negative supercoil which can appear as (1) a R-handed plectonemic helix or (2) a L-handed solenoidal wrapping

Question 12

chromatin

purpose? histones (+tails)? nucleosomes?

Answer 12

• eukaryotic DNA packaged into chromatin
• histones = proteins tightly associated with chromatin & function to package and order DNA
  
  • histone tails are important for gene regulation
• nucleosomes = fundamental structural unit of chromatin composed of core histone proteins bound to DNA