8_12_16 DNA with Gélinas

Question 1

DNA composition

Answer 1

base: purine (A and G) pyrimidine (C, U and T)
bound to structural components
1 5C sugar either ribose (RNA) or deoxyribose (DNA)
and a phosphate group

terms:
nucleoside = base linked to 5C sugar (N-glycosidic bond)
nucleotide= nucleoside + phospate mono, di or tri

3’ to 5’ phosphodiester bond makes 5’ end partially negative relative to 3’ (cut by nucleases)

Question 2

Structure

Answer 2

Commonly a dsDNA double helix, two strands go in opposite directions, right handed, internal H bonds 3 for CG, two for AT

10bp / turn
full turn every 34Å

major and minor grooves coil around helix

Question 3

Coiling

Answer 3

6 feet of DNA in one human cell
supercoil - double helix coils upon itself

neg super coiling has fewer bp/turn, facilitates strand separation, energetically favored, energy for sep in the coils themselves, promoted by histone binding

Topoisomerases allow change in coiling, make swivel points- nuclease/ligase (Topo I just single strand, II does 2)
subset of TopoII is bacterial DNA gyrase, remove supercoils for replication, needs ATP, drug target

Question 4

Even more structure

Answer 4

in prokaryotes, DNA binds to non-histoned proteins, condense in non membrane-bound cell region (nucleoid)

eukaryotes- both histone and non-histone proteins, nucleus, DNA + histone = chromatin

Question 5

Histones

Answer 5

small basic proteins rich in Arg and Lys, arrange into nucleosomes with DNA

Nucleosome- histone octamer with 140bp with a spacer of 1 histone containing 20-80bp

nucleosome futher winds into a solenoid, which loops itself again and aggregates around a protein scaffold which is what you picture when you think of a chromosome

Question 6

Prokaryotic Repilcation

Answer 6

DNAa and DNAb (helicase) initiate and split circular DNA with a topoisomerase in the lead to prep the strands for unwinding, SSB proteins bind to exposed strands to prevent reformation of double helix

Primase lays RNA primers in 5 to 3 with a free 3’ OH
few on leading strand, but always generated at replication fork for lagging strand

Replication with DNAPolIII is mostly 5 to 3 continuous on leading strand and choppy with okazaki frag on lagging strand, always 5’ to 3’

DNAPolIII also has 3’ to 5’ exonuclease activity for MMR

gaps on lagging strand filled by PolI which also removes okazaki frag 5 to 3

ligase with ATP joins

Question 7

Eukaryotic Replication

Answer 7

many replication forks

Polymerases
alpha- primer, initiates on both leading and lagging strands
delta- replication on lagging strand, assoc with PCNA, 3 to 5 exonuclease activity, displaces 5’ end on okazaki frag for degradation, MMR and NER
epsilon- rep on leading strand, assoc with PCNA, 3 to 5 exonuclease act. MMR and NER

nucleosome displaced by rep fork, histones remain loosely assoc with 1 parental strand, new histones made with rep. nucleosomes reform behind rep fork

Question 8

telomeres

Answer 8

ends of linear chromosomes, G rich non coding DNA
forms t-loops with overhang on 3’ end binding back to same strand

protect ends from degradation, recomb and end to end fusion

prevent loss of genetic material with removal of DNA associated with RNA primers

added by telomerases with RNA template for telomeric repeats with rev transcriptase act. (makes DNA from RNA template)