DNA synthesis/packing

Question 1

what is the nucleosome?

Answer 1

DNA looped round a central core of 8 histone proteins

Question 2

what is a chromatosome?

Answer 2

DNA looped round a central core of 8 histone proteins plus H1 linker

Question 3

DNA stability affected by 4 factors

Answer 3

hydrogen bonds
stacking- induced dipole interactions between aromatic bases
cations- positive ions prevents repulsion of Pi backbone
hydrophobic effect- hydrophobic bases buried

Question 4

melting temp of DNA

Answer 4

70-90 degrees C

GC higher- 3 h bonds

Question 5

what is the origin of replication

Answer 5

sequence that attracts Dna A which recruits DNA helicase which unwinds the DNA (PROKARYOTES)
parental strands separated and held in single stranded conformation (Replication bubble)

Question 6

where does the energy for DNA synthesis come from?

Answer 6

3’ OH nucleophilic attack on the alpha phosphate group- releases bipyrophosphate- gives energy for DNA synthesis

Question 7

how does DNA Pol I work

Answer 7

lagging strand- discontinuous replication
DNA pol I binds the okazaki fragments together and fills in gaps between them. removes primer and adds necleotide
Beta clamp- holds it all together
Single stranded binding proteins maintain it in single stranded confirmation

Question 8

which polymerase is the most common

Answer 8

DNA pol III

also most efficient

Question 9

DNA Pol III

Answer 9

3’-5’ exonuclease
5’-3’ polymerase
requires a template
coordinates synthesis of leading and lagging strand
component of replisome
trombone model on leading strand discontinuous replication

Question 10

what does DNA ligase do

Answer 10

catalyse formation of phosphodiester bond between the nick

Question 11

how do the histones bind DNA

Answer 11

histones enriched with positive aas means that it has electrostatic interactions with negative phosphate

Question 12

packing ratio

Answer 12

packing ratio= length of B form/length of nucleus

Question 13

what is the first form of packing

Answer 13

nucleosome- dna looped around core of 8 histone protein

Question 14

how many times does the DNA wrap around the histones?

Answer 14

nearly 2 times

1.65

Question 15

what is the packing ratio normally in the cell and what is it in replication

Answer 15

1000 fold normally

10000 fold in replication

Question 16

how doest gel electrophoresis seperate DNA

Answer 16

based on molecular weight (BP) the more bp the less far it will travel
gel electrophoresis utilises the negativity of DNA. it exposes the gel to a positive current and the DNA gets pulled along. the big the peice of DNA the less far it will travel- resistance from the gel.
supercoiled travels faster than linear which is faster than coiled

Question 17

how is gel visualised

Answer 17

using ethidium bromide intercollates between the bases and fluoresces

Question 18

explain southern blotting

Answer 18

no

Question 19

what is the difference between prokaryotic and eukaryotic origin/ termination of DNA replication

Answer 19

prokaryotes: one origin one termination

eukaryotes multiple origins and multiple terminations

Question 20

how does DNA synthesis happen

Answer 20

3’ hydroxide initiates a nucleophilic attack of the new alpha phosphate group this releases bipyrophosphate- this provides energy for DNA synthesis

Question 21

explain the trombone model of DNA replication

Answer 21

DNA can only be synthesised 5’ to 3’ and lagging strand runs 3’ to 5’ therefore to allow it to be made 5’ to 3’ the trombone model is used.
loop of DNA forms so that the two DNA pol III are moving in the sme direction

Question 22

what is the replisome

Answer 22

only exists when attached attached to replication fork of DNA

Question 23

what does the clamp loader do

Answer 23

assists with loading and clamping the DNA

Question 24

subunits and functions of the DNA pol III prokaryotes

Answer 24

dimer- (two strands of DNA each strand takes a part of the dimer)
α- polymerase element
β-clamp
tau- stability of the dimeric protein- ATPase
γ- clamp loader
epsilon-3’ to 5’

Question 25

what shape is the prokaryotic DNA polymerase

Answer 25

right hand
DNA comes in across palm fingers close slightly if correct match is found->
5’-3’ synthesis happens in palm
positive cation mg2+ draws H+ from OH- initiates nucleophilic attack
fingers draw back- conformational change moves undesired strands out of the way 3’-5’ exonucleases in the base of the palm.

Question 26

what is the overall process of DNA synthesis

Answer 26

origin attracts DNA A-> DNA helicase recruited->DNA pol III-> single stranded binding proteins keep lagging strand seperate->DNA pol I turns primer into DNA-> ligase joins okazaki fragments.