Lecture 2 Flashcards
What is involved in DNA metabolism ?
Replication
Repair + recombination
What does it mean for dna to be semi conservative
One strand is parent
One strand is daughter strand
What did cairn prove
DNA strands are replicated simultaneously bidirectionally from origin
-not only is each strand replicated at once
-each direction of each strand replicated at once
2 forks per replication
Ozaki fragment sizes
Bacteria = 1000-2000bps
Eukaryotes = 150-250 bps
Nuclease Enzyme Definition
Degrade dna
NOT RNA
2 classes
-exonucleases = end
-endonucleases = specific middle sites = fragments
DNA Polymerases
DNA synthesizers
Strand that is growing is 5’->3’
What about the dna polymerase mechanism requires 5-3 directionality ?
The nucleotide is the 3’ hydroxyl at the end of the growing strand
-If the 3’ is always at the end of the strand post addition and
-the 5’ carbon of the incoming dNTP is connected to the phosphorus group that is receiving the attacking nucleophile
5-3 directionality required
DNA polymerase basic reaction
(dNMP)n + dNTP —> (dNMP)n+1 + PPi
PPi is inorganic pyrophosphate
Hydrolyzed by pyrophosphatase —> true source of energy favorability
Can DNA polymerases spontaneously add to dna?
NO
Require primer
Primers = fee hydroxyl group at primer terminus
Processivity definition
Number of nucleotides added to strand before polymerase dissociates
E Coli mistake rate
1/ 10^9 - 10^10
What do correct base pairs depend on?
-hydrogen bonds between bases
-common geometry for complements (will not fit if wrong pair in polymerase active site)
Topoisomerases ability to work on certain dna
Topoisomerase I : relaxes negatively supercoiled dna (+1)
Topoisomerase ii: relaxes both pos and neg supercoiled dna (-2)
Type of structural change induced by nucleosome
?
No Lk change
2 structural components for dna molecule to maintain negative supercoiling
-covalently closed
-circular dna ( or bound on either end )
Cruciform dna
Form at palindromic sequences
Can supercoiled dna be found closer to top or bottom of agarose gel
Bottom
More compact so travels faster even if same number of base pairs
What happens when negatively super coiled dna is treated with Topoisomerase 1 for a medium amount of time?
Long time?
Think about agarose gel
Positively supercoils DNA until relaxed
Only adds +1 Lk each time so needs to relax the dna step wise
Each step = new Lk = new topoisomer
Long time = 100% DNA in relaxed form
Cannot positively supercoiled relaxed dna
E coli have 4 Topoisomerases
I and iii
Relax by removing negative super coils
ii = dna gyrase - needs atp
Eukaryotes type I and ii topoisomerases
Type I : I and III
Type II: Alpha and beta
Cannot underwind dna but can relax towards zero both pls and neg supercoils
Catenanes
DNA circles topologically linked
Chromosomes tangled = relaxed via Topoisomerase IV
Plectonemic coiling
Twisted thread
Main axis but branches coming off
40% of original length
Right handed turns
Solenoidal supercoiling
Garden hose
Works on underwound dna
Tight left handed turns
Better compaction than plectonemic
Twist around histone
describe the consistency of the chromosome structure
Changes throughout cell cycle
Uncondensed throughout interphase
More condensed during prophase
Less condensed after telophase
Phases of cell cycle
G1 (gap)
S (synthesis)
G2 (cohere)
Mitosis
Mitosis phases
Pro
Meta
Ana
Telo
Chromatin
Strands of protein to dna
1:1
Nucleosome
Basic structural unit for packaged dna
DNA wrapped around histone cores
Linking strands
Beads on a string
Why is compaction vital for cells?
Cell diameter : 5-10 mm
DNA length: 10^5 mm long
Requires 10,000 fold compaction
How many base pairs bound tightly to histone core? How many in linking strands?
146
34
Histone core dna remains safe during digestion treatments
Nucleosome made up of
Octamer of histones
Diameter : 10-11 nm
Nucleosome attaches to what kind of dna?
Relaxed dna
Introduces negative supercoiling
But pos supercoiling induced on unlinked dna
Overall Lk is the same!!
Topoisomerases can then relax pos unlinked dna so overall is negative
Epigenetics
Information passed down that is not encoded in DNA
Euchromatin
Chromatin region being actively transcribed
Chromatin immunoprecipitation
Antibodies bind to nucleosome
With or without dna attached
Nucleosome core compaction
7 fold
Need 10,000 fold
Requires use of Topoisomerase II to compact without tangling
If topo II inhibited = antibiotics = kills rapidly dividing cells
Heterochromatin
Highly condensed dna that is transcriptionally inactive
Active and inactive compartments are separated
= TAD + binding site for base of loop
How can E. coli have a 20 minute cell cycle?
New round of replication begins before previous is completed
