chapter 11: DNA replication Flashcards
conservative
two newly synthesized DNA strands are joined together
original strand remains together
semi conservative
each replicated DNA molecule consists of one old and one new strand
dispersive
parental strands are dispersed into two new double helices
Messelson-Stahl experiment
N15 has higher density than N14
- in this experiment:
ecoli is grown in a N15 heavy medium (high density on centrifugal force)
then it is added to an N14 medium
the cells replicate once in N14 ( N15/N14 ; band is in the middle)
then replicated a second time in N14 ( two bands ; N14/N14 (on the low density side) and N15/N14 (in the middle) )
replicated for a third time (more N14/N14 and less N15/N14)
conservative –> semi conservative –> mix of conservative and semi conservative
origin of DNA replication (Ori)
where DNA replication begins
replication fork
At ori the helix is gonna unwound to create replication forks
there are 2 replication forks because replication is bidirectional
Replicon
length of DNA replicated
e coli replicon consists of entire genomes (4.6 million base pairs)
Direction of DNA synthesis is 5’ to 3’
3’ end attaches to 5’ end
biochemical properties of DNA polymerases 1
initiation of chain synthesis: (negative; it can not do it on its own because a primer is required)
5’-3’ polymerization: (positive)
3’-5’ exonuclease activity: (positive)
5’-3’ exonuclease activity: (positive)
molecules of polyemrase/cell: 400
biochemical properties of DNA polymerases 2
initiation of chain synthesis: (negative; it can not do it on its own because a primer is required)
5’-3’ polymerization: (positive)
3’-5’ exonuclease activity: (positive)
5’-3’ exonuclease activity: (negative)
molecules of polyemrase/cell: ?
biochemical properties of DNA polymerases 3
initiation of chain synthesis: (negative; it can not do it on its own because a primer is required)
5’-3’ polymerization: (positive)
3’-5’ exonuclease activity: (positive)
5’-3’ exonuclease activity: (negative)
molecules of polyemrase/cell: 15
exonuclease activity
DNA proofreading
DNA polymerase 3
an enzyme involved in DNA replication
the components of the DNA polymerase III holoenzyme
holoenzyme: active form of DNA pol 3, it contains poly 3 core enzyme complex made up of 3 subunits
- α - 5’-3’ polymerization
- ɛ - 3’-5’ exonuclease
- θ - core assembly
core enzymes: elongate polynucleotide chain and proofreads
t (tau): dimerizes core complex
sliding DNA clamp loader ( Y): loads enzyme on the template (serves as a clamp loader)
sliding DNA clamp: binds the core enzymes and double-stranded DNA, keeps the core enzymes bound to DNA for a longer time allowing it to synthesize long stretches of DNA before it detaches from DNA
OriC DNA composition
has three AT rich 13bp repeasts and five bp repeasts
DnaA
DNA replication initiation in E coli requires dnaA
it is an initiator protein
it binds to the 9bp repeat of the oriC DNA
Helicase
hexamer of dnaB subunits
subsequently recruits DNA pol 3 holoenzyme to bind replication fork and initiate repliaction
requires energy supplied by hydrolysis of ATP ti denature hydrogen bonds between base pairs that stabilize the double helix
assembles around exposed ssDNA
DNA gyrase
cuts DNA to relax positive supercoils; or introduce negative supercoils
driven by energy released during ATP hydrolysis
Single stranded DNA binding proteins (ssbp)
stablize the open conformation of helix
bind specifically to single strands of DNA
Primase
RNA polymerase
recruited to replication fork by helicase
synthesize RNA primer
provide 3’-OH required by DNA pol 3 for elongation
DNA polymerase I
removes primer and replaces it with DNA
DNA ligase
catalyzes formation of phosphodiester bonds
seals nicks and joins DNA fragments
leading strand
continuous DNA synthesis
lagging strand
discontinuous DNA synthesis
steps during DNA replication
- strand separation at OriC (role of DnaA)
- involvement of heicase (main role to separate DNA strands ahead of the replication fork)
- binding to SSBP (help keep the strands separated at the fork)
- relaxation of positive supercoils (DNA gyrase)
- RNA primer synthesis (primase: RNA polymerase)
- DNA synthesis (by DNA pol 3)
- removal of RNA primer and filling in the gaps with DNA (DNA pol 1, RNAse H auxiliary role in removing RNA)
concurrent DNA synthesis
achieved on both strands at single replication fork
lagging strand is looped and spooled out
inverts physical but not biochemical direction (still 5’-3’)
DNA clamp binds to the core enzyme and prevents core enzyme dissociation from template
find picture on presentation and explain what is happening
temp sensitive mutations
produce function protein at 30 degrees celsius but a non-functional protein at 42 degrees celsius
provides a way to study the effect of lethal mutations affecting DNA replication
mutant cells grow at permissive temp and mutant phenotype expressed at restrictive temp to identify the defective function
telomere replication
Telomerase: Ribonucleoprotein having reverse transcriptase activity plus RNA
- add repeats of six nucleotide sequence to 3’ end of DNA: GGGTTG
telomeres
inert chromosomal ends that protect intact eukaryotic chromosomes from improper fusion or degradation
long stretches of short repeating sequences preserve the integrity/stability of chromosomes
Shelterin
protein complex that protects the telomere
extends 3’ loop back on itself
T loop stabilized by protein complexes
loss of telomere effect
loss of telmoeres –> loss of chromosome end protection –> chromosome instability –> aging, cancer, cell death, disease
