dna replication wc: dna replication is semi-conservative strands melt: form templates for copy copy...
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DNA REPLICATIONWC: DNA replication is semi-conservative
strands melt: form templates
for copy
Copy is reverse &complement of template
Copy of other strand
Meselson & Stahl
proved DNA replication is semi-conservative
1) grew E. coli on 15N to tell new DNA from old
• make dense DNA
Meselson & Stahl1) grew E. coli on 15N to tell new DNA from old• make dense DNA
2) Xfer to 14N
Meselson & Stahl1) grew E. coli on 15N to tell new DNA from old• make dense DNA
2) Xfer to 14N• new DNA is light
Meselson & Stahl1) grew E.coli on 15N totell new DNA from old• make dense DNA
2) Xfer to 14N• new DNA is light
3) measure of F1 & F2 DNA by centrifuging in CsCl
Meselson & Stahlmeasure of F1 & F2 DNA by centrifuging in CsClforms gradients when spun@500,000 x g
Meselson & Stahlmeasure of F1 & F2 DNA by centrifuging in CsClforms gradients when spun@500,000 x gDNA bands where is same as CsCl
Meselson & StahlResultsF0 DNA forms HH band
control Parental
Meselson & StahlF0 DNA forms HH band F1 DNA forms one higher band Control Parental F1
Meselson & StahlF0 DNA forms HH bandF1 DNA forms one higher band: HL Control Parental F1
Meselson & StahlF0 DNA forms HH bandF1 DNA forms one higher band: HL F2 DNA forms 2 bands: #1 same as F1#2 same as 14N DNA Control Parental F1 F2
Meselson & StahlF2 DNA forms 2 bands: # 1 same as F1#2 same as 14N DNA# 1 = HL# 2 = LL : DNA replication is semiconservative
DNA replication1) Replication begins at origins of replication
DNA replication1) Replication begins at origins of replicationPolymerases are dumb!
DNA replication1) Replication begins at origins of replicationDNA polymerases are dumb!other proteins tell where to start
DNA replication1) where to begin?2) “melting” DNA
DNA replication1) where to begin?2) “melting” DNA• must melt DNA @ physiological T
DNA replicationmust melt DNA @ physiological THelicase melts DNA
DNA replicationmust melt DNA @ physiological THelicase melts DNAForms “replication bubble”
DNA replicationhelicase melts DNAForms “replication bubble”
SSB proteins separate strands until they are copied
DNA replicationhelicase melts DNAunwinding DNA increases supercoiling elsewhere
DNA replicationhelicase melts DNAunwinding DNA increases supercoiling elsewhereDNA gyrase relieves supercoiling
DNA gyrase
DNA replication1) where to begin?2) “melting”3) “priming” • DNA polymerase can only add
DNA replication “priming”
DNA polymerase can only addprimase makes short RNA primers
DNA replication “priming”
primase makes short RNA primersDNA polymerase adds to primer
DNA replication “priming”
primase makes short RNA primersDNA polymerase adds to primer later replace primers with DNA
DNA replication1) where to begin?2) “melting”3) “priming”4) DNA replication
DNA replication4) add bases bonding 5’ P to 3’ OH @ growing end
DNA replication4) add bases bonding 5’ P to 3’ OH @ growing endTemplate holds next base until make bond
DNA replicationTemplate holds next base until make bond- only correct base fits
DNA replicationTemplate holds next base until make bond- only correct base fits- energy comesfrom 2 PO4
DNA replicationenergy comes from 2 PO4
"Sliding clamp" keeps polymerase from falling off
DNA replicationenergy comes from 2 PO4
"Sliding clamp" keeps polymerase from falling offProof-reading: only correct DNA can exit
DNA replicationProof-reading: only correct DNA can exit Remove bad bases & try again
DNA replication
Only make DNA 5’ -> 3’
Leading and Lagging StrandsOnly make DNA 5’ -> 3’strands go both ways!
Leading and Lagging StrandsOnly make DNA 5’ -> 3’strands go both ways!Make leading strand continuously
Leading and Lagging StrandsMake leading strand continuouslyMake lagging strand opposite way
Leading and Lagging StrandsMake leading strand continuously Make lagging strand opposite waywait for DNA to melt, then make Okazaki fragments
Leading and Lagging StrandsMake lagging strand opposite waywait for DNA to melt, then make Okazaki fragmentseach Okazaki fragment has its own primermade discontinuously
Leading and Lagging Strandseach Okazaki fragment has its own primermade discontinuouslyDNA replication is semidiscontinuous
Leading and Lagging Strandseach Okazaki fragment has its own primermade discontinuouslyDNA replication is semidiscontinuousOkazaki fragments grow until hit one in front
Okazaki fragments grow until hit one in frontRNAse H removes primer & gap is filled
Okazaki fragments grow until hit one in frontRNAse H removes primer & gap is filledDNA ligase joins fragments
Okazaki fragments grow until hit one in front
RNAse H removes primer & gap is filled
DNA ligase joins fragments
Energy comesfrom ATP-> AMP
DNA replicationReal process is far more complicated!Proteins replicating both strands are in replisome
DNA replicationReal process is far more complicated!Proteins replicating both strands are in replisome: feed DNA through it
DNA replicationProteins replicating both strands are in replisome: feed DNA through itlagging strand loops out so make both strands in same direction
lagging strand loops out so make both strands in same directionDNA pol detaches when hits previous primer, reattaches at next primer
Bacterial DNA has one originEuk have ARS ~ every 100,000 bp
- speed DNA replication
Euk have ARS ~ every 100,000 bp - speed DNA replication
ORC (Origin Recognition Complex) binds ARS
A B1 B2 B3
ORC binds ARSlicensing factors ensure each ARS is only replicated once/Sfall off when ARS is replicated, don't reattach until G1
Telomeres• sequences at chromosome ends • humans have 250-7,000 repeats of CCCTAA• special proteins bind them
Telomeres• sequences at chromosome ends • humans have 250-7,000 repeats of CCCTAA• special proteins bind them• can't replicate 5' end of lagging strand since remove primer
Telomeres• can't replicate 5' end of lagging strand since remove primer• telomeres lose ~ 200 bp each S
Telomeres• can't replicate 5' end of lagging strand since remove primer• telomeres lose ~ 200 bp each S• telomerase replaces missing bases
Telomerase• telomeres lose ~ 200 bp each S• telomerase replaces missing basesreverse transcriptase with attached RNA template
Telomerase• telomeres lose ~ 200 bp each S• telomerase replaces missing basesreverse transcriptase with attached RNA template1) RNA bonds leading strand
Telomerasereverse transcriptase with attached RNA template1) RNA bonds leading strand2) Forms template to extend leading strand
Telomerasereverse transcriptase with attached RNA template1) RNA bonds leading strand2) Forms template to extend leading strand3) Translocates6 bases & repeats
Telomerasereverse transcriptase with attached RNA template1) RNA bonds leading strand2) Forms template to extend leading strand3) Translocates6 bases & repeats4) Extend lagging strandwith primer & DNA pol
Telomeres
Aging theory:” mature” cells lose telomerase
Telomeres
Aging theory:” mature” cells lose telomerase
lose DNA each cell cycle
Telomeres
Aging theory:” mature” cells lose telomerase
lose DNA each cell cycle
Die when lose too much
Telomeres
Aging theory:” mature” cells lose telomerase
lose DNA each cell cycle
Telomeres
Aging theory:” mature” cells lose telomerase
lose DNA each cell cycle
Die when lose too much
Cancer cells reactivate telomerase
Telomeres
Aging theory:” mature” cells lose telomerase
lose DNA each cell cycle
Die when lose too much
Cancer cells reactivate telomerase
Can use serum telomerase to diagnose cancer
Telomeres
Aging theory:” mature” cells lose telomerase
lose DNA each cell cycle
Die when lose too much
Cancer cells reactivate telomerase
Can use serum telomerase to diagnose cancer
Can kill cultured cancer cells by inhibiting telomerase
Telomeres
Aging theory:” mature” cells lose telomerase
lose DNA each cell cycle
Die when lose too much
Cancer cells reactivate telomerase
Can use serum telomerase to diagnose cancer
Can kill cultured cancer cells by inhibiting telomerase
Telomerase is symptom cf cause of cancer
DNA repair
DNA is constantly damaged
DNA repair
DNA is constantly damaged
UV makes pyrimidine dimers
DNA repair
DNA is constantly damaged
UV makes pyrimidine dimers• Photolyase uses light energyto cleave dimers
DNA repair
DNA is constantly damaged
UV makes pyrimidine dimers• Photolyase uses light energyto cleave dimers• Also fixed by Nucleotide Excision Repair
DNA repairUV makes pyrimidine dimers• Photolyase uses light energy to cleave dimers• Are also fixed by Nucleotide Excision Repair• NER also fixes chemical damage
Nucleotide excision repair1) XPC finds damage2) Cut bad strand on each
side of lesion, then remove it
Nucleotide excision repair1) XPC finds damage2) Cut bad strand on each
side of lesion, then remove it
3) DNA polymerase fills gap
Nucleotide excision repair1) XPC finds damage2) Cut bad strand on each
side of lesion, then remove it
3) DNA polymerase fills gap
4) Ligase seals it
DNA repairbase excision repair fixes altered and missing bases
DNA repairbase excision repair fixes altered and missing bases1) Enzymes find & remove
bad bases
DNA repairbase excision repair fixes altered and missing bases1) Enzymes find & remove
bad bases 2) DNA pol replaces missing base
DNA repairbase excision repair fixes altered and missing bases1) Enzymes find & remove
bad bases 2) DNA pol replaces missing base3) Ligase seals gap
DNA repair
mismatch repair : fix new DNA to match old DNA
DNA repairmismatch repair : fix new DNA to match old DNA
• DNA is modified t/o cell cycle
mismatch repair1) enzymes find mismatched bases2) replace base on new strand using old strand
DNA repair
Repairing 2 -strand breaks
1) MRN finds broken DNA
DNA repair
Repairing 2-strand breaks
1) MRN finds broken DNA
2) Activates enzymes that ligate broken ends
DNA RepairClinical significance
Important for preventing cancersMany genetic disorders are due to bad DNA repair• bad BRCA1 or BRCA2 predispose to cancer
DNA RepairMany human genetic disorders are due to DNA repair• bad BRCA1 or BRCA2 predispose to cancer• NER defects cause Xeroderma Pigmentosum• 11 genes have "same"phenotype
DNA RepairMany human genetic disorders are due to DNA repair• bad BRCA1 or BRCA2 predispose to cancer• NER defects cause Xeroderma Pigmentosum• 11 genes have "same"phenotype
• bad mismatch repaircauses hereditary nonpolyposiscolon cancer (HNPCC)
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