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)