Aging Genome 1.DNA damage 2.Epigenetic shifts 3.Telomere shortening Cellular level 1.Mitochondria: ROS, DNA damage, other 2.Misfolded proteins 3.Dysfunctional.

AgingGenome

1. DNA damage2. Epigenetic shifts3. Telomere shortening

Cellular level1. Mitochondria: ROS, DNA damage, other2. Misfolded proteins3. Dysfunctional stem cells

Organismal level1. Autoimmune, other defects in immune system2. Defective signaling

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 replicationDNA gyrase relieves supercoilingTopoisomerases : enzymes that untie knots in DNAType I nick backbone & unwind once as strand rotatesType II cut both strands: relieve two supercoils/rxn

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