S Phase: DNA Synthesis. Every cell has Specific DNA Nucleus contains DNA Nucleus contains DNA –Each cell in one body has identical DNA DNA DNA –Set number.

S Phase: DNA SynthesisS Phase: DNA Synthesis

Every cell has Specific DNAEvery cell has Specific DNA

• Nucleus contains Nucleus contains DNADNA– Each cell in one Each cell in one

body has body has identical DNAidentical DNA

• DNADNA– Set number of Set number of

chromosomeschromosomes• Humans have 46Humans have 46• Flies have 4Flies have 4

– Each one is vitalEach one is vital

Synthesis PhaseSynthesis Phase

• After G1 phase of cell After G1 phase of cell growthgrowth

• All of DNA MUST be All of DNA MUST be replicatedreplicated– Try for no errorsTry for no errors

• Each daughter cell gets Each daughter cell gets her own set of her own set of chromosomeschromosomes– My example: encyclopediasMy example: encyclopedias

Synthesis PhaseSynthesis Phase

• After G1 phase of cell After G1 phase of cell growthgrowth

• All of DNA MUST be All of DNA MUST be replicatedreplicated– Try for no errorsTry for no errors

• Each daughter cell gets Each daughter cell gets her own set of her own set of chromomeschromomes– My example: encyclopediasMy example: encyclopedias

3 major steps of DNA 3 major steps of DNA replicationreplication• 1. Enzymes bind to the double stranded 1. Enzymes bind to the double stranded

DNADNA

• 2. DNA is unwound and separated into 2. DNA is unwound and separated into single strandssingle strands

• 3. New complementary strands are 3. New complementary strands are created for both original single strandscreated for both original single strands

11stst Step: Enzymes bind DNA Step: Enzymes bind DNA

• Origin of ReplicationOrigin of Replication– Specific region on DNA that Specific region on DNA that

enzymes will bindenzymes will bind

• A group of enzymes bind A group of enzymes bind originorigin– 1. Helicase1. Helicase: will unzip DNA: will unzip DNA– 2. RNA Polymerase2. RNA Polymerase– 3. DNA polymerase3. DNA polymerase

• ReplisomeReplisome::– Once proteins bindOnce proteins bind– Proteins plus DNA complexProteins plus DNA complex

Origin of Replication

22ndnd Step: unwinding DNA Step: unwinding DNA• Helicase: protein that unwinds DNAHelicase: protein that unwinds DNA

– Binds DNA and moves along in 1 directionBinds DNA and moves along in 1 direction

Pulls two complementary strands apart by Pulls two complementary strands apart by breaking hydrogen bonds between the breaking hydrogen bonds between the two strandstwo strands

• Exposes single stranded Exposes single stranded

DNADNA

22ndnd Step: unwinding DNA Step: unwinding DNA• Helicase: protein that unwinds DNAHelicase: protein that unwinds DNA

– Binds DNA and moves along in 1 directionBinds DNA and moves along in 1 direction

Pulls two complementary strands apart by Pulls two complementary strands apart by breaking hydrogen bonds between the breaking hydrogen bonds between the two strandstwo strands

• Exposes single stranded Exposes single stranded

DNADNA

Step 3Step 3: Synthesize new : Synthesize new complementary strandscomplementary strands

• Original strands=template strandsOriginal strands=template strands– Each single strand is used as a Each single strand is used as a

“template” to make a new “template” to make a new complementary strandcomplementary strand

• 11stst: RNA polymerase synthesizes a : RNA polymerase synthesizes a primerprimer

•Why?Why?

PolymerasesPolymerases• Create new strands of RNA or DNACreate new strands of RNA or DNA• Link nucleotides togetherLink nucleotides together• Move in Move in ONEONE direction: direction: • DNA polymerasesDNA polymerases

– Cannot start from scratchCannot start from scratch– Can only add nucleotides to a DNA or RNA Can only add nucleotides to a DNA or RNA

strand already therestrand already there– Make strands 5` to 3`Make strands 5` to 3`

• RNA polymerasesRNA polymerases– Can start from scratchCan start from scratch

5 prime

5`

3 prime

3`

sugar

P

base

sugar

P

base

PolymerasesPolymerases• RNA polymerasesRNA polymerases

– Can start from scratchCan start from scratch– Take free nucleotides and assemble a new strandTake free nucleotides and assemble a new strand

• DNA polymerasesDNA polymerases– Cannot start from scratchCannot start from scratch– Can only add nucleotides to a DNA or RNA strand Can only add nucleotides to a DNA or RNA strand

already therealready there– Make strands 5` to 3`Make strands 5` to 3`

primer

Step 3: Synthesize new Step 3: Synthesize new complementary strandscomplementary strands

• Original strands=Original strands=template template strandsstrands– Single stranded template strand used as Single stranded template strand used as

“template” to make new strands“template” to make new strands

• RNA polymeraseRNA polymerase– Binds origin of replicationBinds origin of replication– Creates a Creates a primerprimer

•Starting blockStarting block

•A short ~10 bases long segment of RNAA short ~10 bases long segment of RNA

Step 3: Synthesize new Step 3: Synthesize new complementary strandscomplementary strands

• RNA polymerase makes RNA polymerase makes primerprimer

• DNA polymerase takes overDNA polymerase takes over– Adds on nucleotides to the primerAdds on nucleotides to the primer– Synthesizes all of DNASynthesizes all of DNA– creates complementary new strand 5` to 3`creates complementary new strand 5` to 3`

• Last stepLast step: RNA primer is replaced with DNA: RNA primer is replaced with DNA

• DNA replication movieDNA replication movie

Step 3: Synthesize new Step 3: Synthesize new complementary strandscomplementary strands

DNA replication movieDNA replication movie

Helicase unzips in one Helicase unzips in one directiondirection

• Helicase latches onto origin of Helicase latches onto origin of replicationreplication

Step 1b:Step 1b:

• Moves along breaking weak Moves along breaking weak hydrogen bondshydrogen bonds

• Forms areas of single stranded DNAForms areas of single stranded DNA

Note 1c:Note 1c:

• Replication forks formReplication forks form

• Junction where double stranded DNA Junction where double stranded DNA becomes single stranded DNAbecomes single stranded DNA

• Looks like a fork in the roadLooks like a fork in the road

• You’ll find helicase hereYou’ll find helicase here

Replication fork

• Helicases move in one directionHelicases move in one direction

• Another helicase may jump on Another helicase may jump on – Move in other directionMove in other direction

• Why sometimes you see 2 replication Why sometimes you see 2 replication forksforks

helicase

Note 1d:Note 1d:

Note 1e:Note 1e:

• When they get to end, helicases When they get to end, helicases jump off and look for other dsDNAjump off and look for other dsDNA– dsDNA=double stranded DNAdsDNA=double stranded DNA

Problem: Leading vs. Lagging StrandProblem: Leading vs. Lagging Strand

• Recall: helicase unwinds DNA in one Recall: helicase unwinds DNA in one directiondirection

• Polymerase moves in one directionPolymerase moves in one direction

• DNA is two strands in opposite DNA is two strands in opposite directiondirection

• How is this a problem?How is this a problem?

Watch the helicase moveWatch the helicase move

Leading Strand: Slide 2Leading Strand: Slide 2

• DNA polymeraseDNA polymerase jumps on jumps on

• Moves in one directionMoves in one direction– Same directionSame direction as helicas as helicas

• Follows helicase closelyFollows helicase closely

• DNA polymerase continues to follow DNA polymerase continues to follow helicasehelicase

helicase

Leading Strand Leading Strand SynthesisSynthesis

• Normal, continuousNormal, continuous

• Makes new DNA strand all in one Makes new DNA strand all in one motionmotion

• No problemNo problem

• What about lagging strand?What about lagging strand?

Lagging Strand: Slide 2Lagging Strand: Slide 2

• Another DNA polymerase jumps on Another DNA polymerase jumps on top strandtop strand

• It also moves only in one directionIt also moves only in one direction

Lagging Strand: Slide 3Lagging Strand: Slide 3

• However, top strand is in However, top strand is in opposite opposite directiondirection than bottom strand than bottom strand

• DNA polymerase DNA polymerase moves awaymoves away from from helicasehelicase

• DNA polymerase finishes the top DNA polymerase finishes the top strandstrand

Lagging slide 4Lagging slide 4

• OOPS!!OOPS!!

• The helicase has kept movingThe helicase has kept moving

• More open single stranded template More open single stranded template openopen

Lagging 5Lagging 5

• Another polymerase has to jump on Another polymerase has to jump on againagain

• It fills in the space…but it is not It fills in the space…but it is not done…done…

• Helicase is continually opening up Helicase is continually opening up more spacemore space

• Polymerase are just trying to catch Polymerase are just trying to catch upup

• Lagging strand makes “Okazaki Lagging strand makes “Okazaki fragments”fragments”

Okazaki fragments Okazaki fragments Okazaki fragments fragments

• DNA Ligase:DNA Ligase:– A proteinA protein– fills in spaces between okazaki fills in spaces between okazaki

fragmentsfragments

Okazaki fragments Okazaki fragments Okazaki fragments fragments

• Final product: two new ds DNAFinal product: two new ds DNA– Identical to original template DNAIdentical to original template DNA

•one strand: originalone strand: original

•One strand: brand-newOne strand: brand-new

•videovideo

Leading vs. Lagging StrandLeading vs. Lagging Strand

• LeadingLeading– Follows same direction as helicaseFollows same direction as helicase– continouscontinous

• LaggingLagging– Polymerase moves opposite of helicasePolymerase moves opposite of helicase– DiscontinousDiscontinous– Multiple polymerasesMultiple polymerases– Forms Okazaki fragmentsForms Okazaki fragments– Requires ligase enzymeRequires ligase enzyme

But then we face a problemBut then we face a problem

• Scientist noticed: a bacterial Scientist noticed: a bacterial chromosome is almost 10 cm longchromosome is almost 10 cm long

• Bacteria are only around 1 to 10 um Bacteria are only around 1 to 10 um longlong

• Where does the DNA go?Where does the DNA go?

DNA must be condensedDNA must be condensed

• DNA is condensed in organismsDNA is condensed in organisms

• DNA is negatively chargedDNA is negatively charged– Negative charges repel each otherNegative charges repel each other

• Wrapped tightly around Wrapped tightly around nucleosomesnucleosomes

• NucleosomesNucleosomes– made of 4 positively proteins made of 4 positively proteins

histoneshistones

• DNA wrapped around DNA wrapped around nucleosomes forms nucleosomes forms chromatinchromatin

• Tightly wrapped DNA cannot Tightly wrapped DNA cannot have gene expressionhave gene expression