s phase: dna synthesis. every cell has specific dna nucleus contains dna nucleus contains dna...
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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
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