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 of chromosomes Humans have 46 Humans have 46 Flies have 4 Flies have 4 –Each one is vital

Synthesis Phase After G1 phase of cell growth After G1 phase of cell growth All of DNA MUST be replicated All of DNA MUST be replicated –Try for no errors Each daughter cell gets her own set of chromosomes Each daughter cell gets her own set of chromosomes –My example: encyclopedias

Synthesis Phase After G1 phase of cell growth After G1 phase of cell growth All of DNA MUST be replicated All of DNA MUST be replicated –Try for no errors Each daughter cell gets her own set of chromomes Each daughter cell gets her own set of chromomes –My example: encyclopedias

3 major steps of DNA replication 1. Enzymes bind to the double stranded DNA 1. Enzymes bind to the double stranded DNA 2. DNA is unwound and separated into single strands 2. DNA is unwound and separated into single strands 3. New complementary strands are created for both original single strands 3. New complementary strands are created for both original single strands

1 st Step: Enzymes bind DNA Origin of Replication Origin of Replication –Specific region on DNA that enzymes will bind A group of enzymes bind origin A group of enzymes bind origin –1. Helicase: will unzip DNA –2. RNA Polymerase –3. DNA polymerase Replisome: Replisome: –Once proteins bind –Proteins plus DNA complex Origin of Replication

2 nd Step: unwinding DNA Helicase: protein that unwinds DNA Helicase: protein that unwinds DNA – Binds DNA and moves along in 1 direction Pulls two complementary strands apart by breaking hydrogen bonds between the two strands Exposes single stranded Exposes single stranded DNA DNA

2 nd Step: unwinding DNA Helicase: protein that unwinds DNA Helicase: protein that unwinds DNA – Binds DNA and moves along in 1 direction Pulls two complementary strands apart by breaking hydrogen bonds between the two strands Exposes single stranded Exposes single stranded DNA DNA

Step 3: Synthesize new complementary strands Original strands=template strands Original strands=template strands –Each single strand is used as a “template” to make a new complementary strand 1 st : RNA polymerase synthesizes a primer 1 st : RNA polymerase synthesizes a primer Why? Why?

Polymerases Create new strands of RNA or DNA Create new strands of RNA or DNA Link nucleotides together Link nucleotides together Move in ONE direction: Move in ONE direction: DNA polymerases DNA polymerases –Cannot start from scratch –Can only add nucleotides to a DNA or RNA strand already there –Make strands 5` to 3` RNA polymerases RNA polymerases –Can start from scratch 5 prime 5` 3 prime 3` sugar P basebase P basebase

Polymerases RNA polymerases RNA polymerases –Can start from scratch –Take free nucleotides and assemble a new strand DNA polymerases DNA polymerases –Cannot start from scratch –Can only add nucleotides to a DNA or RNA strand already there –Make strands 5` to 3` primer

Step 3: Synthesize new complementary strands Original strands=template strands Original strands=template strands –Single stranded template strand used as “template” to make new strands RNA polymerase RNA polymerase –Binds origin of replication –Creates a primer Starting block Starting block A short ~10 bases long segment of RNA A short ~10 bases long segment of RNA

Step 3: Synthesize new complementary strands RNA polymerase makes primer RNA polymerase makes primer DNA polymerase takes over DNA polymerase takes over –Adds on nucleotides to the primer –Synthesizes all of DNA –creates complementary new strand 5` to 3` Last step: RNA primer is replaced with DNA Last step: RNA primer is replaced with DNA DNA replication movie DNA replication movie DNA replication movie DNA replication movie

Step 3: Synthesize new complementary strands DNA replication movie DNA replication movie

Helicase unzips in one direction Helicase latches onto origin of replication Helicase latches onto origin of replication

Step 1b: Moves along breaking weak hydrogen bonds Moves along breaking weak hydrogen bonds Forms areas of single stranded DNA Forms areas of single stranded DNA

Note 1c: Replication forks form Replication forks form Junction where double stranded DNA becomes single stranded DNA Junction where double stranded DNA becomes single stranded DNA Looks like a fork in the road Looks like a fork in the road You’ll find helicase here You’ll find helicase here Replication fork

Helicases move in one direction Helicases move in one direction Another helicase may jump on Another helicase may jump on –Move in other direction Why sometimes you see 2 replication forks Why sometimes you see 2 replication forks helicase Note 1d:

Note 1e: When they get to end, helicases jump off and look for other dsDNA When they get to end, helicases jump off and look for other dsDNA –dsDNA=double stranded DNA

Problem: Leading vs. Lagging Strand Recall: helicase unwinds DNA in one direction Recall: helicase unwinds DNA in one direction Polymerase moves in one direction Polymerase moves in one direction DNA is two strands in opposite direction DNA is two strands in opposite direction How is this a problem? How is this a problem?

Watch the helicase move

Leading Strand: Slide 2 DNA polymerase jumps on DNA polymerase jumps on Moves in one direction Moves in one direction –Same direction as helicas Follows helicase closely Follows helicase closely

DNA polymerase continues to follow helicase DNA polymerase continues to follow helicase helicase

Leading Strand Synthesis Normal, continuous Normal, continuous Makes new DNA strand all in one motion Makes new DNA strand all in one motion No problem No problem What about lagging strand? What about lagging strand?

Lagging Strand: Slide 2 Another DNA polymerase jumps on top strand Another DNA polymerase jumps on top strand It also moves only in one direction It also moves only in one direction

Lagging Strand: Slide 3 However, top strand is in opposite direction than bottom strand However, top strand is in opposite direction than bottom strand DNA polymerase moves away from helicase DNA polymerase moves away from helicase

DNA polymerase finishes the top strand DNA polymerase finishes the top strand Lagging slide 4

OOPS!! OOPS!! The helicase has kept moving The helicase has kept moving More open single stranded template open More open single stranded template open Lagging 5

Another polymerase has to jump on again Another polymerase has to jump on again

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

Helicase is continually opening up more space Helicase is continually opening up more space Polymerase are just trying to catch up Polymerase are just trying to catch up

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

DNA Ligase: DNA Ligase: –A protein – fills in spaces between okazaki fragments Okazaki fragments fragments

Final product: two new ds DNA Final product: two new ds DNA –Identical to original template DNA one strand: original one strand: original One strand: brand-new One strand: brand-new video video video

Leading vs. Lagging Strand Leading Leading –Follows same direction as helicase –continous Lagging Lagging –Polymerase moves opposite of helicase –Discontinous –Multiple polymerases –Forms Okazaki fragments –Requires ligase enzyme

But then we face a problem Scientist noticed: a bacterial chromosome is almost 10 cm long Scientist noticed: a bacterial chromosome is almost 10 cm long Bacteria are only around 1 to 10 um long Bacteria are only around 1 to 10 um long Where does the DNA go? Where does the DNA go?

DNA must be condensed DNA is condensed in organisms DNA is condensed in organisms DNA is negatively charged DNA is negatively charged –Negative charges repel each other Wrapped tightly around nucleosomes Wrapped tightly around nucleosomes Nucleosomes Nucleosomes –made of 4 positively proteins histones DNA wrapped around nucleosomes forms chromatin DNA wrapped around nucleosomes forms chromatin Tightly wrapped DNA cannot have gene expression Tightly wrapped DNA cannot have gene expression