Lecture #3 Flashcards
Base Review
Bases: Cytosine, Thymine, Adenine, Guanine
Nucleoside: Base + sugar
- Cytidine, Thymidine, Adenosine, Guanosine
Nucleotide: Base+ Sugar+ Phosphate
- Mono, Di, Tri Phosphate
- Nucleoside monophosphate
- CMP, TMP, AMP, GMP
Nucleotide Addition
Nucleotides are always added in a 5’ and 3’ direction. (always added to 3’ carbon)
How is DNA replicated?
Semiconservative: - Template each parent strand - hybrids created Dispersive: - Strands copied in short segments - Mixture of parent and daughter Conservative: - Parent molecule remains intact - original + daughter
“Most beautiful experiment in biology”
- Matt Meselson
- Frank Stahl (both grad students)
Two sets of e.coli grown- 15N media (heavy)
- 14 N media (light)
- Incorporated into DNA
Isolated from E. coli - mix w/ cesium chloride
Centrifuge (way to separate molecules/ DNA based on density) - density gradient forms
- equilibrium density centrifuge
The Experiment 1
- Grow bacteria in 15N media
- transfer to 14N media
- Grow another 20 min.
- Extract DNA
- centrifuge
- Heavy or light? (two bands: conservative. one band: semi-conservative).
The Experiment 2
Heat DNA before centrifuging to denature the helices.
- heavy or light? (one band: dispersive. two bands: semi: conservative)
Characteristics of replication
DNA is replicated VERY quickly
- 1000nt/sec for bacteria (circular chromosome) –> fully extended
- 100nt/sec for human (highly complex chromosomes)
Each strand acts as a template for the synthesis of a new strand.
The helix needs to be separated in order for replication to occur one of the first things needed to happen.
Opening of DNA Double Helix
The double Helix is VERY stable
- Numerous hydrogen bonds
G-C pairs more stable
100 degrees C is needed to denature the DNA
In vivo the helix is broken apart by “Initiator Proteins”
- Break the hydrogen bonds
Replication begins at Replication Origins
replication begins at replication origins
- One in bacteria, 10,000 in humans
- Why?
- A/T rich regions
Opening of the helix creates a “bubble”
- Proteins bind and act on DNA
-Replication fork formed, bi-directional replication
Replication occurs on the top and bottom strands
Replication Fork is Asymmetrical
Replication occurs in a 5’ –>3’ direction
- ALWAYS? Yes
Replication occurs in an asymmetrical manner
Leading strand: continuous replication
Lagging strand: discontinuous replication
Lagging strand synthesis
Generates Okazaki fragments - small sequences of DNA - later joined together Depends on which "end" of the replication fork - Right end-top strand - left-end bottom strand
stud figure in slide
Replication Machinery
- initiator protein
- DNA helicases
- single-strand DNA biding proteins
- primase
- sliding clamp
- clamp loader
- DNA polymerase
- Endo (within)/ Exo (end of DNA) nucleases
- Telomerase
Step 1: Recognition of the Replication Origin
Initiator protein used
- E. coli: DNA
- Eukaryotes: origin recognition complex: ORC
DNA sequence specific
Step 2: Loading the Pre-replication complex
Late “M” phase + Early G1 phase of the cell cycle (right before synthesis starts)
DNA helicase is loaded
- Release tension on the end
Step 3: Recruitment of the Replisome
DNA helicase Clamp Loader - RFC: Replication Factor C Sliding Clamp - PCNA: Proliferating Cell Nuclear Antigen Topoisomerase Single Stranded BInding Protein (SSB) - RPA: Replication Protein A Primase DNA Polymerase RNase H DNA Ligase
The Replisome
DNA Helicase- *unwinds the DNA/splits double helix*
- Recruited to the DNA by the ORC
- upstream of the polymerase
- Ring shaped
- 5' --> 3' or 3' --> 5'
- Located at the front of the replication fork
Pries apart the helix and unwinds DNA
- Breaks H bonds
ATP dependent
Selects for ssDNA over dsDNA
- Narrowest point is only 13AThe Replisome: Topoisomerase
- Relieves positive supercoiling generated by the helicase
- can break one or both strands
The Replisome: Single Stranded Biding Protein (SSb; RPA)
- Sequence independent
- INteracts with DNA through electrostatic interactions with the backbone
- cooperative
The Replisome: The Clamp Loader (RPA)/ The Clamp (PCNA)
The Clamp Loader (RPA)
- Loads the clamp onto the DNA; ATP dependent
The Clamp (PCNA)
- Ensures that the DNA polymerase remains attached to the template DNA –> sliding clamp
The Replisome: Primase and RNase H
Primase:
- RNA polymerase, deposits an RNA primer complementary to the DNA templates. Does not have deoxyribose. Cannot start a DNA replication without a primer.
RNase H:
- Recognizes RNA/ DNA hybrids; removes RNA primer
The Replisome: DNA Polymerases/DNA Ligase
DNA Polymerases
- Pol (alfa): extends the RNA primer another 20 nt.
- Pol (symbol) and Pol (symbol): extends primer on the leading strand and at Okazaki fragments.
- check slides for symbols*
DnA Ligase
- Joins the Okazaki fragments by ligating the 3’ OH w/ the 5’ phosphate
ATP dependent
Filling in the Okazaki Fragments
DNA polymerase adds to the RNA primer
- Ends at the next RNA primer
RNase H degrades the RNA primer
DNA polymerase replaces the sequence with DNA
DNA ligase joins the two ends together
know image on this slide
The Job of DNA Polymerase
Some DNA polymerases are more processive than others
- The # of nucleotides added each time a polymerase sits down/binds
- sitting down is the rate limiting step
- varies for each polymerase
DNA Polymerase contains two domains
- Polymerization
- Editing/ Exonuclease
Addition of nucleotides using DNA Polymerase
All Polymerases add nucleotides in a 5’ –> 3’ direction
Incoming nucleotide
- 5’ end contains a triphosphate
Addition of nucleotides using DNA Polymerase
Formation of phosphodiester bond
- Tyrosine in polymerase interacts with base
- Arginine and lysine in polymerase interact with phosphates
Coordinated by two metal ions
- Usually Mg2+
- Neutralizes the (-) charges
Addition of nucleotides using DNA Polymerase
- Energy required for phosphodiester bond is supplied by…
- Release of pyrophosphate
- Hydrolysis of pyrophosphate by pyrphosphatase
Translocation
- Hydrogen bonds between polymerase and template broken.
- Electrostatic interactions between polymerase and template remain.
What if a mistake is made?
DNA polymerase contains a 3’ –> 5’ exonuclease domain
- higher affinity for ssdNA for dsDNA
A problem is noticed due to a change in geometry
- Incoming nucleotide doesn’t fit well
(This property helps ensure that the nutation rate is 1 every 10^10 bp)
What happens if the Polymerase was 3’ –> 5’
If nucleotides were added to the 5’ end
- Phosphate is released from the 5’ end.
- Proofreading results in a monophosphate end.
- No energy is available to join the new nucleotide.
But what happens at the end of the chromosome?
End Replication Problem
- Cap is left at the end of the chromosome
- Lagging strand
Telomerase (Its own RNA primer, own polymerase) recruited to the ends of the chromosomes
- Contains an RNA template
- Binds the 3’ end of the template DNA
- Uses its RNA template to extend the 3/ end of the template DNA
- Final Okazaki fragment is generated
