DNA Replication

Proposed model of DNA replication - Conservative

• The original double strand serves as a template for a new molecule of DNA

• ORIGINAL DNA: It opens up and splits → each strand used as a template to generate new strand

• FIRST REPLICATION: there is a old strand that came together and a new strand that came together

• SECOND REPLICATION: The old strand splits up → there is a new entity in second replication. The new strand splits → there are new entities

Proposed Model of DNA Replication - Dispersive

• DNA Strand both double stranded would break up to multiple fragment and each fragment serves as a template → fragments come together → combination of old and new in both entities

• Both DNA molecules break down into fragment and serve as a template for the new fragment

Proposed Model of DNA replication - Semi conservative

• The original strand unwinds and is used as a template to generate the new strand

• ORIGINAL DNA: Original strand breaks apart → as they break apart → serve as a template to get new strand

• FIRST REPLICATION: No breaking apart, just a combination of old and new strand

• SECOND REPLICATION: New strands just used red as a template

Meselson and Stahl’s experiment

• Needed a way to distinguish the old from new strand. They used 2 isotopes of Nitrogen: 14N and 15N (this was used since bases that make up rungs of ladder are nitrogenous bases)

• There are heavy and light form of Nitrogen

Steps of Meselson and Stahl’s experiment

• 1st: grow bacteria in heavy 15N for many generations. This made the DNA HEAVY.

• 2nd: Took a sample of those bacteria in 15N and switched the rest to 14N. The rest left out was left in 15N. New DNA contained 14N which was lighter.

• 3rd: Ran the collected DNA on an equilibrium density gradient centrifugation to distinguish between heavy and light strands.

Meselson and Stahl’s Equilibrium Density Gradient Centrifugation

• Take acquired DNA and put in tube of different levels of density as you move down the tube

• If you put DNA on top and spin it in centrifuge → DNA travels down and stops where it meets identical density → establish density gradient using different amounts of sugar (create increased density in solution)

• At the bottom, it is heavier material (more sugar)

• As you go up, it is slightly lighter and there is less sugar

• This allows it to separate DNA molecule

Details of Meselson and Stahl’s experiment (0 mins)

• One sample was kept at 15N and the other was switched to 14N

• Sample at 0 mins: found double stranded molecule with 15N and sunk down further (from sample with 15N)

• Interpretation: All bacteria is heavy

Details of Meselson and Stahl’s experiment (20 mins)

• Sample that was transferred to 14N was rested in intermediate zone (not too light nor heavy)

• This is slightly slower and lighter

• Interpretation: it contained both parent 15N and 14N in strands

Details of Meselson and Stahl’s experiment (40 mins)

• Found strand in intermediate zone that had both 14N and 15N

• Found band with just 14N which is lighter

• It is semi conservative since there is a band that is light meaning there were 2 strands that were generated using 15N and strands that had a single copy of 14N

Meselson and Stahl demonstrated DNA replication is semiconservative

• After 2 rounds of replication, if replication were dispersive, you expect the weight of DNA to be: all in intermediate range where every strand has a mixture of old and new DNA

• The fact that some of the DNA was light and some was of intermediate weight after 2 rounds of replication suggested semi-conservative replication - dispersive replication will produce DNA that contains both light and heavy DNA - all intermediate weight

Several modes of DNA replication

• There are several ways that semi conservative replication can take place differing based on whether the DNA is circular or linear

  • Theta replication

  • Rolling Circle replication

  • Linear eukaryotic replication

Several modes of DNA replication: Theta replication

• Takes place in circular DNA/plasmid such as e. coli, bacteria, and prokaryotes

• Expect organisms that undergo replication to have circular plasmids

• 1st: 2 strands that make up plasmid need to melt (unwind/separate)

• Unwinding starts at origin of replication where polymerase will start where it will bind and begin to do job of replicating

• It needs to unwind before origin of replication so all machinery can get there and ready to do its job

• There is a slow unwinding and it’s called a replication bubble

Theta replication (2nd)

• 2nd: the unwinding continuous and bubble gets large. Location where there is unwinding is called replication fork.

• If there is unwinding on both ends → bidirectional replication

• If unwinding is on one side → unidirectional

• In this case, it is bidirectional

Replication bubble

• proteins come in and do their job

• proteins decide whether DNA replication is unidirectional (travels 1 way) or bidirectional (bubble opens and DNA replication is going to take place around)

Theta replication (3rd)

• 3rd: DNA synthesis proceeds in 5-3’ direction

  • DNA will be built in where polymerase will actually bind DNA moving in 5’ to 3’ prime direction

  • physical pieces that the nucleotides are being added in 5’-3’ prime direction so it synthesizes the strand in that direction

  • DNA is antiparallel

  • Original template is moving in opposite direction

Theta replication summary

• In replication, bubble forms and its bidirectional and need to split from one another

Several Modes of DNA replication: Rolling Circle replication

• 1st: Single strand break creating a 3’ OH and 5’ Phosphate group

• DNA polymerase: move from 5’ to 3’ direction

  • Next one that attaches to phosphate and C (5’ C) which is attached to another ring

  • There is a reliance on OH group in order for synthesis to occur

  • Critical elements required for function of DNA polymerase is 3’ OH (which comes off 3’ C)

• When there is a plasmid in circle → no access to 3’ OH group so you create a nic → break DNA to expose 3’ OH

• Polymerase can come in and find 3’ OH group → attach and synthesize

• Unidirectional

• Bacteria uses this

Several Modes of DNA replication: Rolling Circle replication (2nd)

• 2nd: New nucleotides are added on the 3’ OH break using the inner strand as a template

• Now that the 3’ OH group establishes (in yellow area), DNA polymerase synthesizes new strand (red) → displaces original strand that existed → polymerase is a chunky protein moving along old DNA and using it as a template to generate new strand

• By displacing old DNA, it can be used as a its own template to generate new strand

Several Modes of DNA replication: Rolling Circle replication (3rd)

• 3rd: As the new strand gets elongated, the original strand gets displaced, eventually rolling off the entire plasmid. This can continue for several rounds until many copies are made.

  • Purpose of rolling circle: repeat and generate new strand and it is a continuous cycle

Several Modes of DNA replication: Rolling Circle replication (4th)

• 4th: the linear fragment that rolls off can be used as a template for a new strand

  • Run into position where every strand is being displaced every single time and now circularized and used as a template to generate a double strand

  • Every time it goes around original strand, it gets displaced to make more copies

Several Modes of Replication: Linear Chromosomes

• Too large to have a single replication origin, so linear chromosomes in eukaryotes need multiple origins

• Linear chromosomes: complex and linear and lots of nucleotides and many sites of replication (unique and need to melt DNA)

• Origin 1, 2, 3 initiate replication, meet up when bubbles merge together and have replication of entire strand

• Bidirectional

Stages of DNA replication of Eukaryotic and Prokaryotic

• Initiation

• Unwinding

• Elongation

• Termination

Stages of DNA replication Prokaryotic: Initiation

• Initiation: DnaA protein binds to the origin of replication (oriC)

  • Initial bubble that forms and refers to unwinding part of DNA (melting)

  • Opens a small site for Helicase and single-strand binding protein to enter and being unwinding the 2 strands

AT rich region

• Area where replication, initiation needs to begin so by bending → causes unwinding of location → can throw machinery for work to take place → establish opening of entire strand

• Seen in initiation

DnaA box

• Location where it has high tendency to bind proteins (DnaA proteins) and when binding to it → causes conformational change where DNA twist where releases its hold on upstream region

• Seen in initiation

Protein Helicase

• responsible for unzipping inner wrongs and undoing hydrogen bonds between base pairs in middle

• Seen in initiation

Stages of DNA replication Prokaryotic: Unwinding

• Unwinding: DNA replication requires a single strand template. Multiple enzymes

  • Unwind whole thing

  • Helicase undo H bonds between base pairs

    • Strands separate: need something to hold DNA from not collapsing

    • The second you unwind, it wants to go back → it keeps it open to allow continuous replication by using SSN proteins → stay bound to unwounded DNA so machinery can do their job

    • Supercoiling is prevented during unwinding with DNA Gyrase

DNA Helicase

• breaks hydrogen bonds between bases

• involved in unwinding

Single-strand binding proteins

• bind to single strands once they form to stabilize the structure

• involved in unwinding

DNA gyrase

• known as topoisomerase

• prevents supercoiling and upstream torsional strain the builds up during unwinding

• Forces torsional strain upstream → take wound DNA force apart, push it out → prevent you from going further

• Need to unwind a bit so replication can continue

Kinds of DNA Gyrase: Topoisomerase 1

• Creates single stranded breaks in DNA

• take 2 strands that are there → clip one of them and twist it back to the other one → undoes a single turn to relax DNA

Kinds of DNA Gyrase: Topoisomerase 2

• Creates double stranded breaks in DNA

• Breaking the double strand, passing the double helix through it and resealing again

• Does something similar to 1 but will clip both strands and turn them around each other → undo a single turn → both functioning a way to relax DNA before replication occurs

• It is needed since it will seize to continue since it will not be able to unwind and there will be too much strain upstream

Stages of DNA replication Prokaryotic: Elongation

• Elongation: single strands of DNA are used as template to make a new strand.

Primers

• DNA polymerase can’t start replicating on its own. It needs a free 3’ OH to start adding nucleotides to. To bypass this issue primase is used to generate a short 10bps of RNA which ends in a free 3’-OH group. The RNA is later removed and replaced with DNA.

• Elogation

DNA polymerase 3

• Replicates DNA using the RNA 3’-OH group. Has very high processivity (holds on to the DNA and doesn’t release until its done). Works on leading strand.

  • Has 5’ → 3’ activity for replication

  • Has 3’ → 5’ exonuclease activity for backing up and replacing errors

  • Elongation

DNA polymerase 1

• Removes RNA primer and replaces it with DNA. Working on the lagging strand

  • Has 5’ → 3’ activity for replication

  • Has 3’ → 5’ exonuclease (enzyme that removes nucleotide, specifically bad ones) activity for backing up and replacing errors

  • Has 5’ → 3’ exonuclease activity to remove primers and replace with DNA

Stages of DNA replication Prokaryotic: Elongation (2)

• Poly 1 removes the RNA primer and replaces it with DNA

• But a single break remains between the DNA generated by Poly 1 (replacing RNA primer) and poly 3.

• This break is sealed by the enzyme Ligase

Stages of DNA replication Prokaryotic: Elongation (3)

• DNA polymerase 1 can add/remove but not give DNA.

• Once there is a new strand, DNA polymerase 1 budges all RNA primers off and its gone.

• It cannot stitch together stands before and after primer

• Little gap (no stitching) remains unglued (Ligase glues the gap back together)

Stages of DNA replication Prokaryotic: Termination

• Termination:

  • 1 - Some DNA replication terminate when 2 replication forks meet

  • 2 - Others need assistance of termination proteins Tus. The Tus protein binds to a sequence known as a Ter sequence. Upon binding, the complex blocks replication.

    • Tus behaves like a barricade which prevents polymerase/hell case from moving any further past that point. It is like a clamp and acts like a physical barrier.

One strand must be synthesized in fragments

• Structure is antiparallel - 2 strands run in opposite orientations (5’ to 3’ versus 3’ to 5’)

• The new chains are synthesized in what for them is the 5’ to 3’ direction

• The template is read in what for it is the 3’ to 5’ direction

• Fragments: DNA polymerase can replicate in 5’ to 3’ direction

One strand must be synthesized in fragments 2

• Polymerase can synthesize on leading strand without problem

• The primer is generated on leading strand

• Okazaki fragments: this strand is laying down 5’ to 3’ direction → then polymerase can bind and build in this direction

Eukaryotes DNA replication

• Origin

• Cell cycle

• licensing

• DNA polymerase

• Nucleosome

Origin

• multiple origins in eukaryotes in long linear strand

• Unique sequences that are associated with yeast called autonomously replicating sequence (every time you see it → it indicates origin of replication. They are AT rich)

Cell Cycle

• Has specific proteins that need to be turned on and off to cycle

• There are checkpoints where DNA is checked to make sure it is correct

  • more dependent on timing

Licensing

• Takes place to ensure replication forks initiate at the same time and doesn’t initiate more than once per cycle

• Step 1: origins are licensed. Licensing proteins (origin recognition complex - ORC) get attached to the origin.

• Step 2: Initiation machinery (MCM2 - helicase) binds and starts replication. Licensing factor is removed as the replication fork moves away from origin. Licensing factor is removed to ensure that replication can’t occur more than once per cycle.

• MCM - minichromosomal maintenance. Has helicase activity and unwinds a small stretch of DNA at the beginning of replication.

• Geminin Binds to Cdt1, which is used in replication initiation, and degrades it at G1. Therefore, replication can not re-initiate.

DNA polymerase

• DNA polymerase in Eukaryotes

  • Alpha: has primase activity and initiations DNA synthesis by generating a short primer

  • Delta: takes over after the primer has been laid down (by alpha DNA polymerase) on the lagging strand

  • Epsilon: functions like Delta but works on leading strand

• There is a small binding pocket in these polymerases that doesn’t allow incorrect bases to enter. If there is a lesion and an incorrect base needs to be paired, then these polymerases detach, and a trans lesion polymerase comes in and adds an incorrect base just to bypass the error. Error can always be fixed later.

Nucleosome

• Eukaryotic DNA is complex and packaged for stability with histone proteins. It has 8 histone proteins, with DNA wrapped around it. During replication, the DNA needs to unwrap, and histones need to be removed so that all DNA can be replicated.

  • 1: original nucleosome is removed

  • 2: some old histones find their way to their location on the new strand

  • 3: new histones are made and attach to old histones