CH 11 — Wave 4 (EXAM 1 CONTENT FINISHES HERE)

DNA Replication

Genetic material is copied. Very quick, very accurate. Relies on complementarity of DNA strands. 1 molecule —> 2 molecules. 2 complementary DNA strands come apart and each serves as the template strand for synthesis of new complementary DNA strands.

Daughter strands

2 newly made DNA strands

Parental Strands

2 original DNA strands

3 original proposals for DNA replication

Conservative model, semiconservative model, dispersive model. Proposed in the late 1950’s after Watson and Crick Model dropped.

Proving the semiconservative model experiment

First, grow bacteria in the presence of nitrogen 14 for many generations to make sure their DNA only contains nitrogen 14 in it. then, for one generation, grow the bacteria in the presence of nitrogen 15 (20 min). Due to the different masses of the isotopes, the DNA would have a different weight/density in the conservative and the semiconservative model. To be able to determine the density of the DNA to know the right model, they lysed the bacteria and isolated the plasmid DNA and used an ultracentrifuge to separate the DNA by weight. They created a CsCl gradient via a swinging bucket rotor with tubes. If the DNA is heaver you move farther in the gradient, as the DNA would move until it hit an equilibrium density with the Cesium Chloride and it wouldn’t move anymore. If the conservative model was correct, there would be 2 bands: 1 lighter and 1 heavier. For semiconservative model, there would only be 1 14/15 band in the middle of where the conservative model’s bands would be. They visualized the DNA by putting ethidium bromide in the DNA prior and then shined UV light on the tube with the gradient and DNA. As each generation went on, more of the nitrogen would be in the DNA. You CANNOT tell the difference between the semiconservative model and the dispersive model if you only use 1 generation of the bacteria.

Bacterial DNA Replication 101; E. Coli

DNA replication is bidirectional (2 replication forks) to go faster; there is an OriC and a ter (terminator) where the replication stops and it is at the opposite side of the OriC. the OriC is around 275 bp of DNA.

OriC in E. Coli

only one of these per cell on their main chromosome. Contains AT-Rich regions, DNaA boxes (5), and lots of GATC (DpnI) sequences. A box is just a sequence in dsDNA.

How replication starts using all 3 OriC parts in bacteria

DNaA proteins bind to DNaA boxes and to each other (additional proteins bind) to bend DNA and strands separate at the AT-rich region (has less hydrogen bonds so it is easier to melt). Topoisomerase II cuts and untangles the DNA and reseals them. Replication needs all the adenines on the GATC sequences to be methylated on both strands of DNA, and they are not methylated after just being made, so it is a regulation to slow down DNA replication to prevent another replication from happening immediately. The enzyme that methylates adenine at the 7th position is called DNA adenine methyltransferase or methylase or DAM. Another regulation is that the DNaA protein concentration after replication gets really low because most of them bind to the cell wall after replication so they are unusable. the E. Coli needs time to make another sufficient amount of them.

helicase/DNaB

composed of 6 subunits, travels along DNA in 5’ to 3’ direction and uses energy from ATP to unwind the DNA for the replication to occur.

DNaA boxes/proteins

Use ATP hydrolysis to create kink in the region that causes the melting of AT-rich region causing the AT-rich area to open/split, then helicase attaches/unwinds DNA and further causes DNA to open. there are some other proteins that bind to the DNaA proteins (or DNaA proteins bind to DNaA proteins as well as the DNaA boxes) such as Hu or DNaC.

Primer

RNA primer is laid by DNA primase. There are a lot of these on the lagging strand and only one on the leading strand. The 5’ side of the RNA primer and the nucleotides that follow it until the next RNA primer make 1 okazaki fragment and there are a lot of okazaki fragments throughout the lagging strand. usually 10-12 nucleotides in length.

DNA polymerase III

has 5’-3’ polymerase/synthesis activity (due to alpha subunit) and 3’-5’ exonuclease proofreading activity (from epsilon subunit). Has beta subunit that is the clamp subunit which helps the protein stay on the parent strand to keep adding nucleotides instead of falling off. keeps it running for 500000 nucleotides not 20. Needs to have pre-existing 3’ OH group to add new nucleotides onto so it needs a RNA primer before it does its work. Has 10 subunits. since the 4 million bp bacterial chromosome is replicated bidirectionally, this enzyme only has to do 2 million bp. The enzyme falls off after 500000 bp due to beta subunit clamp so 4 primers are needed for the leading strand.

DNA polymerase I

has 5’-3’ polymerase and 3’-5’ exonuclease proofreading activity and has 5’-3’ exonuclease activity to replace RNA nucleotides from the primers with DNA nucleotides. Only 1 subunit—makes it good for using in vitro because it only needs 1 subunit to be synthesized (easy to make) and has all the necessary activities such as polymerase, proofreading, and RNA replacing activities.

DNA ligase

seals in the gaps between the DNA nucleotides synthesized by DNA polymerase and the DNA nucleotides that replaced RNA primer nucleotides.

DNA polymerases II, IV, and V

for DNA repair and replication of damaged DNA.

DNA gyrase

removes positive supercoils that occur due to DNA replication ahead of the actual replisome complex

primosome

DNA primase and helicase

SSBs or single strand binding proteins

prevent DNA from reannealing prematurely .

Replisome complex

primosome and 2 molecules of DNA polymerase III. the 2 DNA polymerase III’s dimerize. the DNA polymerases work together with the lagging strand looped way out in looped out model so that both can be together.

avg density of DNA

1.7 g/cm³

holoenzyme DNA polymerase III

all 10 subunits of DNA pol. III