(Transcribed from Dr. Cadilla’s lecture, 17 Mar 2000 by Brian Buschman)
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The human genome is composed of transcription units which are transcribed to RNA and regulator units. In eukaryotes the transcription units code for a single peptide where as in prokaryotes they may each code for multiple peptides. In prokaryotes transcription and translation occur simultaneously whereas in eukaryotes the steps are compartmentalized. The compartmentalization requires that they occur sequentially because they must move between compartments.
DNA is composed of four bases held in order by a backbone made of deoxyribose and phosphate groups. It’s 5’ end has a carbon and it’s 3’ end an –OH group. The four bases the two purines, A and G, have two heterocyclic rings, and the pyramidines, C and T, have only one ring.
Deoxyribose is a derivative of ribose where the –OH at carbon two has been replaced with –H.
Nucleosides are units made up of one of the four nitrogenous bases bound to a deoxyribose or ribose.
Nucleotides are the base, the sugar and between one and three phosphate groups attached to the sugar.
The secondary structure of DNA is a double helix which is held together by hydrogen bonds between the bases and by the Van der Waals interactions from the stacking of the bases in the helix. The helix has two grooves that appear, the larger major groove and the smaller minor groove.
In the structure there are three bonds between G and C and only two between A and T. This means that G, C rich regions will require a higher temperature to denature then A, T rich regions.
DNA is found primarily in three forms:
1) B-DNA is the traditional Watson-Crick model. It has about 10 bases to the turn and is a right-handed helix.
2) A-DNA is a slightly dehydrated version of B-DNA. It is still right handed but is fatter and more compact.
3) Z-DNA is a hard to find version where the helix is left handed.
DNA can be denatured by high temperatures, high pH, urea, salt or very high concentrations of other ions. As it denatures it’s viscosity decreases and it’s A260 increases. The A260 is the peak absorbance of DNA/RNA and is used to determine concentration in solution.
When undergoing annealing, putting it back together, it’s slow to start but once the first bases are lined up again then the process proceeds quickly.
DNA becomes supercoiled in the process of compaction and while helicase is working to unwind the double helix to form the replication fork. It would take a lot of energy to twist the whole molecule to relax this tension which is why the cells have topoisomerases which can cut the molecule to take care of this. Type 1 only cuts one strand, putting a nick in the DNA, and allows the DNA to freely unwind and relax the tension. It requires no ATP. Type 2 topoisomerases cut both strands and allow entire double helices to pass through. This is most common in gyrase of bacteria which adds supercoils for compaction. Type 2 topoisomerases do not require ATP if relaxing a stress but do require ATP is adding supercoils like gyrase does.
DNA is compacted into units of chromatin which use histones to help organize the DNA.
1) Euchromatin is the decondensed state of the DNA where transcription or replication are taking place.
2) Heterochromatin is the condensed version where it’s inactive. In heterochromatin about 50% of the mass is DNA and 50% associated proteins.
Histones are small proteins that function in helping organize the DNA into nucleosomes. Four of the types of histones, H2A, H2B, H3 and H4, together form a core that DNA will wrap itself around two and a half times. This helps compact DNA as it takes about 146 bases to do this. Those units are connected by linker DNA that are supported by a H1 histone. The together form a series that looks like a bunch of beads on a string. This series is called the 10nm protein because of it’s 10nm width.
These strings are then supercoiled to form a larger protein called the 30nm protein for the next level of compaction.
The 30nm proteins are then thought to be wrapped back and forth and be connected to a scaffold protein for more compaction but this level is still not very well understood.
DNA replication is semiconservative in that when it is done one new strand is paired with one old strand.
Replication begins when DNA is unwound by helicase (with topoisomerase type 1 releasing the supercoils) beginning at the origin of replication. In E. coli it is the oriC sequence. In prokaryotes the DNA is circular and there is only one origin of replication. The replication fork proceeds into both directions and they meet on the other side. In eukaryotes there are many origins of replication since the DNA is longer and replication is about ten times slower.
During replication the template strand is read in the 3’ to 5’ direction and the new strand is synthesized in the 5’ to 3’ direction.
The process of synthesis used nucleotides that have three phosphates (dATP, dCTP, dGTP, dTTP) where the energy for placing them comes from hydrolysis to remove pyrophosphate (two attached phosphate groups).
When the replication fork is opened up single-stranded DNA-binding (SSB) proteins quickly bind to them which prevent the strands from joining back together and protects then from nuclease cleavage.
The next step is the placement of a primer made of RNA which is required of DNA polymerases. After the primer is bound synthesis continues 5’ to 3’ as helicase continues to do it’s job.
The polymerization of DNA becomes a problem since it is only synthesized 5’ to 3’ and DNA is antiparallel. One strand is able to follow directly behind helicase but for the other strand polymerization would have to work 3’ to 5’ for this to happen. In this case the one strand, the leading strand, follows directly behind the helicase and replicates in one big long strand. The other strand, the lagging strand, is replicated in a number of shorter strands, Okazaki fragments, working in the opposite direction of helicase.
The polymerization is carried out by DNA polymerases. The polymerases are classified in part by their processivity, which is the number of bases they will lay before falling off the template strand. With high processivity thousands of bases may be laid before stopping while with low processivity only a few. High processivity is optimized for replication while low is best for repair. DNA polymerases usually also have an endonuclease activity for either proofreading, removal of the primer or correcting mutations in the DNA.
Pol III has a high processivity and lays down the new DNA. It also has 3’ to 5’ endonuclease activity for proofreading as it goes so it can correct minor errors.
Pol I has a low processivity and multiple jobs. It first has 5’ to 3’ endonuclease activity and 5’ to 3’ polymerization activity to replace the RNA primer with DNA. It also has 3’ to 5’ endonuclease activity for proofreading.
Pol II has an intermediate processivity and functions in repairs.
Ligase is the enzyme which is the last to follow along and fix the nicks that are remaining to form a complete unbroken strand.
Pol a works as the primase, synthesized and places the primer.
Pol d has high processivity and synthesizes the leading strand and had 3’ to 5’ endonuclease activity for proofreading.
Pol e has high processivity and synthesized the lagging strand. Also having 3’ to 5’ endonuclease activity.
Pol b removes and replaces the RNA primer.
Pol g replicated mitochondrial DNA.
The lagging strand of eukaryotic DNA is unable to reach all the way to the end of the template strand. This would result in the lagging strand loosing bases with every replication. This is solved by placing a sequence called a telomere at the end of the lagging strand. This allows room for the Pol e to synthesize all the way to the end of the main part of the lagging strand and then a telomerase places the bases of the telomere.
Telomeres become shorter the older cells become. This raised the question in cloning as to whether the cloned animal will have the “older” looking DNA or not. I have to confess I don’t remember what she said the answer was. I think Dolly got full length telomeres but it’s not really important for this class anyway.
Antiviral drugs often make use of analogs of nucleosides where the base is normal but the sugar is modified slightly. This will cause them to get taken into the DNA but in the case of AZT (the anti-AIDS drug) the polymerases will fall off when the get to this analog. This will slow the replication of rapidly duplicating cells. The problem is that it works to kill both infected and good cells.
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