Personal_Knowledge_Base/Biology_and_Chemistry/20240831141411-genetics_dna_replication.org

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#+title: Genetics: DNA Replication
#+filetags: :genetics:lecture_notes:
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Two important facts about DNA replication is:
1. It is *semiconservative*
2. It is *bidirectional*


Semiconservative means that when each strand is separated for replication, each strand is used as a template for a new strand.

This fact was determined by Messelson and Stahl. They grew /E. coli/ cells in an environment with heavy $^{15}N$ instead of the more naturally occurring, lighter $^{14}N$ isotope. This effectively makes the nitrogen on the DNA of the cells heavier than normal.

They put the DNA from the cells in a Cesium chloride density-gradient centrifugation process. The heavy DNA sits lower in the solution than lighter DNA.
After several generations in the heavy nitrogen, they transferred them to an environment with lighter nitrogen and they sat there for one generation of replication.
Once put in the density-gradient again, there appeared to still be some heavy DNA, as well as "hybrid" DNA, whicih sat in between the heavy position and the light position.

This essentially confirmed semiconservative replication.

Replication is also bidirectional. A "bubble" forms in the DNA and each replication fork grows away from each other.



* Origins of Replication

In /E. coli/ cells, the replication origin is called the /ori C/.
The /ori C/ has two elements:
    - First is a 13 base-pair-long sequence that exists in 3 tandem repeats. They are very AT-rich, and because of having only 2 H-bonds, they serve to separate first, before the rest of the other DNA.
    - Second is a 9 base-pair-long sequence that exists in 4 repeats with some other sequences interspersed within. These sequences facilitate the binding of proteins such as *DnaA proteins*.
In Yeast, and other eukaryotes, origin sites are referred to as *Autonomously Replicating Sequences, or ARS.*
In SV40 virus, the origin site is 64 bp-long sequence.
    - It is very AT-rich.
    - serves as a protein binding site
    - T-antigen binds to palindrome 27bp long.


* Bidirectional Replication
- Shnos and Inman made features, almost like physical markers, on a bacteria plasmid, by heating the DNA to a point to where only the AT-righ regions denatured.
- They used these 'landmarks' to visually see which direction the replication fork was travelling.
- from these landmarks, they determined it was bidirectional.


* The Players

- *DNA Polymerase I*
  * Also known as Kornberg's enzyme, after the person who first purified it.
  * Needs a template and a primer
  * Directions is *always* 5' --> 3'
  * Utilizes 5' triphosphates of each deoxynucleoside. (dNTPs)
  * 2 phosphates get cut off when attached to the backbone, releasing a lot of energy. (See [[id:d4ec4698-96e7-4cce-93a3-27e7b9eb5965][Genetics: DNA and the Molecular Structure of Chromosomes]] For more information)
  * *Pyrase* recycles the phosphates.

  This enzyme incredibly has 3 different functions:
  1. 5' --> 3' polymerase
  2. 5' --> 3' exonuclease, meaning it can remove objects in front of it that may be blocking it. Important for removing primers, which will be disussed below.
  3. 3' --> 5' exonuclease, meaning it can clip out something incorrect that it placed. The base can drift away bit must receive 3 phosphates before it can bind again.


- *DNA Polymerase III*
  * Also needs a template and a primer.
  * Contains 20 different proteins.
  * $\alpha$ subunits are responsible for polymerase activity.
  * $\epsilon$ subunits are responsible for proof-reading.
  * $\theta$ subunits bind with $\alpha$ and $\epsilon$ to create the *minimal core*. This in itself does not have function, but it is necessary for structure.
  * t subunits link both of the domains together; they are responsible for dimerization.
  * $\beta$ subunits physically grab the DNA template. This makes DNAP III much more stable on the strand than DNAP I.


- *Lagging Strand/Leading Strand and more Players*
  * composed of *Okazaki fragments*.
  * results in covalent breaks between the fragments (since DNAP III cannot remove things in its path like DNAP I, as long as it's in an environment with more than one strand).
  * These breaks are remedied by *DNA Ligase.*
  * Each fragment must also be primed.
  * Leading strand does not have these fragments and thus must only be primed once (however, leading strand on side may be lagging strand on the opposite side. Remember that replication is bidirectional).
  * *DNA primase* lays down this primer, *which is made of RNA*. These provide the 3' OH needed by DNAPs.
  * DNAP I can go back and remove these primers afterword, using its exonuclease function.


* Unwinding the DNA Strands

- Before replication can take place, DNA must first be unwound, at an estimated rate of 3,000 revolutions per minute. This is accomplished by *DNA Helicase*.
- Opened DNA is kept open by *single-strand DNA-binding proteins, or SSB proteins*.
- *Topoisomerases* remove positive supercoils (also by introducing negative supercoils) created by the rapid unwinding of the DNA strand.
    * These come in two classes: Class I and Class II
    * Class I removes supercoils by nicking just one strand (no ATP).
    * Class II breaks the double-strand and pulls each side out of the coil and reattaches them, thus solving two coils at once. It breaks the strand and literally pulls it through the coil.



* Eukaryotic Differences

- DNA replication restricted to S-phase.
- Multiple replicons per chromosome.
- Two or more polymerases at each fork; RNA primers have to be removed by *RNase H1/RNase FEN-1*. the polymerases lack a 5' --> 3' exonuclease activity.


* Problems with Telomeres

- Occurs on lagging strand.
- At the end of a DNA strand, when RNA primer is cleaned up, there is nothing there to build it up again.
- This creates a *3' overhang*.
- *Telomerase* makes the 3' end longer, using its very own RNA template.
- The RNA template is the same in every telomerase, making it a very repetitive and predictable sequence.
- The lengthening of the 3' end allows for a primer to be put in. Not as much real estate is lost.
- However, there will still be some sort of overhang left over in the end.