Hi friends. Hope you all are having great times. This post is from a webinar organized by my department which I held across 2 different days. Hope you enjoy it.
Day 1
To remind us of what we know, there are 4 macromolecules in living organisms and the DNA is one of them.
Macromolecules here include the carbohydrates, proteins, lipidd and nucleic acids.
They are all made up of smaller subunits and as for the DNA, it is made up of subunits called nucleotides. Just the same way proteins are made up of amino acids, carbohydrates are made up of sugar subunits and lipids are made up of fatty acids.
Each of the nucleotides in the DNA is made up of 3 things: a phosphate group, a 5-carbon sugar and a nitrogenous base(either adenine, guanine, thymine or cytosine)
Photo from Tochukwu Agboeze
This DNA is double helical and consist of 2 strands which are aligned antiparallel or running in opposite direction to each other, with one seeming to point downward and the other upward in a 5' to 3' direction.
One other thing we need to know is that when the two strands align, they follow the complementary base pairing rule whereby adenine pairs with thymine while guanine pairs with cytosine. With this, knowing the sequence of one strand means that you can predict the other.
These base pairs are joined by hydrogen bonds with A and T held by 2 hydrogen bonds while G and C are held by 3 hydrogen bonds.
What we've done so far is just a brief insight on the DNA and its features.
I'll go on to discuss DNA synthesis through DNA replication in bacteria and the stages
involved.
DNA replication means producing identical molecules of DNA from an original copy.
It is very essential for bacterial cells before they divide so that new daughter cells can also get a copy of the DNA.
During DNA replication, the 2 strands of the DNA helix are separated where each strand serves as a template for the synthesis of a complementary strand according to the base pairing rule.
This replication is said to be SEMICONSERVATIVE which means that each new DNA retains one original strand and has one new copy.
To remind us, bacteria which are prokaryotes have circular DNA unlike eukaryotes that have linear DNA.
For this, the replication machinery employed in both is different with differing enzymes/proteins but despite these differences, the overall process or stages passed through is similar in all organisms.
GIF from Tochukwu Agboeze
This is how the replication takes place in bacteria circular DNA prior to cell division unlike that seen in the eukaryotic linear DNA.
To take a deep dive into how this DNA replication actually accomplished, there are 4 stages involved:
1. The pre-priming stage
2. The priming stage
3. Elongation and
4. Termination
To give us a head start, the first stage prepares the DNA for replication, the second involves the synthesis of RNA primers, the third adds DNA sequences while the last stage rounds up the process by removing RNA primers and replacing them with DNA sequences.
These stages which I'll breakdown after this are passed through with the help of a huge enzyme complex called replisome.
Some of these enzymes are DNA polymerase III and I, Helicase, Primase and DNA Ligase.
The replication of chromosomal DNA proceeds bidirectionally meaning that the two replication sides move in opposite direction. It begins at a single point, the origin of chromosomal replication (aka OriC) and the DNA is replicated until the two sides meet on the other side called the replication termination site(ter) where the two are then released.
It is worthy of note that the OriC (origin of replication) is usually rich in A-T base pairing and since adenine pair with guanine using only 2 hydrogen bonds, this A-T rich segment of the DNA become single stranded easily than G-C regions which are paired with 3 hydrogen bonds. This is considered very important for initiation of replication
Let's take a deeper look at the stages, starting with the pre-priming stage.
During the pre-priming stage, a bacterial initiator protein known as DNaA triggers replication by binding to the regions in the OriC.
Once this happens, it paves way for an enzyme known as helicase to start unwinding the DNA
This enzyme HELICASE will now break the hydrogen bonds joining the 2 strands of the double helix together.
GIF from Tochukwu Agboeze
This is what the HELICASE does
While this is going, an enzyme known as DNA gyrase help to avoid the supercoiling or over-winding of the other sequence that are ahead by reducing the torsional strain.
In order to help keep the strands that have been separated from snapping back together, a protein known as single strand binding protein (SSB protein) does the job by holding the strands back.
Once this template of single stranded DNA is prepared, we move onto the priming stage, where an enzyme known as primase help to synthesize an RNA primer which is a short strand of RNA which is complementary to the single strand DNA. This RNA strand paves way for the addition of DNA nucleotides.
The essence of using RNA as a primer here is because unlike the DNA polymerase, it can initiate addition of nucleotides without an existing 3' hydroxyl group.
Once the RNA primer is synthesized, we move on to the elongation stage where the enzyme DNA polymerase III add nucleotides in the 5' to 3' direction.
GIF from Tochukwu Agboeze
This is a representation of what we've treated so far.
Day 2
Tochukwu Agboeze:
We discussed following back then:
-DNA replication in bacteria cells.
-The pre-priming stage of DNA replication.
-The priming stage
-A brief intro to the elongation stage
Let's go:
So after the initiation of replication by the DNaA protein, the unwinding of the DNA by helicase and the synthesis of RNA a primers, the elongation stage starts...
This stage is where the addition of DNA nucleotides begins with the help of an enzyme called DNA polymerase III.
When it comes to DNA replication, this particular enzyme could be referred to as the builder because it catalyzes DNA synthesis by adding nucleotides to the growing chain.
DNA polymerase III is an enzyme complex and consist of 3 core enzymes. These core enzymes (each of them) bind to the strand of DNA and will be responsible for catalyzing the synthesis of DNA.
Structure of the DNA polymerase III
From the picture above, other could-be-strange structures can be seen apart from the core enzymes (speaking of the B-clamp and the T-complex clamp loader. So what are those?
The B-clamps (in red) are subunits that hold the core enzymes in place on the DNA strand while they perform their actions, just the same way an object is held in place by a clamp. For example, a metre rule (as core enzyme) supported on a bench or table (as the DNA strand) by a clamp ( as B-clamp).
The other structure (T complex clamp holder) is a complex protein which is responsible for loading the B-clamps onto the DNA. It uses those structures that look like tentacles to load the B-clamps that hold the core enzymes onto the DNA strands.
Out of these three core enzymes in the DNA polymerase III enzyme complex that can be seen in the picture above, two of them replicate one of the DNA strands while the remaining one replicates the other strand.
Thus, we can now say that the two strands of the DNA are bound by a single DNA polymerase III that contains all these (the core enzymes, T-complex clamp holder and B-clamps.
As we noted from the past lecture, DNA synthesis by DNA polymerase progress in the 5' to 3' direction in both strands that run in opposite directions (one strand seeming to be heading forward while the other one backward). One of the strands here is known as the leading strand while the other one is known as the lagging strand.
Here's the difference between the two strands...
As the DNA double-strand is unwounded or separated the leading strand is replicated continuously towards the same direction as the unwinding DNA ahead of the core enzyme while the lagging strand is replicated in the opposite direction.
As a result the lagging strand is synthesized discontinuously in the 5' to 3' direction (i.e in opposite direction to the movements of the unwinding DNA) and this will lead to the production of a series of fragments called okazaki fragments.
These Okazaki fragments are about 1000 to 2000 nucleotides long in bacteria and approximately 100 nucleotides long in eukaryotes.
GIF from Tochukwu Agboeze
As we can see above...
This production of series of Okazaki fragments occurs because the enzyme primase makes many RNA primers along the template strand as the DNA unwind. DNA polymerase III then extends these primers by adding the nucleotides.
Remember: RNA primers make way for the addition of DNA nucleotides by the DNA polymerase
Thus, while the leading strand requires only one RNA primer to initiate synthesis as it proceeds continuously with the unwinding DNA, the lagging strand has many RNA primers that must eventually be removed. SEE HOW👇
Onto the removal of RNA primers: yes, this should happen since RNA strands are not part of the DNA. They are just needed to make way for DNA synthesis by the DNA polymerase III.
After the lagging strand has been synthesized by the formation of Okazaki fragments, another enzyme known as DNA polymerase 1 removes the RNA primers due to its special ability to snip off nucleotides one at a time starting at the 5' end and while moving towards the 3' end of the RNA primer.
This unique ability of the DNA polymerase 1 is known as exonuclease activity.
Note that an exonuclease is an enzyme that can remove nucleotides from a strand starting at one end (in this case DNA polymerase 1 which removes RNA nucleotides).
Finally, the okazaki fragments are joined by the enzyme DNA ligase which acts as a gluer. It forms phosphodiester bonds between the 3' hydroxyl group of the growing strand and the 5' phosphate group of an Okazaki fragment.
One thing I’m yet to mention which the DNA polymerase III is capable of is that it is capable of proof reading.
Which means that it is capable of of replacing mismatched DNA base pairs immediately after it has been added, while the proper nucleotide is put in place (for example, if adenine is mistakenly paired with guanine, DNA polymerase III corrects the mistake)
Note that this removal of mismatched base pairs must occur before the next base is added.
Onto the last thing we have here ...The termination of DNA replication.
After the replication, a protein called Tus (termination utilization substance) is required to bind to a terminus region which contains multiple copies of 20 base pair sequence called ter, ending replication for good.
When the replication is complete, it could be possible that the two circular daughter DNAs may be intertwined together forming what we call CATENANES. This is obviously a problem if each daughter is to inherit a single DNA.
For this problem to be solved, it requires an enzyme called topoisomerase IV which breaks the DNA molecules so that the strands can be separated.
Ehmm...I guess that's that for DNA replication from me. Feel free to add some other things you know which we've not talked about.
Question time:
Christian Presido:
The DNA polymerase II and III, biko(please) differentiate their functions.
Tochukwu Agboeze:
The DNA polymerase III is the enzyme responsible for catalyzing the addition of DNA nucleotides as well as proofreading
while...
DNA polymerase II is only capable of proof reading but cannot catalyze the addition of DNA nucleotides
Christian Presido:
On the B clamp, T-complex clamp holder and the core enzyme, what is simply their function?
Tochukwu Agboeze:
The B-clamp holds the core enzyme in place on the DNA strand.
The T (pie) complex clamp loader loads the B-clamp which is carrying the core enzyme onto the template DNA strand.
The core enzyme catalyzes the addition of DNA nucleotides.
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