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Generation of DNA nucleotides

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Generation of DNA nucleotides

Postby gs99 » Thu Dec 31, 2009 4:24 pm

My question relates to the base molecules (Adenine, Cytosine, Guanine, Thymine) in DNA.

The chromosomes are replicated in Synthesis part of cell cycle Interphase.
About 204 billion atoms are required to complement the two sides of the human genome.

There is much information about how certain things work in the cell (eg. how proteins are generated). But I'm curious about the chemical level:

How and where are the base molecules generated?

Is there a special protein or enzyme that combines the needed atoms into each molecule?

How does the polymerase obtain the needed molecules to generate the new strands?
(Does it send a message to a protein that gets the requested molecule. Or does polymerase utilize supply tanks with spigots that inject the proper molecule?)

Does the cell know in advance that there are sufficient materials available?

Any comments welcome,

George
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Re: Generation of DNA nucleotides

Postby kolean » Thu Dec 31, 2009 10:39 pm

gs99 wrote:How and where are the base molecules generated?

From this article on Nucleotides in Wikipedia, it will tell you that nucleotides can be salvaged from other complexes, or made de novo (from scratch):
http://en.wikipedia.org/wiki/Nucleotide
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Re: Generation of DNA nucleotides

Postby gs99 » Fri Jan 01, 2010 4:04 am

Thanks for that web page.

Method 1. "salvaged from other complexes"
In this page, it states:
"Salvage pathways are used to recover bases and nucleosides that are formed during degradation of RNA and DNA."
http://en.wikipedia.org/wiki/Nucleotide_salvage
Question: Since DNA replication is being done in the cell cycle immediately following formation of the cell, how could there be any degradation of DNA or RNA?
Is it possible this method is used (only) later in cell life, after may genes have been expressed?

Method 2. "de novo"
I failed to mention that I'm not a chemist major; much of the web page is too technical for readers like me.
I have written computer programs; I'm accustomed to look for objects that have properties and methods (actions).

So what are the material objects needed to start the process?
Molecule x and enzyme y

What is the first method (action)? What does enzyme y do to molecule x? Perhaps the web page calls this Reaction 1?

There are many web pages that provide great drawings of transcription and translation to explain those things clearly.

I'm looking for the steps needed to generate one molecule of A, C, G, or T, in simple words.
Have you seen this kind of information?

Then, one of the other questions was: How is the newly formed molecule attracted to move when and where it is needed?
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Re: Generation of DNA nucleotides

Postby kolean » Fri Jan 01, 2010 5:48 am

Wow, so many questions. See if I can tackle a few:
gs99 wrote:Question: Since DNA replication is being done in the cell cycle immediately following formation of the cell, how could there be any degradation of DNA or RNA?

DNA replication immediately following formation of the cell is for cells that are only for reproducing themselves (such as stem cells and immune cells) when it is called for in the organism. If the cell has any function in the organism, it will only proceed onto DNA replication when it is needed to be replicated. Most cells have a function to do and perform it, and may not go on to replicating itself. Sometimes the cells just wear out or parts wear out, and the cell proceeds to salvage the parts.

gs99 wrote:How is the newly formed molecule attracted to move when and where it is needed?

Proteins in the cell do pretty much everything in the cell. If the protein is not an enzyme catalyzing a reaction to go a certain way (due to environmental conditions and the needs of the cell), it is providing transport or storage for the molecules. Nucleotides are made and transported to the nucleus where they are confined to this part of the cell and are used by the proteins when duplicating the DNA during the S phase.
gs99 wrote:I'm looking for the steps needed to generate one molecule of A, C, G, or T, in simple words.
Have you seen this kind of information?

Google 'Biosynthesis of Nucleotides'. It is really a complicated biochemical reaction with many steps. And yes, each step has a specific enzyme that takes a basic starting molecule (like the amino acid aspartate) and modifies it in steps, till it is a nucleotide (a nitrogenous base attached to a deoxyribose sugar, which is then phosphorylated three times). It may be too technical for you. You could possibly find it completely explained for you in a biochemistry book that would show the biochemicals and what the enzymes catalyze in each of the reaction steps. And each nucleotide is unique in their biosynthesis, so A, T, G, and C are different.
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Re: Generation of DNA nucleotides

Postby gs99 » Fri Jan 01, 2010 8:30 pm

1. Are you saying that DNA replication (S phase) is done only if more cells are needed? That makes sense.
The new cell enters phase G1, then G0 in which the cell does its main job.
If more of this cell are needed, it goes into Phase S, G2 and Mitosis.
This page mentions four resources needed, under "DNA Replication"
http://www.biology-online.org/1/5_DNA.htm
Phase S and G2 seem to be needed only before Mitosis.

2. Until I understand more chemistry, I'm looking for an overview explanation.
"After Helicase splits the DNA, Polymerase adds about 6 billion A, T, G, or C molecules, resulting in two identical DNA strands."
Is that correct?
In a computer program, subroutines would be needed that repeat the steps needed for each nucleotide, such as:
>Read the nt. on original strand (eg. C)
>Determine its complement (eg. G)
>Obtain a new molecule of the complement (eg. G)
>Attach new molecule to new strand

3. To do DNA replication efficiently, I'm wondering if (my imagination):
There are multiple copies of Polymerase, so multiple chromosomes could be processed at the same time.
Perhaps microtubules are utilized in some way to organize the "production area".
There are "bucket brigades" of proteins carrying each type of nt. into one or more temporary dispensers connected to the Polymerase.
Polymerase causes dispenser to release a molecule of needed nt.
This would be similar to the spider releasing various types of web string material.
Have you seen any pictures of stained components to shed light on this system (like those that illustrate Mitosis)?

4. Comments about the chemical formulas for nucleotide metabolism (involving things like IMP, AMP, and GMP)
http://themedicalbiochemistrypage.org/n ... m.html#top
It's amazing to me that they discover what enzymes do in each step; this has taken much observation and testing.
Question: Is there only one occurrence of steps processed for a given amount of source material?
Or is it possible that multiple "strings" (possibly same or different nt. molecule) are being processed at different stages within the cytoplasm? This would be like various experiments being conducted at the same time in a lab or classroom.

5. I noticed this interesting statement: "Purine Nucleotide Biosynthesis, The major site of purine synthesis is in the liver."
http://themedicalbiochemistrypage.org/n ... olism.html
What products are produced in the liver; how are they transported to the cells?
Are the Final stages to produce each type of molecule always performed in the cell?

That's all for now.
Appreciate any comments on whatever.
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Postby JackBean » Fri Jan 01, 2010 9:26 pm

2. well, yes, basically, that's correct

3. in bacteria, there is only one replication start, but probably there are more polymerases in the cell.
In human, there are multiple replication starts on each chromosome, which are fired consequently (several of them at a time).

4. I'm not sure, if I understand your question correctly, but there are definitelly several copies of each enzyme/protein/molecule in each cell.

5. livers are said to be the factory of the body, there is produced almost everything, what is needed in the body ;)
http://www.biolib.cz/en/main/

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Re: Generation of DNA nucleotides

Postby gs99 » Sat Jan 02, 2010 1:16 pm

4. Evidently many steps are needed to make the nucleotide molecules.
Note: When I first asked this question, I thought the nt. molecules were made in the cytoplasm (see next item).
I'm thinking 1.5 billion molecules of each nt. are needed. When the nt. molecules are assembled into DNA they're handled individually. But it doesn't seem plausible that they're generated individually...
So I thought there must be ways of multiple concurrent production.
I envisioned many students with there own containers doing various steps.
Does this occur in the cytoplasm? How would the reactions be "protected" from each other?

5. It's reasonable that large volumes of stuff be made in the liver.
And obviously there are all kinds of reactions happening there!
But why can't I find web pages that clearly say this? I'm looking for articles that state:
a) Liver makes what products of interest to this subject.
b) They are transported to cells how (eg. red blood cells carry oxygen).
c) Cells receive products when ready to do replication.
d) Cells do something to the products if the products from liver are not what's needed by the polymerase to do replication.

There are many web pages that clearly describe the mechanics of gene expression (transcription, mRNA, translation). Pictures with stained components help explain mitosis. Where are there similar explanations of DNA replication and nucleotide generation?
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Postby JackBean » Sat Jan 02, 2010 6:32 pm

4. First of all, there are needed 3 billions of nts in EACH cell ;)
Second of all, as kolean wrote, I think, the cells in our body do not divide usually, so you do not have to generate 3 billion × 10^14 nucleotides ;)

5.
http://en.wikipedia.org/wiki/Liver#Physiology
d) if the cell needs 3 BILLIONS of nucleotides. Do you think, it really matter if it gets e.g. one guanine more or less?

I recommend to look for books Biochemistry by Voet & Voet or by Stryer, Tymoczko and someone... there you can find many pictures and probably also CDs with some animations ;)
http://www.biolib.cz/en/main/

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Re: Generation of DNA nucleotides

Postby gs99 » Sat Jan 02, 2010 10:52 pm

Yes there are many cells in my body that are NOT in the mood to divide today.
However, there are many times in the history of an organism when cells are prone to divide, correct?

And evidently, certain cells of the human do continue to divide:
"Other types of cells, such as epithelial cells, continue to divide throughout an organism's life and rarely enter G0."
Under heading "In relation to the cell cycle" at http://en.wikipedia.org/wiki/G0_phase

Obviously my question relates only to those cells that DO decide to activate Phase "Synthesis" in preparation for Mitosis.


4.2
I was thinking of the requirements of EACH cell that divides.
"two nucleotides on opposite complementary DNA or RNA strands that are connected via hydrogen bonds are called a base pair"
"The (human genome) is estimated to be about 3 billion base pairs long"
http://en.wikipedia.org/wiki/Base_pair

Doesn't that mean 6 billion nucleotides in the DNA are required for EACH dividing cell?
That comes to about 1.5 billion of each nt (A, T, C, G).
Am I looking at this incorrectly?


5.
The article you mention
http://en.wikipedia.org/wiki/Liver#Physiology
does not include the terms DNA, nucleotide, or purine.
I'm not sure how it helps at all.

A linked article mentions several proteins provided by the liver, but I don't see any devoted to exporting nt stuff.
http://en.wikipedia.org/wiki/Proteins_p ... _the_liver

I looked for book "Biochemistry" by Voet & Voet. Not in local libraries, including the Community College.
It is for sale online, so I'll need to start saving money for that.

Does anyone know if that book explains clearly in one place how:
a. the liver produces either nt molecules or things later used for nt construction
b. how those things are transported from liver to cells
c. what (if anything) the cell does to those things to enable DNA replication

There is a place for technical jargon; there is also a place for explanation in common language.
For example when writing computer programs, instructions are written in code structures of various languages (eg. Visual Basic).
It's considered a good professional thing for the programmer to insert comments WHY certain code was utilized.
The comments do not affect how the program executes, but are helpful to anyone reading the program to understand what's going on.

I'm just trying to understand "what's going on" to generate nucleotides for replication.

Thanks JackBean, for your help in this mystery.
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Postby kolean » Sun Jan 03, 2010 12:01 am

Wanted to add that I think the liver is for the salvage pathway, or the primary processing of ingested DNA/RNA. In your first link, the article said that all nt synthesis starts with PRPP that can be made from ingested nts.

You also can not 'see' nucleotides being processed by staining slides. The chromosomes that you can see during mitosis is when the linear chromosomes are all condensed into a thick rope, and the stain shows up nicely on slides. The other visual of the 'beads-on-a-string' DNA is thru the use of the electron microscope. Crystallography and looking at the sequence of the protein is how its structure is proposed.

This is a cool animation that I found last year:
http://www.hhmi.org/biointeractive/dna/ ... n_vo2.html

I can also see that the amount of nucleotides is mind bending for you. If you think about all the cells you ingest everyday, you can see why there would even be a surplus of nts in the fluid outside of the cells. In fact you can even get a metabolic disease from the processing of purine metabolism called Gout.
from the Wikipedia on Gout . . . .
Uric acid is a product of purine metabolism, and in humans is normally excreted in the urine. Purines are generated by the body via breakdown of cells in normal cellular turnover, and also are ingested as part of a normal diet. The kidneys are responsible for approximately two-thirds of uric acid excretion, with the liver responsible for the rest.

You also have to take into account that the nitrogenous bases that make up the nts, are also used for alot of metabolic pathways (especially ATP - the energy currency of the body/organism, and it can be converted into other NTPs (nucleotide tri phosphates). There is alot going on in a system that has many checks and balances to keep it at homeostasis, with many functions to perform.
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Postby JackBean » Sun Jan 03, 2010 10:25 am

Well, you already have one strain of DNA and replicates only the second one, so divide your number by 2 :wink:
Anyway, read, what kolean wrote. Also notice, that billion molecules is only 10^-15 mole, that is really, really, really just a little ;) Notice, that in EACH living cell is every minute produced RNA of at least several thousands if not millions of nts ;)

5. that should just show, that livers are really the factory of the body ;)

Well, you do not have any library around? You don't have to buy Voet just because of one problem ;)

Unfortunatelly, the nature did not provide any comments and that's why we study all these things ;)


Asking why in science is a little problematic. You may try to answer, maybe your answer may fit the reality a little, if you're happy, but may never be sure, what is really reason and what consequence ;)
http://www.biolib.cz/en/main/

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Postby gs99 » Sun Jan 03, 2010 8:41 pm

JackBean,
You said "Well, you already have one strain of DNA and replicates only the second one, so divide your number by 2"

Could you please clarify that?

In the newly-formed daughter cell from Mitosis, what is in the DNA?
One double helix strand of DNA.
3 billion base pairs in human genome, each pair having two nucleotide molecules.
That makes 6 billion nucleotides, give or take a guanine.
Is that correct?

The nucleotides can be A, T, C, or G.
Assuming random assignments, about 1.5 billion of each.
Note: The A count equals T count, and C count equals G count.
(A+T) is probably different than (C+G).
But my focus is not in exact accounting.
(Rumor has it that there is some "junk DNA" on chromosome 22 that has final subtotals of each nt type.)

To replicate the human DNA, how many "new" nucleotides are needed?
(This is part of the job's "bill of materials".)

What number do I divide by 2?
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