If you want to join a DNA strand, you’ll need to pay a fee 💵 👉energy provided in the form of d-something-TP 😬 If you can’t afford it, there is another way 😅 DNA LIGASE provides ATP to pay 🤗
Yesterday we looked at the Meselson-Stahl experiment and how it answered the question: when double-stranded DNA gets copied, who gets to keep the originals? Answer – each copy gets an original of 1 strand (semi-conservative replication). More here: http://bit.ly/2kMWca1
But there were still a lot of questions – like how does DNA get copied? It involves “paragraph writers” (DNA polymerases) and paragraph joiners (DNA ligases). First, the gist, then we’ll look in more detail 👉 It’s energetically costly to link DNA letters together – DNA Polymerases (DNA Pol) writes DNA chains by connecting letters that come with money in (left) hand, whereas DNA LIGASES connect DNA chains that have already been written but got separated. Since they’ve already been written, they’ve already given up their money so they need someone to pay for them -> Ligases require external energy (usually from ATP). Rambly detail time… (& be sure to look at pics! they should 🤞help)👇
DNA is the biochemical “language” genetic information is written in & it’s made up of long chains of “letters” called NUCLEOTIDES (nt). There are 4 DNA nt -> A, T, C, & G & they’re made up of 3 main parts – a deoxyribose sugar & phosphate(s) form the generic “backbone” part & then each letter has a unique “nitrogenous base” (“base”) which has 1 ring (the pyrimidines C & T) or 2 rings (the purines A & G).
I like to picture them as tiny little cartoons where the sugar’s 5-sided ring forms the core body & various groups stick off of its arms & legs. The “right arm” (as in the right of your screen/paper) is the “1’” position (the ‘ is pronounced “prime”) & this is where the base attaches.
The “left arm” (5’ position) is where the phosphate(s) link on. The 5’ position is actually more like an elbow because there’s a “linker” from the 4’ “shoulder”
The “right leg” (2’ position) is where DNA & RNA differ. DNA just has a hydrogen (-H) here but RNA has a hydroxyl -OH, hence the D for Deoxyribose in DNA (the NA stands for Nucleic Acid). (The other difference between DNA & RNA is that RNA has a “U” instead of a T)
&, finally, the “left leg” (3’ position) has a hydroxyl (-OH) group.
Nucleotides link together left arm (5’ phosphate) to left leg (3’ OH) through PHOSPHODIESTER BONDS. You can link up as many as you want to get a chain, one end of which will have a free 5’ phosphate (the 5’ end) & the other end of which will have a free 3’ hydroxyl (the 3’ end).
This linkage is energetically costly because it involves a large decrease in entropy – entropy is a physics word for randomness or disorder & the universe likes it – molecules like to be free to move around (high entropy) & don’t like to be held down (low entropy).
So these linkage reactions require “help” in the form of energy (from “nTPs”) & enzymes (protein or RNA molecular reaction speeder-uppers) to act as a sort of “mediator” holding reactants together in optimal positions to react, stabilizing reaction intermediates & providing a friendly environment
There are 2 main types of DNA linkers -> DNA Polymerases (DNA Pols) & DNA Ligases. Pols “write” DNA by connecting the nucleic acids as they go, whereas ligases just connect DNA that’s already been written.
DNA Pol adds on nucleotides that come with money in (left) hand. Free nucleotides that come to join come as TRIPHOSPHATES -> they have 3 phosphate groups – so we call them dATP (deadenosine triphosphate), dCTP, dGTP, & dTTP
These triphosphates are like energy money because they’re like compressed springs -> PHOSPHATE (PO₄³⁻) has a central phosphorous(P) atom connected to 4 oxygen(O) atoms. It has “extra” electrons (e⁻) so it’s ➖ charged⚡️
Like charges repel, so phosphates don’t like to be next to each other -> Bonds between the phosphates are considered “high energy” meaning they have ⬆️ CHEMICAL POTENTIAL ENERGY 🎢 👉 takes effort (in the form of energy (E)) to bring & hold them together (like compressing a spring) 😓 👉 when they’re broken apart that E’s freed to be used for other things like paying cost of linking nucleotides together 👍
The more phosphates in a row, the more potential E 👉 ATP > adenosine DIphosphate(ADP) > adenosine MONOphosphate(AMP)
When DNA Pols link together nucleotide triphosphates (NTPs), they kick off 2 phosphates as a molecule of pyrophosphate (PPi). This PPi is then hydrolyzed (split by water) with the help of pyrophosphatase to give you 2 individual orthophosphates (Pi) 👉 so even though you tied down the DNA, you get a large entropy increase from splitting up those “high energy” phosphates.
The loss of these phosphates has another consequence – the DNA letters are only bridged by a single phosphate group – so if the chain gets broken, there will only be 1 phosphate left. Such breakage can happen “accidentally” from things like UV light or “purposefully” like if a cell “erases” DNA letters to fix them or if we add restriction endonucleases (REases) to DNA in a test tube to cut it so we can “paste it” somewhere else) see yesterday’s post 👉http://bit.ly/2TFaLMT
Pasting it back together will still require energy, but now you no longer have that phosphate money there, so you have to provide it. DNA Pol can’t do this, so you need a different helper – a DNA LIGASE.
Even if everything goes right, no typos, UV damage, or anything, your cells still need ligases because DNA Pol has a couple restrictions 👉 it can only write one way & it can’t start from nothing – it has to be “primed” – it needs a 3’ OH to add onto
This leads to a “lagging strand” problem when replicating DNA – DNA Pol can only copy in 1 direction but the 2 strands in double-stranded DNA are antiparallel – meaning one goes 5’->3’ & the other 3’->5’. So one strand (the lagging strand) has to be written in pieces called Okazaki fragments that are then stitched together by ligases
Wouldn’t those fragments have triphosphates at their 5’ end though? They would, except that’s where that 2nd “limitation” comes in -> because DNA Pol has to be primed, the fragments “start” with RNA primers put on by primase that get chewed off, leaving a monophosphate when they do
The ligase reaction occurs by a 3-step process involving nucleophilic substitutions 👉 NUCLEOPHILES (Nü) are molecules that have “extra electrons (e⁻)” which gives them more negativity than they can handle 👉 they “love nuclei” because opposites attract & that’s where the positive protons are 😍
ELECTROPHILES are also “unhappy” with the amount of e⁻ they have😒 BUT they want more, more more! (they “love” e⁻) 😍
In the 1st step, the nucleophile is the end (terminal) amino of a lysine (one of protein’s amino acid letters) in the ligase’s active site & the electrophile is the phosphorus in ATP’s 1st (α) phosphate (the one closest to the sugar). Because this phosphorous (P) is surrounded by electronegative (electron-hogging) oxygens, it’s partly positive & thus electrophilic.
So the lysine goes on the nucleophilic attack -> it grabs onto that α P & kicks off the other 2 phosphates as inorganic pyrophosphate (PPi), which can get hydrolyzed to 2 Pi to give you a further entropic benefit (as is the case with the DNA Pol reaction)
So, at this point, you have AMP stuck to the ligase (a covalent enzyme-adenylate intermediate)
Next, you have another nucleophilic attack – this time from the free 5’ end of the DNA. It takes that AMP from the lysine
Then the free 3’ end gets jealous – it wants in on the fun too, so it attacks the phosphate, kicking off the AMP & forming a phosphodiester bond.
So, I may wear the cape, but DNA LIGASE is the real superhero of this story!