When DNA letters get married, who pays for the wedding? Linking DNA letters (deoxynucleotides, dTs) together is energetically expensive. When DNA Polymerases (DNA Pols) link dTs together (based on a template strand), the incoming letter “pays” but when DNA ligases stitch together strands that have been broken post-payment, it relies on “philanthropic donations” –  external energy has to be provided, typically in the form of ATP.  

note: updated & video added 11/14/21

Marriage comes with sacrifices, even when you’re a molecule and “marriage” involves sharing pairs of electrons to form strong covalent bonds. No longer can they just go about doing their own thing, traveling on a whim & rocking the single life. Instead, they’re tied down and can only move with their extended family. So it better be worth it. And paying a sort of dowry in the form of energy money is a way to make the marriage worthwhile. 

The reason for the high price is a concept called entropy, which is a physics word for randomness or disorder – the more freedom a molecule has to move, the higher its entropy. And the universe likes entropy – in fact the second law of thermodynamics is that the entropy is always increasing. So in order to bring some order to things that want to be disordered, you have to invest energy. 

Sometimes the invested energy comes from a lot of little gains, like binding energies that come from when molecules stick to something that offers attractive interactions that allow it to “relax” a little. Doing a wall sit requires some serious energy, but if you slip a stool under there to sit on you’ve suddenly freed up that energy to do other things (like explain to your coach how the stool just magically got there and you weren’t trying to cheat…) Similarly, each weak interaction a molecule makes with another molecule that has charge, shape, etc. that it likes, releases a little bit of energy that can be used to counter the entropic urge. 

But sometimes those little bursts aren’t enough. Sometimes you’ve gotta use some hard “energy cash.” When it comes to energy money, the typical cellular currency is ATP (Adenosine TriPhosphate) which is actually one of the RNA letters (nucleotides). Through processes like cellular respiration, cells are able to break down (catabolize) molecules like sugars into smaller, recyclable, parts. This is kinda like the opposite of the don’t-tie-me-down problem, you’re increasing entropy when you break apart the molecules, so you’re doing entropically-favorable things, and generating energy that gets “stored” in the form of ATP.

It’s useful to have that one universal currency so all sorts of molecules can evolve to use that one “coin” instead of having to be able to accept all different “currencies” – ATP is kinda like an arcade token in this sense. But the reason ATP “stores energy” is because of the TP (triphosphate) part, not the A, so any nucleotide triphosphate (NTP) (RNA letter) or deoxynucleotide triphosphate (dNTP) (DNA letter) can provide energy if the molecules can “take that coin.” 

quick note on nucleotide nomenclature. I often use the term “nucleotide” to refer to nucleotides (RNA letters) and deoxynucleotides (DNA letters). They’re really similar – both have a sugar (ribose in the case of RNA and deoxyribose in the case of DNA) sugar & phosphate(s) foring agaric generic “backbone” part & then each letter has a unique “nitrogenous base” (“base”) which has 1 ring (the pyrimidines C & T (U instead of T in RNA)) or 2 rings (the purines A & G). The bases stick out from the backbone and allow for specific, letter-to-letter interactions. The letters A and T (or U) and C & G complement each other – they can form weak bonds that can hold complementary DNA strands together through base pairing. 

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) &, finally, the “left leg” (3’ position) has a hydroxyl (-OH) group.

But, as I was saying, it’s the phosphates that matter energy-wise… A phosphate (PO₄³⁻) is a phosphorus (P) atom, surrounded by 4 oxygens (O) atoms. It’s negatively charged, and like charges repel each other, so sticking three of them in a row like you have in a triphosphate is like clamping a stiff spring – breaking them up is like unclamping the spring, releasing the potential energy that what held in these “high-energy” bonds to be used for things like paying the linkage cost. 

“High energy” refers to them having high chemical potential energy (like you have high kinetic potential energy (energy to move) when you’re at the top of a roller coaster. And just like it takes work to pull a roller coaster car up to the top of the tracks, it takes effort (in the form of energy) to bring & hold phosphates  together (like compressing a stiff spring). The more phosphates in a row, the more potential, (like being at the top of a higher roller coaster) so, energy-wise, dNTP > dNDP (diphosphate) > dNMP (monophosphate).

So, free nucleotides that come to DNA Pol come with money in hand – as triphosphates –  in addition to their deoxyribose sugar (with its 3’ hydroxyl (-OH) group and their nitrogenous base (“base”)(A, T, G, or C), they have 3 phosphate groups (at their 5’ position) – so we call them dATP (deadenosine triphosphate), dCTP, dGTP, & dTTP. 

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). And DNA-DNA polymerases can travel along DNA and use 1 DNA strand as a template for making a second strand.

I like to think of DNA Pol kinda like a train that’s running on a “half track” (single-stranded DNA) and laying down the other half of the track (complementary strand) ahead of it as it goes, one piece at a time – encounter an A, lay down a T, see a G, better put in a C. Sound easy?

Well, not so fast… It’s gotta “get paid” and this imposes some limitations. You see, even if Pol “knows” what letter to lay down (because the bases are specifically attracted to each other thanks to their complementary chemical structures) actually getting them to link together through a phosphodiester bond is “hard” because it involves a large decrease in entropy. Those base-base attractions which are the basis of knowing what to add aren’t enough to keep it tied down (at which point it still has to get linked to its neighbor)

So, while enzymes (reaction mediator/speed uppers) like DNA Pol are able to help out by holding the letters together in optimal positions to react, stabilizing reaction intermediates, providing a friendly environment, etc, the reaction still needs “money” and those little bursts of binding energy released as the letter cozies up, aren’t enough. Thankfully, those letters DNA Pol is adding come with money in (left) hand! 

If you look at a phosphodiester bond, you’ll see that there’s only one phosphate in between the sugars in the backbone. This is because, although the letters come to DNA Pol in their triphosphate form, when DNA Pol links them together it kicks 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, decreasing it’s entropy, you get a large entropy increase from splitting up those “high energy” phosphates. 

DNA Pol helps hold an incoming nucleotide (of the triphosphate variety) close to the growing chain it needs to be added to & in the right position. The 3’ hydroxyl (OH) group then goes in for the attack! It latches on to the 1st phosphorus (P) group of the incoming nucleotide -> that P now has too many bonds, so it kicks out the other 2 phosphate groups as the inorganic phosphate molecule pyrophosphate (PPi) (energy boost 1). Pyrophosphate is then hydrolyzed (broken by the addition of water) into 2 molecules of orthophosphate (Pi) (energy boost 2). And it uses magnesium (Mg²⁺) to help stabilize all the negatively-charged groups and keep the strands from repelling each other. The Mg²⁺ also helps pull that H away from the O in the hydroxyl to make it more attack-y. So the Mg²⁺ is really important and it’s included in PCR reactions (where we copy DNA in a test tube). more here: http://bit.ly/PCRmovie 

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). Pasting it back together will still require energy, but now you no longer have that phosphate money there (it’s already been spent), so you have to provide it using external energy. 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. Much more here: https://bit.ly/tsunekookazaki

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. Molecules are made up of atoms (individual carbons, hydrogens, etc.) and atoms are made up of smaller parts called protons (which are positively-charged) and neutrons (neutral) that hang out together in a dense central nucleus and are surrounded by a “cloud” of negatively-charged electrons they interact with other atoms through. The # of protons defines an element (e.g. carbon always as 6 and hydrogen always has 1). But the # of electrons can change (which is how you get charged particles (ions) & the # of neutrons can also change (which is how you can get radioactive isotopes). 

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⁻)

It’s a match made in o-chem heaven – nucleophiles can share an e⁻ pair with an electrophile to form a new covalent bond. more here http://bit.ly/nucleophilefileshttp://bit.ly/sn1vssn2 

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). Even though phosphate is negative overall, 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, to summarize: 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, uniting an end with a 3’ OH and an end with a 5’ triphosphate. Ligases “just” connect DNA that’s already been written, uniting an end with a 3’ OH and an end with a 5’ monophosphate. And, while it sounds like ligase has an easier job since it just has to stitch together and not write, it requires external energy to pay for it, whereas DNA Pols get energy from the incoming letters. 

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0

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