Good morning, everyone! It hasn’t been very long since you heard from me last, but I hope you all had a good night! Although I don’t normally get up at this hour on Thursdays, I have a test to study for, so I’m here to bring you the wonders of DNA replication. Today’s subject: prokaryotes!
We know that, when one cell splits into two, it has to copy its DNA so that each of the daughter cells will have a copy. We also know that the mechanism for this is a pretty straightforward one: the strands of the double helix are pulled apart, and each strand is used as a template to make its complementary strand. (This is called semiconservative, since each daughter copy has one old strand and one new one.)
High school biology students are also probably aware that DNA replication occurs at origins of replication, and replication forks move away from origins at both directions. In E. coli, the origin is OriC.
As mentioned in my post on transcription, unwinding the double helix creates tension (positive supercoiling) that can stop replication if it’s not alleviated. Thus, an enzyme called DNA gyrase (a type II topoisomerase) uses ATP to introduce negative supercoiling.
Other proteins are also important to the process of replication: helicase, an ATP-dependent enzyme, binds to a single-stranded region of DNA and then unwinds the rest of it by disrupting the hydrogen bonds between base pairs. SSBs, or single-stranded binding proteins, keep the single strands from reannealing before our polymerase can do its magic.
And, speaking of the polymerase, let’s introduce our main player! The main part of replication is carried out by an enzyme (or, I guess, a class of enzymes) called DNA polymerase. These use the single-stranded template of DNA to synthesize a complementary strand by assembling dNTPs in the proper order.
However, there’s a problem with this enzyme: it can only add nucleotides in a 5′ to 3′ direction. That’s no problem for the 3′-5′ strand, whose complementary strand (the “leading” strand) runs in that direction, but a little bit of a problem arises when dealing with the opposite template. In fact, the best our DNA polymerases can do is bend the 5′-3′ strand around and replicate it in small (1000-2000 bp) backwards. This leads to the creation of fragments called Okazaki fragments, which are then sealed together in the final product to form a whole “lagging” strand.
Another limitation of DNA polymerases is that they can’t start synthesizing a complementary strand from scratch. This is easily alleviated, though, by an enzyme called primase, an enzyme that synthesizes short stretches of RNA called “primers.” Primers give the DNA polymerase a starting point, and they’re replaced with DNA in the final product.
Now, you’re noticing that I’m referring to polymerases in the plural, implying that we don’t have just one. That’s true for both prokaryotes and eukaryotes (unlike RNA polymerase, which is the only prokaryotic RNA polymerase). To keep things a little ([coughs up a lung] A LOT) simpler, we’re going to start by looking at just prokaryotic replication, and there’s no better place to start than the polymerases.
Prokaryotes have five DNA polymerases that are designated with numerals I through V. I, II and V mostly just deal with DNA repair, so we won’t talk about them too much. Wikipedia describes IV as “an error-prone polymerase involved in non-targeted mutagenesis.” III is the one that carries out most DNA replication; it’s not very abundant in cells, but it’s extremely processive (good at hanging on to the DNA template) in comparison to the others.
DNA polymerase III holoenzyme is composed of seventeen different subunits, each of which confers it some sort of functionality. The most noteworthy of these is the gamma-complex, which acts as a “clamp loader” by helping clamp the beta-dimer rings of the enzyme (which help keep it on the DNA) to the strand using ATP.
Now, once we actually get our polymerase III going and making DNA, DNA polymerase I gets its time to shine. This enzyme carries out the very important job of replacing the RNA primers with DNA. It’s also special because, in addition to being able to do this, is can remove nucleotides from a DNA strand in both the 3′ to 5′ and 5′ to 3′ direction.
The 3′ to 5′ exonuclease activity is useful because it means that this enzyme can quite literally check its work as it goes. If it discovers that it’s made a mistake, all it has to do is back up and chew the incorrect base off of the growing strand. Pretty nifty, if you think about it.
After polymerase I does its thing, an enzyme called ligase comes in an seals the nicks in the backbone left behind by all of this priming and Okizaki fragment-forming nonsense.
Finally, we need to terminate our DNA replication. In prokaryotes, this isn’t too difficult: it just requires the Ter sequence (GTGTGTTGT). Tus protein, a contrahelicase, binds here and basically just goes, “Nope nope nope, no helicase is passing through here!” The replication forks end, and replication is terminated.
See, not too bad, huh? Now we get to the really fun stuff—those stupid, show-off eukaryotes!
See a lot of errors? Feel the need to point them out? Please have mercy—I’m posting this without proofing it, because I’m kinda in a hurry here.