Posts Tagged ‘replication’
a DNA dialogue 6: Okazaki fragments, as promised
Canto: Okay, so first off, why are Okazaki fragments so called?
Jacinta: Well as anyone would guess, they’re named after someone Japanese, in this case two, the husband and wife team Reiji and Tsuneko Okazaki, who discovered these short, discontinuously synthesised stretches of DNA nucleotides in the 1960s.
Canto: Yes their story is intriguing – tragic but also inspiring. Reiji, the husband, was born in Hiroshima and died in 1975 from leukaemia, related to the 1945 A-bomb. He was only 44. Tsuneko Okazaki continued their research and went on to make many other contributions to genetics and molecular biology, as a professor, teacher, mentor and director of scientific institutes. Her achievements would surely make her a Nobel candidate, and she’s still alive, so maybe…
Jacinta: Now the key to Okazaki fragments is this lagging strand. Its directionality means that the DNA primase, followed by the DNA polymerase, must work ‘backwards’, away from the replication fork, to add nucleotides. This means that that they have to have periodic breaks – but I’m not sure exactly why – in creating this lagging strand. So the entire replication process is described as semi-discontinuous because of this fundamental difference between the continuously created leading strand and the stop-start ‘fragmentary’ (at least briefly) lagging strand.
Canto: But we need to know why this ‘backward’ movement has to be stop-start, and I’d also like to know more about this primase and polymerase, thank you.
Jacinta: Well the Okazakis and their team discovered this semi-discontinuous replication process in studying the replication of good old Escherichia coli, the go-to research bacterium, and it was a surprise at the time. Now, I’m looking at the explanation for this necessarily discontinuous process in Wikipedia, and I confess I don’t really understand it, but I’ll give it a go. Apparently the Okazakis ‘suggested that there is no found mechanism that showed continuous replication in the 3′ to 5′ direction, only 5′ to 3′ using DNA polymerase, a replication enzyme’, to quote from Wikipedia. So they were rather cleverly hypothesising that there must be another mechanism for the 3′ to 5′ lagging strand, which must be discontinuous.
Canto: And another way of saying that, is that the process must be fragmentary. And they used experiments to test this hypothesis?
Jacinta: Correct, and I won’t go into the process of testing, as if I could. It involved pulse-labelling. Don’t ask, but it has something to do with radioactivity. Anyway, the test was successful, and was supported by the discovery shortly afterwards of polynucleotide ligase, the enzyme that stitches these fragments together. Now, you want to know more about primase, polymerase, and now ligase no doubt. So here’s a bit of the low-down. DNA primase is, to confuse you, an RNA polymerase, which synthesises RNA from a DNA template. It’s a catalyst in the synthesis of a short RNA segment, known as a primer. It’s extremely important in DNA replication, because no polymerase (and you know how polymerase keeps getting associated with primase) can make anything happen without an RNA (or DNA) primer.
Canto: But why? This is getting so complicated.
Jacinta: I assure you, we’ve barely scratched the surface….
Canto: Well, Socrates was right – there’s an essential wisdom in being aware of how ignorant you are. We’ll battle on in our small way.
a DNA dialogue 5: a first look at DNA replication
Jacinta: So let’s scratch some more of the surface of the subject of DNA and genetics. A useful datum to remember, the human genome consists of more than 3 billion DNA bases. We were talking last time about pyrimidines and purines, and base pairs. Let’s talk now about how DNA unzips.
Canto: Well the base pairs are connected by hydrogen bonds, and the two DNA strands, the backbones of the molecule, run in opposite, or anti-parallel, directions, from the 5′ (five prime) end to the 3′ (three prime) end. So, while one strand runs from 5′ to 3′ (the sense strand), the other runs 3′ to 5′ (the antisense strand).
Jacinta: Right, so what we’re talking about here is DNA replication, which involves breaking those hydrogen bonds, among other things.
Canto: Yes, so that backbone, or double backbone whatever, where the strands run anti-parallel, is a phosphate-sugar construction, and the sugar is deoxyribose, a five-carbon sugar. This sugar is oriented in one strand from 5′ to 3′, that’s to say the 5′ carbon connects to a phosphate group at one end, while the 3′ carbon connects to a phosphate group at the other end, while in the other strand the sugar is oriented in the opposite direction.
Jacinta: Yes, and this is essential for replication. The protein called DNA polymerase should be introduced here, with thanks to Khan Academy. It adds nucleotides to the 3′ end to grow a DNA strand…
Canto: Yes, but I think that’s part of the zipping process rather than the unzipping… it’s all very complicated but we need to keep working on it…
Jacinta: Yes, according to Khan Academy, the first step in this replication is to unwind the tightly wound double helix, which occurs through the action of an enzyme called topoisomerase. We could probably do a heap of posts on each of these enzymes, and then some. Anyway, to over-simplify, topoisomerase acts on the DNA such that the hydrogen bonds between the nitrogenous bases can be broken by another enzyme called helicase.
Canto: And that’s when we get to add nucleotides. So we have the two split strands, one of which is a 3′ strand, now called the leading strand, the other a 5′ strand, called the lagging strand. Don’t ask.
Jacinta: The leading strand is the one you add nucleotides to, creating another strand going in the 5′ to 3′ direction. This apparently requires an RNA primer. Don’t ask. DNA primase provides this RNA primer, and once this has occurred, DNA polymerase can start adding nucleotides to the 3′ end, following the open zipper, so to speak.
Canto: The lagging strand is a bit more complex though, as you apparently can’t add nucleotides in that other direction, the 5′ direction, not with any polymerase no how. So, according to Khan, ‘biology’ adds primers (don’t ask) made up of several RNA nucleotides.
Jacinta: Again, according to Khan, the DNA primase, which works along the single strand, is responsible for adding these primers to the lagging strand so that the polymerase can work ‘backwards’ along that strand, adding nucleotides in the right, 3′, direction. So it’s called the lagging strand because it has to work through this more long, drawn-out process.
Canto: Yes, and apparently, this means that you have all these fragments of DNA, called Okazaki fragments. I’m not sure how that works…
Jacinta: Let’s devote our next post on this subject entirely to Okazaki fragments. That could clarify a lot. Or not.
Canto: Okay, let’s. Goody goody gumdrops. In any case, these fragments can be kind of sewn together using DNA ligase, presumably another miraculous enzyme. And the RNA becomes DNA. Don’t ask. I’m sure all will be revealed with further research and investigation.
References
Leading and lagging strands in DNA replication (Khan Academy video)