Thread: DNA synthesis - lagging strand

I have a question regarding the DNA synthesis that takes place on the lagging strand of a DNA molecule.

I'm aware that replication on the lagging strand is discontinuous due to the anti-parallel nature of DNA's structure, and that primase lays down RNA primers, to whose free 3' DNA polymerase adds nucleotides in the direction opposite of the DNA polymerase on the leading strand.

However, what I fail to understand is how the very first RNA primer on the lagging strand "works", for lack of a better word. How does primase know where to place the primer? Also, more importantly, how does DNA polymerase know when to stop extending the nucleic acid chain, since there are no RNA primers ahead of it to "bump" into? Where does it stop?

Also, consequently, I know that in a replication "bubble" there are two replication forks, and that on each strand, one end acts as a lagging strand and the other as a leading strand. How does this work? How are the strands that are being separately and in different ways later attached; that is, where does each one stop being replicated?

Any help is greatly appreciated, as I have a test on this material on Friday. Many thanks in advance!

Dear Pupate,

Sorry I will have been late with regards your test. However...

Firstly, in terms of how the primase "knows" where to place the primer: DNA contains specific sites for the origin of replication. Such DNA sequences normally contain, i) a binding site for the Origin Recognition Complex (ORC), which is essentially a large initiator protein; ii) an AT-rich stretch of DNA (A-T base pairs share only 2 H-bonds, so the sequence will be relatively 'easy' for the helicase to open); iii) a binding site for proteins that help to attract ORC to the origin DNA.

Initiator proteins bind at this site -> attracts DNA helicase -> helicase unwinds DNA, thus exposing sufficient ssDNA for primase to synthesise RNA primer. Ergo, the site at which the primase commences is determined ultimately by the origin of replication sequence in the DNA. However, I very well may have misinterpreted this, and would strongly advise you to check with somebody else.

Secondly. There are RNA primers ahead of the DNA polymerase to "bump into". The synthesis of each Okazaki fragment ends when the DNA polymerase encounters the RNA primer attached to the 5' end of the previous fragment.

Thirdly - I think that what happens as the two replication forks approach each other is not v. well understood. However, it is known that the replication machines are disassembled during the process.

I hope that this helps,

Best wishes,

Tridimity 8)

Ref. Alberts et al 'Molecular Biology of the Cell' 5th Ed. (2008) p. 266-288

I see some hard core biology goin on here, but I think you are too plate, tridimity. Haha! I have another question tho now, when helicase 'unwinds' dna, does it start rotating? And in which direction does it rotate, clockwise to 5' or to 3' ?

Originally Posted by Mark Ian

I see some hard core biology goin on here, but I think you are too plate, tridimity. Haha! I have another question tho now, when helicase 'unwinds' dna, does it start rotating? And in which direction does it rotate, clockwise to 5' or to 3' ?

I don't know what direction the rotation occurs, but it does occur ahead of the replicating fork. An enzyme called topoisomerase cuts and repairs the DNA backbone to unwind knots that form and relieve torque generated by the unwinding. I like to think of it as twisting and untwisting rather than in terms of rotation though lol.

Edit: Also, DNA normally exists in sort of a twisty glob, so during replication there are a lot of proteins involved in keeping the parts being replicated from going back into tertiary structures that would make unwinding difficult.

The video below is informative:

http://www.dnatube.com/video/1216/Me...imase-Helicase

cool video! I had no idea that topoisomerase existed, thanks!