Enzymes Involved in DNA Replication and Repair

This post should have a lot more new stuff in it, I hope. Or maybe I don't, because that involves more typing and therefore more thinking and more work...

To be familiar with the major enzymes involved in DNA replication in E. coli.

From my previous posts, you should have a good handle on how DNA replication works, so I won't go over that again. Instead, I'm just going to list off a bunch of enzymes and how they aid in this process:

• Helicase separates the DNA strands so that DNA Polymerase can get in and start replicating.
• Topoisomerase I (a.k.a. gyrase) stops DNA from overwinding by creating and resealing single-stranded nicks. (There's also a topoisomerase II, which can create double-stranded nicks. Should be easy enough to remember- topoisomerase I nicks one strand, topoisomerase II nicks two.)
• Primase creates short RNA primers. DNA Polymerase cannot do its job without these primers.
• DNA Polymerase does the actual replication stuff. Specifically, DNA Polymerase III is the one that does the chromosomal replication in E. coli.
• RNase H removes the primers created by primase.
• DNA Ligase seals off gaps in the lagging strand.
• A clamp loader uses ATP to load a beta clamp onto the DNA. The beta clamp holds the DNA polymerase in place so that it won't have to dissociate and reassociate between bases (dissociative synthesis); rather, it can stay on and do its job smoothly (processive synthesis). (Processive synthesis is MUCH more efficient and rapid than dissociative synthesis.)

To understand the polymerase and exonuclease activities of the different DNA polymerases.

E. coli has five different DNA polymerases:

1. DNA Polymerase I is pretty versatile, but it's mainly used for repairing DNA and processing Okazaki fragments. It has three sites: a 5' -> 3' polymerase site, which is where most of the copying and stuff takes place; a 3' -> 5' exonuclease site, which can remove the last base that was added if there's a problem with it; and a 5' -> 3' exonuclease site, which can remove stuff in front of it and is particularly useful for removing RNA primers. The 5' -> 3' exonuclease site is unique to DNA Polymerase I.
2. DNA Polymerase II also has roles in DNA repair.
3. DNA Polymerase III is the one that replicates the chromosomal DNA.
4. 5. DNA Polymerase IV and V are the only two that do not have the 3' -> 5' exonuclease site, and therefore lack proofreading function. They are instead "translesion" DNA polymerases that can replicate through damaged DNA.

Since the 5' -> 3' site on DNA Polymerase I is pretty unique, I'll just talk about it a bit more. As DNA Polymerase I moves along the lagging strand, eventually it reaches a one-base gap and then the RNA primer of the next Okazaki fragment. This one-base gap is called a nick. DNA Polymerase I can simultaneously remove bases ahead of the nick while adding new bases. This process is known as "nick translation." A nick is left at the end, which can be sealed with DNA ligase.

Also, just a quick note on the DNA polymerase reaction itself. Even though there's only one phosphate group per base in DNA, deoxynucleotriphosphates (dNTPs) are used for the synthesis reaction. A new base is added from the previous 3'-OH nucleophilically attacking (I think that's what it's called...) the second phosphate group on the dNTP. This releases pyrophosphate. Pyrophosphate is then broken down by pyrophosphatase, so that the DNA polymerase reaction cannot be reversed. DNA polymerase also has two Mg2+ ions, attached to highly conserved aspartic acid residues, that help to facilitate this process. One Mg2+ ion deprotonates the 3'-OH group, making it more nucleophilic. The other Mg2+ ion facilitates the departure of the pyrophosphate product. (The magnesium ions also have some roles in the 3' -> 5' exonuclease activity, but apparently we don't get to cover that yet. Or something.)

To understand how the DNA polymerases replicate DNA with high fidelity.
To be familiar with the different steps in DNA replication that contribute to the overall accuracy of the process.

As mentioned before, DNA polymerase has proofreading functions, eliminating most errors quite quickly. The structure of DNA polymerase also helps to ensure accurate replication. DNA polymerase is shaped like a three-fingered "hand" which closes as bases are added. If an incorrect base is added, the "hand" cannot close as well, allowing the base more time to dissociate. In the event that a base is added, the next base is even slower to associate. The previous mismatched pair is then repositioned into the 3' -> 5' exonuclease site, and the incorrect base is removed.

After replication has occurred, there are still ways of detecting and patching up errors. An incorrect base pairing will cause the width of the helix to change at that point. This can be detected by a protein called MutS. MutS can also recognise the parent strand from its methyl groups: you see, a newly created strand will not have any methyl groups, but will gain them over time. Two more proteins then become involved: a protein called MutL recruits another one called MutH, which nicks the DNA strand near the mutation. Exonucleases then remove a bit more DNA, including the site of the mutation. DNA Polymerase III and DNA Ligase then come in to fix things up.

To be aware that the use of DNA polymerases in the laboratory has been fundamental to the development of Recombinant DNA Technology. 

This is something that I'm probably going to end up covering in the next few posts on BIOC2001. PCR is a good example: see an earlier post of mine on this topic.