Sunday, August 3, 2014

Science Sunday: DNA Replication (Part 3)--Enzymes

Posted by Rebekah

 Please click here for “DNA Replication (Part 2)

Note: this post describes DNA replication as understood today.

While reading this post, it is important to note that DNA replication in eukaryotes is somewhat different than in prokaryotes (although they do have a lot in common). However, as I mentioned in my last post (yes, it’s been a long time), much of the knowledge we have concerning DNA comes from research involving bacteria. As a result, Lord willing I will be concentrating on DNA replication in E. coli.
OriC Up Close

Origins of DNA Replication

Origins of DNA replication are crucial in that they attract replication enzymes.

But how do they work?

In E. coli, the origin of replication (which is known as oriC) is characterized by an adenine and thymine rich sequence. A-T bonds require less energy to denature than G-C bonds; hence, making it a logical design. This sequence is approximately 245 base pairs (bp) long, and is partitioned by three 13-bp sequences which are then followed by four 9-bp sequences (these are called13-mer and 9-mer sequences respectively).

Replication Enzymes

So, how does it all begin?

An enzyme called DnaA attaches to the 9-mer repeats; The DNA then bends, and the AT-rich 13-mer repeats hydrolyze (break). Thus, resulting in an open complex where the double stranded DNA has begun separating.   

And this is where the drawing I left in my last post comes in handy…

An enzyme called DnaC (not pictured) caries another enzyme called DnaB to oriC. DnaB is a kind of helicase protein, which separates the two complementary stands of DNA by hydrolyzing the hydrogen bonds connecting complementary nucleotides.

This to me (yes, this is my opinion here), reminds me of a zipper: helicase is analogous to a slider and the complementary strands are analogous to the two chains of teeth (See here to learn about the structure of a zipper).

Now, in order to keep the two strands from reannealing (joining again), proteins called single stranded binding proteins (SSB) attach to the unwound strands of DNA.

Unfortunately, all this unwinding causes torsional strain on the DNA; which in turn can lead to supercoiling (kind of like a rubber band when twisted too much). Not surprisingly then, there is another enzyme that relieves this strain—topoisomerase. Topoisomerases do so by catalyzing the cutting and rejoining of the “supercoiled” DNA; hence, causing the DNA to rotate and remove the coil.

But which enzyme is responsible for the synthesis of new DNA daughter strands?

The DNA polymerase III (pol III) holoenzyme!

Note: holoenzymes are enzymes with lots of proteins (as well as other compounds) that help it (the enzyme) do its job.

However, in order to begin work, DNA polymerase needs a 3′-hydroxyl (-OH) group (Lord willing I’ll try covering DNA’s molecular structure later). In order to fix this problem, DnaA, several proteins, and an enzyme called primase unite at oriC and form a complex called the primosome. Primase then synthesizes an RNA primer that provides the very much needed 3′-hydroxyl group. Simply put, an RNA primer is a short stretch of RNA (somewhere between 12 and 24 nucleotides long), and “RNA” (ribonucleic acid) is a lot like DNA, but it uses the nucleotide uracil (U) instead of thymine (T). (Note that there are also several other differences between DNA and RNA not mentioned here).

But how are these RNA primers turned into DNA?

Well, pol III finishes its job once it runs into the RNA primer; subsequently leaving a single-stranded gap between the last DNA nucleotide (of the new daughter strand) and the first RNA nucleotide of the primer. In turn, DNA polymerase III is replaced by an enzyme called DNA polymerase I (pol I), which is attracted to the DNA-RNA single-stranded gap.  DNA polymerase I is special in that it is capable of exonuclease activity. This means it can remove the RNA nucleotides one at a time. Note that, as DNA pol I removes the RNA nucleotides, it replaces them with the necessary DNA nucleotides. All this is done in the 5’ to 3’ direction.

Once the primer has been completely removed, however, there remains a DNA-DNA single-stranded gap. In order to close this gap, an enzyme called DNA ligase steps in, and forms the phosphodiester bond necessary to close this gap.

The Replisome
We have now learned about many of the enzymes associated with DNA replication in E. coli; nonetheless, it would be erroneous to assume that these enzymes act independently from each other. In fact, research now indicates that these proteins and enzymes are all part of larger protein complexes called replisomes.

To be continued… 

Reece, Jane B., et al. Campbell Biology. 9th Global Edition. “Many Proteins Work Together in DNA Replication and Repair.” Boston: Pearson, 2011. 357-365. Print.
Sanders, Mark F., and John L. Bowman. Genetic Analysis: An Integrated Approach. 1st ed. N.p.: Benjamin Cummings, 2012. Print.

WARNING: Due to several reasons, I do NOT recommend Campbell Biology for your homeschool. However, due largely impart to its prevalent use in colleges and universities (and even Wikipedia), I chose to use it as a reference.