In order for cells to replicate they have to first copy their DNA in a process called replication. In actively dividing cells replication occurs during the synthesis (S) phase of the cell cycle and later the newly synthesized DNA strands are separated into new cells in mitosis and meoisis.

Problem Solving in Replication

If a cell wants to divide it has to solve several “problems” of replication. Such as how does it make a perfect copy of itself, where should it start replicating, etc.? We will think about these problems sequentially and see how the cell solves each. In this way, we will have a story of how our cells overcome replication challenges. This makes memorizing and remembering the different steps of replication far easier since they won’t be quite as abstract.

Problem 1: Copying What I Have

Our DNA is an instruction kit that tells cells what to do and when. In order for a new cell to “know” what to do it needs an identical copy of the instruction kit. This means that a cell needs to create a perfect copy of its DNA and pass it on to the next cell. In order to do this, our cells use their current DNA strand as a template.

By creating a complementary strand from our current DNA the cell is able to generate two sets of its DNA. However, it never creates a fully new strand. Scientist term this property of DNA semi-conservative, meaning one of the two strands are conserved (basically old) and one of the strands is newly synthesized.

Application: Semi-Conservative Replication

The MCAT likes to ask questions about the semi-conservative replication in tricky ways. Instead of asking for a strict definition they often want you to apply this information to rounds of replication. Let’s look at an example question to see how we can apply this information.

A cell was grown in a medium with green fluorescent protein (GFP) tagged nucleotides and allowed to undergo one round of replication. At this point, the two cells were transferred to a new medium containing red fluorescent protein (RFP) tagged nucleotides and each cell was allowed to undergo two more rounds of replication. At the end of the second round of replication in the RFP medium, what percentage of double-stranded DNA molecules contain both GFP and RFP nucleotides?

During each round, the cell will create a new strand with available nucleotides. So the first cell will create strands that contain GFP only since that is what they have access to. When the two cells are transferred to the RFP medium they will create RFP containing DNA. Since replication is semi-conservative the next two strands will contain both GFP and RFP strands. However, in the second round, there will be one that is completely RFP and another that is comprised of both RFP and GFP.

We could draw out all of the strands but we don’t need to since they will be identical to what is already drawn out. Therefore 50% of the strands will contain both GFP and RFP after two rounds of replication in the new RFP medium.

Problem 2: Gaining Access and Getting Started

Now that our cells have overcome the first challenge of replication they need to overcome the next. Where to start replication. Thankfully our DNA has specific sequences that act as little road signs saying start here. These nucleotide sequences called origins of replication help recruit and position replicative enzymes that will eventually synthesize new strands of DNA.

Since eukaryotic DNA is so big it has multiple origins of replication and in eukaryotes, replication is carried out by multiple sets of enzymes working in different spots. In contrast, prokaryotes only have one origin of replication since their genome is considerably smaller.

Now that our cells know where to start replication they need to get access to the base pairs in order to copy them. To do this the two strands of DNA need to be separated. This process is facilitated by helicase an enzyme that rips apart complementary strands exposing them to other replicative enzymes.

Problem 3: Reannealing of Strands

At this point, we are ready to begin synthesizing new DNA strands using the exposed strand as a template. However, the cell encounters another problem: keeping the separated strands from reannealing. Since the hydrogen bonds between complementary base pairs spontaneously reanneal the cell needs something to keep the strands from coming back together. Here it uses single-stranded-DNA binding proteins (ssBPs) that block the hydrogen bonding slots on base pairs keeping the two strands separated.

Problem 4: Hooking On To DNA

Now that helicase has opened the DNA and the ssBPs have stabilized the two DNA strands it is time for DNA synthesis. Except DNA polymerase, which synthesizes new DNA strands can’t hook onto DNA by itself. Instead, it requires the help of another enzyme called primase that adds an RNA primer to the DNA so DNA polymerase can begin synthesizing.

This introduces another problem: now there is RNA in our newly synthesized DNA. Thankfully we have an additional enzyme in our eukaryotic cells called RNase H that will scan the DNA, find any RNA molecules. From there a different DNA polymerase comes along and replaces the

Problem 5: Replication is Unidirectional

Finally, DNA synthesis begins proceeding from the 5′ end of DNA moving towards the 3′ end opening 3′. up a replication fork. Here DNA polymerase moves down the DNA strand and starts adding nucleotides starting with the 5′ end. As it moves down the strand DNA polymerase catalyzes the formation of phosphodiester bonds forming the backbone of the new DNA strand.

At this point, you’d think the cell has finally overcome all of its challenges, but replication is unidirectional and only proceeds from 5’→3′. This isn’t a problem for the strand that heads into the replication fork since it can continue to continuously synthesize as the fork is opened up by helicase. This strand is called the leading strand since it leads to the replication fork.

However, the strand heading away from the replication fork, called the lagging strand, has to consistently start and stop since it runs out of template to work on. This leaves gaps in the DNA backbone where DNA polymerase has started and stopped multiple times. Each fragmented piece of DNA is called an Okazaki fragment and is later joined by yet another enzyme called DNA ligase that joins together the backbone of adjacent nucleotides.

Another issue with unidirectional replication is copying the 5′ end of the DNA. Since DNA polymerase can’t start at the very end of a DNA strand it can’t copy the DNA there. Thankfully eukaryotes have telomeres or nonsense DNA at the end of its strands that protect the genes from being lost.

Problem 6: Building Tension

As synthesis occurs helicase continues to force apart the two strands this causes the downstream DNA helix to coil more and more tightly. As this occurs it becomes harder and harder for helicase to open up the two strands. To overcome this problem topoisomerase comes along and nicks the DNA causing it to unravel and release the tension in downstream of the replication fork.

Replication As A Whole

Now that we have seen the individual steps of replication let’s look at all of the steps together and how replication proceeds in eukaryotes then in prokaryotes.

Eukaryotic Replication

To begin multiple sets of replicative enzymes align themselves on the various origins of replication. To start the nitrogenous bases are torn apart by a helicase and single-stranded binding proteins attach to the exposed ends preventing the DNA strands from reannealing. At this point, primase comes and lays down an RNA primer which allows DNA polymerase to begin synthesizing the new DNA strand. Synthesis progresses from 5’→3′ and deoxy-tri-phospho-nucleotides (dNTPs) are incorporated. Later RNase H will remove the RNA primers and another DNA polymerase will come and replace the gaps with DNA.

On the leading strand, synthesis proceeds continuously. While on the lagging strand Okazaki fragments are formed and the gaps in the DNA backbone are joined together by DNA ligase. As replication continues and the strands are forced apart tension builds downstream of the replication fork. This tension makes it harder and harder for helicase to unwind the DNA so topoisomerase comes and nicks the DNA allowing it to unravel relieving the built-up tension. Lastly, DNA polymerase is unable to fully synthesize the 5′ end of the DNA thankfully our DNA has telomeres at the end so no genes are lost in the process. However, telomeres will get shorter and shorter as DNA is replicated multiple times. To combat this telomerase comes and extends the telomeres keeping them long enough to prevent gene loss.

Prokaryotic Replication

In prokaryotes the steps are nearly identical so I will highlight the differences rather than rehash the replicative process in prokaryotes.

Replication in a prokaryote begins as it does in eukaryotes by opening up a replication fork at the origin of replication. Unlike eukaryotes, prokaryotes only have one origin of replication so only one set of replicative enzymes are necessary. From here the strands are opened up with helicase and held open by ssBPs then primase comes and lays down an RNA primer. Prokaryotes also use DNA polymerase but there are actually different types.

Eukaryotes use DNA polymerases with greek letters following them (e.g. ⍺, β, 𝛄, etc.) while prokaryotes have roman numerals to denote their DNA polymerases (I, II, III, etc.) Specifically, DNA polymerase III synthesizes the new strand in the same fashion as in eukaryotes. The only major differences from here on out are the enzymes used and the lack of telomeres or telomerase. Instead of using RNase H prokaryotic cells have DNA polymerase I both remove and replaces RNA primers with DNA in one step. Additionally, prokaryotic DNA is circular so it doesn’t need telomeres since the 5′ end of DNA can be synthesized when it loops back around to the beginning.