Question

Read thoroughly.

View the two responses and answer the question below. Basically making a comparison and stating similarities.

Reflect on the Wright et al. (2014) paper. Did you and your classmates tend to make the same mistakes or different errors from the students that Wright interviewed? Please reference an aspect of the Wright paper in your replies. Discuss whether you and your peers, made some of the same errors or different ones when making your descriptions.

Response 1:

DNA replication is a biological process of producing two identical replicas of DNA from one original DNA molecule. This process is described as semi-conservative because each DNA molecule is made up of one old strand of DNA and a new one. DNA replication occurs in all living organisms acting as the basis for biological inheritance. The first step in DNA replication is a separation of the two strands. This unzipping is done by an enzyme called helicase and results in the formation of a replication. Each separated strand acts as a template for creating a new strand of DNA. An enzyme called primase starts the process and makes a small piece of RNA called primer which marks the starting point for the construction of a new strand of DNA. After a starting point for the construction of a new strand of DNA is complete, an enzyme called DNA polymerase binds to the primer and starts to make a new strand of DNA. The DNA polymerase can only add DNA bases in one direction, from the 5’ end to the 3’ end. One of the new strands of DNA, the leading strand is made continuously in the 5’ to 3’ direction. The other strand, the lagging strand, runs in the opposite direction than the leading strand, therefore, it can’t be made in this continuous way. Due to the difference in the direction, the DNA polymerase can only make this strand in a series of small sections called Okazaki Fragments. After each fragment is started with an RNA primer, DNA polymerase adds a short row of DNA bases in the 5’ to 3’ end - this process is repeated. After a new DNA is made, the enzyme called exonuclease removes all the RNA primers from both strands of DNA and another DNA polymerase enzyme fills in the gaps that are left behind with DNA. Finally, the enzyme DNA ligase seals up the fragments of DNA in both strands to form a continuous double strand.

Response 2:

Replication is the method of constructing new copies of DNA from existing DNA strands. It happens within the nucleus and is the semiconservative method which implies that every new DNA molecule has one parent strand and one newly shaped strand. DNA synthesis starts at ORI (origin of replication) that are the AT bases rich sequences and terminates at the terminator. A DNA sequence with ORI and terminator makes a replicon that's capable of its replication. Helicase enzymes unwind the spiral of DNA using the energy of ATP. The single-stranded binding proteins then bind to the only strands of DNA to stop the rewinding. The ribonucleic acid primase enzyme makes primer, the short RNA strands that are elongated by DNA polymerases. DNA polymerases can't begin the formation of the latest DNA strands de novo and need a primer. The elongation of primer happens in 5' to 3' direction solely because the new nucleotide is added to free 3' OH of existing primer. The lagging strand is made within the direction opposite to the growth of replication fork in a semi discontinuous manner to permit the synthesis in 5' to 3' direction solely. The ribonucleic acid primers are removed by the exonuclease activity of DNA enzyme and therefore the DNA ligase enzymes join Okazaki fragment of the lagging strand along.

Answer 1

Introduction

DNA replication, or the copying of a cell's DNA, is no simple task! There are about 333 \text{billion}billion  base pairs of DNA in your genome, all of which must be accurately copied when any one of your trillions of cells divides^11start superscript, 1, end superscript.

The basic mechanisms of DNA replication are similar across organisms. In this article, we'll focus on DNA replication as it takes place in the bacterium E. coli, but the mechanisms of replication are similar in humans and other eukaryotes.

Let's take a look at the proteins and enzymes that carry out replication, seeing how they work together to ensure accurate and complete replication of DNA.

The basic idea

DNA replication is semiconservative, meaning that each strand in the DNA double helix acts as a template for the synthesis of a new, complementary strand.

This process takes us from one starting molecule to two "daughter" molecules, with each newly formed double helix containing one new and one old strand.

Schematic of Watson and Crick's basic model of DNA replication.

DNA double helix.

Hydrogen bonds break and helix opens.

Each strand of DNA acts as a template for synthesis of a new, complementary strand.

Replication produces two identical DNA double helices, each with one new and one old strand.

In a sense, that's all there is to DNA replication! But what's actually most interesting about this process is how it's carried out in a cell.

Cells need to copy their DNA very quickly, and with very few errors (or risk problem such as cancer). To do so, they use a variety of enzymes and proteins, which work together to make sure DNA replication is performed smoothly and accurately.

DNA polymerase

One of the key molecules in DNA replication is the enzyme DNA polymerase. DNA polymerases are responsible for synthesizing DNA: they add nucleotides one by one to the growing DNA chain, incorporating only those that are complementary to the template.

Here are some key features of DNA polymerases:

They always need a template

They can only add nucleotides to the 3' end of a DNA strand

They can't start making a DNA chain from scratch, but require a pre-existing chain or short stretch of nucleotides called a primer

They proofread, or check their work, removing the vast majority of "wrong" nucleotides that are accidentally added to the chain

The addition of nucleotides requires energy. This energy comes from the nucleotides themselves, which have three phosphates attached to them (much like the energy-carrying molecule ATP). When the bond between phosphates is broken, the energy released is used to form a bond between the incoming nucleotide and the growing chain.

DNA polymerization reaction

New strand: 5' ACTG... 3' Template strand: 3' TGACAT 5'

At the end of the new strand, a 3' hydroxyl group is exposed on the final nucleotide of the strand. This hydroxyl group will undergo a chemical reaction with a phosphate group in an incoming nucleotide, resulting in the formation of a new bond (and the addition of the nucleotide to the end of the chain). Specifically, the next nucleotide exposed on the template is an A. The nucleotide complementary to an A is a T, so when a T pairs with the A on the template strand, it will undergo a reaction with the 3' hydroxyl at the end of the chain and be added on.

The nucleotide consists of a sugar with a chain of three phosphates, a base, and a hydroxyl attached. The 3' hydroxyl exposed at the end of the growing strand will form a bond to the innermost phosphate in the chain of the new nucleotide, a reaction that release a two-phosphate unit called pyrophosphate. The pyrophosphate will be further cleaved into two individual phosphate ions. The result of the reaction is the addition of the T nucleotide to the growing strand of DNA. The 3' hydroxyl of the T nucleotide is now exposed at the end of the chain. This hydroxyl can participate in a new reaction with the phosphate of the next nucleotide to be added to the chain.

In prokaryotes such as E. coli, there are two main DNA polymerases involved in DNA replication: DNA pol III (the major DNA-maker), and DNA pol I, which plays a crucial supporting role we'll examine later.

Starting DNA replication

How do DNA polymerases and other replication factors know where to begin? Replication always starts at specific locations on the DNA, which are called origins of replication and are recognized by their sequence.

E. coli, like most bacteria, has a single origin of replication on its chromosome. The origin is about 245245245base pairs long and has mostly A/T base pairs (which are held together by fewer hydrogen bonds than G/C base pairs), making the DNA strands easier to separate.

Specialized proteins recognize the origin, bind to this site, and open up the DNA. As the DNA opens, two Y-shaped structures called replication forks are formed, together making up what's called a replication bubble. The replication forks will move in opposite directions as replication proceeds.

Bacterial chromosome. The double-stranded DNA of the circular bacteria chromosome is opened at the origin of replication, forming a replication bubble. Each end of the bubble is a replication fork, a Y-shaped junction where double-stranded DNA is separated into two single strands. New DNA complementary to each single strand is synthesized at each replication fork. The two forks move in opposite directions around the circumference of the bacterial chromosome, creating a larger and larger replication bubble that grows at both ends.

How does replication actually get going at the forks? Helicase is the first replication enzyme to load on at the origin of replication start superscript, 3, end superscript. Helicase's job is to move the replication forks forward by "unwinding" the DNA (breaking the hydrogen bonds between the nitrogenous base pairs).

Proteins called single-strand binding proteins coat the separated strands of DNA near the replication fork, keeping them from coming back together into a double helix.

Primers and primase

DNA polymerases can only add nucleotides to the 3' end of an existing DNA strand. (They use the free -OH group found at the 3' end as a "hook," adding a nucleotide to this group in the polymerization reaction.) How, then, does DNA polymerase add the first nucleotide at a new replication fork?

Alone, it can't! The problem is solved with the help of an enzyme called primase. Primase makes an RNA primer, or short stretch of nucleic acid complementary to the template, that provides a 3' end for DNA polymerase to work on. A typical primer is about five to ten nucleotides long. The primer primes DNA synthesis, i.e., gets it started.

Once the RNA primer is in place, DNA polymerase "extends" it, adding nucleotides one by one to make a new DNA strand that's complementary to the template strand.

Leading and lagging strands

In E. coli, the DNA polymerase that handles most of the synthesis is DNA polymerase III. There are two molecules of DNA polymerase III at a replication fork, each of them hard at work on one of the two new DNA strands.

DNA polymerases can only make DNA in the 5' to 3' direction, and this poses a problem during replication. A DNA double helix is always anti-parallel; in other words, one strand runs in the 5' to 3' direction, while the other runs in the 3' to 5' direction. This makes it necessary for the two new strands, which are also antiparallel to their templates, to be made in slightly different ways.

One new strand, which runs 5' to 3' towards the replication fork, is the easy one. This strand is made continuously, because the DNA polymerase is moving in the same direction as the replication fork. This continuously synthesized strand is called the leading strand.

The other new strand, which runs 5' to 3' away from the fork, is trickier. This strand is made in fragments because, as the fork moves forward, the DNA polymerase (which is moving away from the fork) must come off and reattach on the newly exposed DNA. This tricky strand, which is made in fragments, is called the lagging strand.

The small fragments are called Okazaki fragments, named for the Japanese scientist who discovered them. The leading strand can be extended from one primer alone, whereas the lagging strand needs a new primer for each of the short Okazaki fragments.

The maintenance and cleanup crew

Some other proteins and enzymes, in addition the main ones above, are needed to keep DNA replication running smoothly. One is a protein called the sliding clamp, which holds DNA polymerase III molecules in place as they synthesize DNA. The sliding clamp is a ring-shaped protein and keeps the DNA polymerase of the lagging strand from floating off when it re-starts at a new Okazaki fragment^44start superscript, 4, end superscript.

Topoisomerase also plays an important maintenance role during DNA replication. This enzyme prevents the DNA double helix ahead of the replication fork from getting too tightly wound as the DNA is opened up. It acts by making temporary nicks in the helix to release the tension, then sealing the nicks to avoid permanent damage.

Finally, there is a little cleanup work to do if we want DNA that doesn't contain any RNA or gaps. The RNA primers are removed and replaced by DNA through the activity of DNA polymerase I, the other polymerase involved in replication. The nicks that remain after the primers are replaced get sealed by the enzyme DNA ligase.

Summary of DNA replication in E. coli

Let's zoom out and see how the enzymes and proteins involved in replication work together to synthesize new DNA.

Illustration shows the replication fork. Helicase unwinds the helix, and single-strand binding proteins prevent the helix from re-forming. Topoisomerase prevents the DNA from getting too tightly coiled ahead of the replication fork. DNA primase forms an RNA primer, and DNA polymerase extends the DNA strand from the RNA primer. DNA synthesis occurs only in the 5' to 3' direction. On the leading strand, DNA synthesis occurs continuously. On the lagging strand, DNA synthesis restarts many times as the helix unwinds, resulting in many short fragments called “Okazaki fragments.” DNA ligase joins the Okazaki fragments together into a single DNA molecule.

Helicase opens up the DNA at the replication fork.

Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA.

Topoisomerase works at the region ahead of the replication fork to prevent supercoiling.

Primase synthesizes RNA primers complementary to the DNA strand.

DNA polymerase III extends the primers, adding on to the 3' end, to make the bulk of the new DNA.

RNA primers are removed and replaced with DNA by DNA polymerase I.

The gaps between DNA fragments are sealed by DNA ligase.

DNA replication in eukaryotes

The basics of DNA replication are similar between bacteria and eukaryotes such as humans, but there are also some differences:

Eukaryotes usually have multiple linear chromosomes, each with multiple origins of replication. Humans can have up to 100,100,100, comma000000000origins of replication^55start superscript, 5, end superscript!

Most of the E. coli enzymes have counterparts in eukaryotic DNA replication, but a single enzyme in E. coli may be represented by multiple enzymes in eukaryotes. For instance, there are five human DNA polymerases with important roles in replication^55start superscript, 5, end superscript.

Most eukaryotic chromosomes are linear. Because of the way the lagging strand is made, some DNA is lost from the ends of linear chromosomes (the telomeres) in each round of replication.