Cell Biology Chapter 6 (Exam 2)

DNA replication is how…

All life on earth replicates itself

DNA replication occurs…

Only at specific times during the cell cycle, primarily during the S phase.

What are the 2 sides of DNA?

5’ to 3’
3’ to 5’

(Complementary to each other)

What is DNA replication?

A semi-conservative process where a cell makes two identical copies of its genome before cell division.

Why is DNA replication semi-conservative?

Each new DNA strand consists of one original strand (template) and one newly made daughter strand.

Each original strand acts as a template to join with the new complementary strand

Semi-conservative replication can be done…

Over and over again

Where does DNA replication begin?

The origins of replication.

What are origins of replication?

Specific sequence of bases that are highly rich in A-T pairs.

What happens to the origins of replication?

Specialized proteins recognize them and pry open the DNA strand at these locations

What are the proteins that split open the origins of replication?

Initiator Proteins

Why are origins of replication A-T rich?

They are held together by only 2 Hydrogen bonds (as opposed to 3). Because of that, they are physically easier for the cell to pull apart

Why are there many origins of replications all over the chromosomes?

To help the ripping and replication of the DNA strand occur faster by prying open many places at once.

In order to replicate DNA, what do you need?

Replisome - a huge protein complex

What IS replisome? What does it consist of?

A massive protein complex (group of proteins) joined together through quaternary regulation.

It mainly has these components:

DNA Helicase, DNA Polymerase, DNA Primase, Sliding clamp, Topoisomerases.

What does replisome DO? How?

For the leading strand: it copies it continuously in a straight line, moving behind the helicase.

For the lagging strand: Because this strand runs in the opposite direction, it copies it backwards in discontinuous chunks called Okazaki Fragments

DNA helicase

An enzyme (type of protein) that breaks the hydrogen bonds between the base pairs to separate the two strands. Uses a lot of ATP to do so and is very quick

DNA polymerase

“Make polymer” (makes DNA)

Makes DNA during the S phase (DNA replication)

It creates the phosphodiester (phosphate-sugar) backbone in the 5’ to 3’ direction

Phosphate-sugar backbone = nucleotide

Where does DNA polymerase elongate the new strand of DNA?

The 5’ end is already fixed in place. It continuously elongates the strand by adding to the 3’ end.

Always creates the phosphodiester backbone in the 5’ to 3’ end.

It adds the new nucleotides to the 3’ backbone

While DNA polymerase is creating the new strand, what is at the 3’ end and why is it important?

A 3’ OH.

This is important because without this, DNA polymerase will be unable to attach new nucleotides to the 3’ end.

What is a phosphodiester bond?

The bond that holds together the phosphate-sugar backbone of DNA

How is DNA polymerase able to build the nucleotides on the 3’ end?

By breaking the high-energy phosphoanhydride bonds between the 3 phosphates. This releases energy and gives ability to build nucleotides on the 3’ end.

What issue comes up for DNA polymerase after the DNA is opened at the origin of replication?

One strand is easy to make (already in 5’ to 3’ direction)

The other strand is difficult to make (in 3’ to 5’ direction - opposite to how DNA polymerase adds new nucleotides)

DNA is antiparallel

How many origins of replications exist in prokaryotic DNA?

1

What is the leading strand?

The new strand that is synthesized easily in the 5’ to 3’ strand based off of its template strand, which is in the 3’ to 5’ direction.

What is the lagging strand?

The new strand that is synthesized in the 3’ to 5’ direction based off of its template strand, which is in the 5’ to 3’ direction

How is the lagging strand synthesized?

DNA primase lays down many RNA primers which are 3’ OH groups. DNA polymerase adds a short stretch of DNA to the 3’ OH group away from the fork until it bumps into another 3’ OH.

What are Okazaki fragments?

Short fragments of newly synthesized DNA that is used to synthesize a 3’ to 5’ strand from the 5’ to 3’ template strand, since DNA can only synthesize new DNA in the 5’ to 3’ direction.

What does DNA polymerase’s active site absolutely need?

A 3’ OH. It fits into it and it carries out the process of synthesizing a new DNA strand.

What is DNA primase?

Places RNA primers the original strands of DNA so DNA polymerase has a starting point for making the new strand off of the original strand

What are RNA primers?

A short, temporary strand of RNA that is the starting point for DNA replication

How do RNA primers help DNA polymerase create a new strand of DNA?

By providing the 3’ OH from its end which DNA polymerase needs in its active site to begin the process of creating the new DNA strand

What happens to RNA primers after they are needed?

Nuclease will remove them. This leaves a gap

What happens to the gap that is left after RNA primer leaves?

DNA polymerase instantly fills them with permanent nucleotides

When is the only time DNA polymerase adds the phosphodiester bonds between the nucleotides it makes?

Only when it physically builds a new nucleotide. So when a nick is created where RNA primer left, it cannot magically create just a new bond if the two pieces of nucleotide sequences already existed.

What happens to the nicks after RNA primer is removed?

DNA ligase glues the disconnected bases together

How many RNA primers does the leading strand require?

1 - the leading strand is built continuously in the same direction the fork opens. DNA polymerase just goes on the one primer and continuously builds new DNA

How many RNA primers does the lagging strand require?

Thousands - it is built backwards in discontinuous chunks. A new RNA primer for every Okazaki fragment

What does DNA polymerase do at the same time it adds each nucleotide?

It simultaneously checks itself. It does not wait until the entire strand is done to proofread

What direction does DNA polymerase check itself?

3’ to 5’

How does DNA polymerase check itself in the 3’ to 5’ direction?

After it makes a single new nucleotide, the 3’ end is the last part made, hence it is exposed. Therefore, it will go from the 3’ tip to the 5’ tip when checking

How does DNA polymerase know if the new nucleotide it made is right or wrong?

By checking the shape of the new nucleotide. If the wrong bases pair, it will create a bump/kink in the strand. Because of this, DNA polymerase cannot move forward. It will check the strand in the 3’ to 5’ direction, realizes its mistake, and cut off the bump where the wrong nucleotide was. Then, DNA polymerase will try again.

What are the 2 jobs of DNA polymerase?

Making the new DNA strand (polymerizing) and checking the new DNA strand

Makes then checks, makes then checks,…

Because DNA polymerase has 2 different jobs, they have…

2 different active sites

If one active site changes, the other will not be able to work

What is the sliding clamp and what is it used for?

A donut-shaped clamp that clamps the entire replisome (that includes all of the important proteins needed for DNA replication) securely onto the DNA strand

Uses ATP to snap the clamp shut around DNA

Allows DNA polymerase to stay on the DNA strand securely, making DNA replication much faster and efficient

What are Single Stranded DNA binding proteins?

When DNA gets unzipped, it wants to naturally form the double helix again due to the complementary base pairs. SSBs coat the single strands, which helps keep the strands from zipping back up again so the replisome can copy them.

When DNA is in a single strand, it can form a hairpin loop where an A near a T on the same strand can attempt to connect. This will jam the DNA polymerase. Because of this, SSBs will bind along the backbone, which keeps it straight

Single-stranded DNA is also unstable, and the cell mistakes it for an attack. SSBs acts as a shield so nucleases do not destroy them.

Which strand has more SSBs?

The lagging strand - takes longer to be synthesized

What are topoisomerases?

Detangle the knots, twists, and mechanical tension in the double helix during replication and transcription

Why are topoisomerases needed?

As the DNA helicase unwinds many sections of the double helix at OoR, the parts before and after them become hypercoiled. Topoisomerases relieve the hypercoiling, or the replisome will not be able to move along the strand anymore.

They work on the topology (shape) of DNA

Topology =

Shape

Topoisomerases fix the…of the double helix

shape

How does Topoisomerase Type 1 relieve the hypercoiling?

It cuts just 1 of the two strands in one place by cutting the phosphodiester bond. Once cut, the tension unwinds. After unwinding, T1 will create a new phosphodiester bond between the broken strands

Will fix the strand after it breaks it!

No ATP needed

Used for hypercoiling along a stretch of a chromosome

How does Topoisomerase Type 2 relieve the hypercoiling?

It cuts both of the DNA strands. While both ends are kept ahold, a window opens up for a separate section of the DNA helix to pass through the window, which unwinds the DNA. After this, T2 will create two new phosphodiester bonds to seal the parts of the backbone it cut.

Does use ATP

Can be used when two sister chromatids are tangled together (2 separate DNA molecules)

Untying a literal knot in a chromosome (need to cute both)

Severe supercoiling

Why are topoisomerases a major target for certain drugs/treatments?

If you can break toposiomerase after it cuts the DNA but before it can paste it back together, the cell’s chromosomes stay broken and die. This is helpful to kill harmful cell types.

How do antibiotics target bacterial topoisomerase?

Wait for topoisomerase to cut the prokaryotic DNA. It binds to its specific active site and completely stops it from remaking the phosphodiester bonds in the DNA. This creates a permanent break in the DNA causing bacterial death

How do antibiotics target cancer cell topoisomerase in chemotherapy?

T1/T2 cut the cancer cell’s DNA. The drug will immediately bind to the active site of T1/T2 and prevent it from making the new phosphodiester bonds needed to reseal the broken strand(s). This breaks the chromosome and triggers apoptosis.

What happens if you do not have Topoisomerase?

The cell dies

What are telomeres?

Protective caps made of repetitive DNA sequence and proteins found at each end of linear chromosomes

Over time, telomeres…

Shorten each time a cell replicates.

Once they become very short, the cell can no longer divide and dies, which leads to natural aging

What is telomerase?

An enzyme that elongates telomeres by adding repetitive DNA sequences back on the ends of the chromosomes.

Where are telomerases present and not present?

Not present in normal human cells. They are only present in cell that require dividing forever, such as stem cells, cancer cells, sperm cells, and egg cells.

What are stem cells?

Cells that can divide and turn into many different types of cells. They start without a specific job and transform into active cells with jobs when the body needs repair.

Why do stem cells have long telomerase?

If they lacked telomerase, they would hit their division limit very quick. Your body would lose its ability to repair itself.

New born babies have long…

Telomeres

Why do babies have long telomeres?

Its early embryo have telomerase activity to increase telomere length from the egg and sperm cells. Once the baby is built, telomerase is switched off and the telomere now gets shorter and shorter each time its cells divide.

Cancer cell have active…

Telomerase

DNA replication lab methods

PCR (Polymerase Chain Reaction)

DNA sequencing

How can DNA polymerase still make mistakes even though it checks itself too?

DNA polymerase works very fast. It is bound to make mistakes. However, it goes back and checks itself. DNA polymerase checking itself, however, is only 99% accurate. A mistake left behind can become a mutation once the cell divides.

As you age, mutations…

Build up in cells. The mutated DNA copies over and over again if the mistake was not fixed immediately

What is DNA Mis-Match Repair Complex?

A group of proteins that follows directly behind DNA polymerase and fixes any mistakes left behind (mistake = distortion in shape, bump due to 2 bases that do not belong next to each other)

What happens if the wrong bases get put on a strand by DNA polymerase

No hydrogen bonds form between them, causing them to stick out on the strand and create a bump/distortion

How does DNA Mismatch Repair complex fix the mistake in DNA?

Binds tightly to the bump. It recruits an enzyme to slice into the new DNA where the mistake is and scoop it out. Then, a new DNA polymerase reforms the gap and DNA ligase glues the backbone together by adding a new phosphodiester bond

What are purine groups?

G and A

What is depurination?

A type of way DNA can get damaged and eventually lead to mutation

A purine group falls off due to an unstable bond connecting it to the backbone. This leaves an empty space

What is deamination?

A type of way DNA can get damaged and eventually lead to mutation

A chemical reaction removes an Amine (NH2) group off a base (usually C). This makes C to transform into U (only found in RNA). This alters genetic code because U bonds with A.

What is Thymine Dimers?

A type of way DNA can get damaged and eventually lead to mutation

Induced mutations caused only by exposure to UV radiation (tanning beds/sunlight). Two T’s form a tight covalent bond between them. Creating bonds require energy.

How do the two thymines in thymine dimers create a bond between themselves?

Creating bonds require energy. That energy is from UV radiation (from sunlight or tanning beds, etc.)

What is UV light?

A carcinogen. Causes cancer because it forces two Thymines to stick together on one DNA strand (side by side)

DNA damage ultimately:

Changes the shape of DNA by changing its structure

What happens if we get mutations through damaged DNA

Repair mechanisms take place

What are the 3 repair mechanisms?

Excision repair, Non-homologous end joining, homologous recombination

How are repair mechanisms activated and controlled?

Through signaling cascades. Cells make a decision and repair DNA through this.

What is excision repair?

A type of DNA repair mechanism.

Fixes damage that affects only one strand of the DNA double helix. It uses the opposite, undamaged strand as a template. Fixes mistakes such as depurination and deamination. It slices the wrong base out. Then, DNA polymerase fills in the missing nucleotide, and DNA ligase seals the backbone shut.

What type of repair is used for single-strand damage?

Excision repair.

What type of repair is used for Double-strand break damage?

Non-Homologous end joining and Homologous recombination (when both antiparallel backbones break)

What is Non-Homologous end joining?

When both sides of the backbone are broken. To fix this, a protein complex cleans up the broken ends to stitch together using nuclease. Then ligase seal the ends back together to make a sealed chromosome.

What can happen during non-homologous end joining?

While jagged ends are being cleaned by nuclease, it can accidentally lose bases. This is okay, because it is a type of quick and dirty clean up that is simply used to prevent cell death or cancer from double strand breaks.

Primarily before DNA replication

Which type of DNA repair is most common?

Non-homologous end joining

What is homologous recombination?

Very precise type of repair for double strand breaks. Occurs after DNA replication because it needs its sister chromatid to act as a blueprint. It takes the broken chromosome and sends it to its homologous (sister) chromosome. It uses it as a template for DNA polymerase to copy the missing gene information and order perfectly.

What are human diseases that are a result of proteins that are supposed to fix DNA not working?

Werner Syndrome

What happens if you break the proteins responsible for fixing wrong/broken DNA?

Your cells will get a collection of the wrong DNA

What is in common with individuals who have diseases caused by protein mutation due to repair pathways not functioning correctly?

Their symptoms

What are the symptoms of individuals who have diseases from protein mutations due to repair pathways not functioning correctly?

Cancer - common with mutated DNA

Sensitive to Carcinogens - extreme IR/UV sensitivity (they break DNA)

Accelerated aging (getting older faster due to DNA degrading quickly over time)

What is Werner Syndrome?

Rapid, accelerated, premature aging. The cell lacks a certain protein that cannot efficiently repair double-strand breaks of DNA. The body cannot repair DNA, leading to rapid cell death.

Explain signaling cascades, which help cells know how to respond to certain changes

Signaling molecule (ligand) activates the receptor. then the signaling proteins are activated, then the effector proteins are activated, and it chooses what to carry out (cellular response)

What is an example of a cellular response that can be carried out by signaling cascades?

Histone Tail Modifications - Goal = alter gene expression

When is DNA polymerase needed?

During S phase - DNA replication

What is the main enzyme needed during Transcription?

RNA Polymerase (DNA to RNA)

When is needed during Translation?

The ribosome assisted with tRNA

What is happening during G1?

High transcription and translation to help the cell grow

What is happening during S phase?

DNA replication with DNA polymerase