UNIT 7 BIOL401

DNA

What was the structure of DNA thought to be? What was the problem with this original description, and why did it not correlate to the concept of inheritance?

A short molecule made up of repeating sequences of 4 nucleotides (ACTGACTGACTG…). Nucleotides are connected by phosphate groups.

It didn’t account for the diversity of life since there are minimal combinations of 4 nucleotides. It lacked the complexity to account for the variety of traits that are passed down from parents to offspring. 

What is the “one-gene-one-polypeptide” hypothesis? Why does this hypothesis better address the concept of inheritance?

It started as the one-gene-one-enzyme hypothesis because enzymes were shown to be inherited just as morphological traits were. It suggests that each gene is responsible for encoding one polypeptide.

The one-gene-one-polypeptide hypothesis better addresses the concept of inheritance because it connects genes to proteins. Proteins are polymers known to be made of 20 amino acids. If, as suggested by the hypothesis, polypeptides are inherited, then their complexities can account for the diversity of heritable traits.

describe Griffith’s experiment and how it demonstrated that genes that can be transferred between organisms, resulting in changes to that organism.

Griffith attempted to induce immunity to the virulent pneumonia strain in mice. He injected mice with either non-virulent R-cells, virulent S-cells, heat-killed S-cells, or live R-cells and killed S-cells. To his surprise, the last group of mice died, and active, virulent S-cells were found in the mice’s blood. Griffith noted that a molecule released from the dead S-cells had been taken up by the R-cells and that that molecule had “transformed” them into the virulent strain. He called the molecule the “Transforming Principle” but didn’t know what it was.

Describe MacLeod and McCarty’s experiment and how they demonstrated that DNA contains genes that can be transferred between organisms, resulting in changes to that organism.

They also tested S. pneumoniae like Griffith. They separated all the cellular components of a dead, virulent S cell, including proteins, DNA, lipids, etc. Each was added to a separate test tube containing non-virulent R-cells, and only the tube containing the DNA fraction resulted in transformation (it killed mice). This demonstrated that DNA was the “transforming” factor and therefore carries genetic information.

Describe Hershey and Chase’s experiment how they demonstrated that DNA contains genes that can be transferred between organisms.

They tagged T2 bacteriophages with either radioactive phosphorus or radioactive Sulfur. Sulfur is an element found only in proteins, and phosphorus is found only in DNA. Next, they treated E.coli with either type of bacteriophage, allowing enough time for the phages to infect a host cell. Some of each culture was allowed to go on and lyse to prove that the cells were infected.

The remaining infected E.coli were blended to separate the empty phage coatings from the host-cell membrane. Whatever material the phage had injected would have to be the molecule carrying genetic information.

The samples were centrifuged, leaving cells in the pellet and phage coats in the supernatant. Radioactive P always ended up in the pellet, and radioactive S always ended up in the supernatant. This meant DNA was the molecule that Phages injected into the E.coli and therefore the molecule of inheritance.

What is the structure of DNA now known to be?

Which bases are paired in a DNA molecule? What type of bond allows bases to pair up?

A linear polymer of stacked nucleotides forming a double helix with antiparallel strands. Nucleotides are joined by phosphodiester linkages, and the 2 strands are held together by H-bonds between complementary bases.

A + T and C + G.

Outline briefly how the structure of DNA was determined.

X-ray crystallography was a key player in generating images of DNA.

Franklin created Photo 51, which outlined the dimensions of DNA and showed its helical structure, leading to Watson and Crick’s creation of the double helix model.

Dimensions:

1. 0.34 nm is the space between adjacent bases

2. 3.4 nm is the length of a full turn of the helix

3. 2 nm is the diameter

Watson and Crick also used Chargaff’s data to infer the bases that would pair up and that they would interact by H bonding.

In their model, antiparallel strands created the most stable molecule.

What are Chargaff’s rules? How did Chargaff’s data demonstrate that DNA could have different base compositions, meaning that DNA could have different genes?

The ratio of A/T = 1 and G/T = 1

also; A+G/T+C = 1

He found these rules by examining the base compositions of several different species. He saw that the compositions varied between species, meaning that DNA could have variable gene sequences.

What are chromosomes? Describe the components of a chromosome.

What is the advantage of having DNA packed into chromosomes during cell division?

A structure of highly condensed chromatin containing genes, one or more origin, telomeres, and a centromere.

Protects our DNA from damage while chromosomes are being moved during mitosis.

What is a karyotype? Are the numbers of chromosomes species-specific? What is a homologous pair?

A visual of the complete set of chromosomes inside a cell, paired up and ordered based on morphology.

Yes

Homologous chromosomes are pairs that contain the same genes at the same locations. One is inherited from the mom and the other from the dad.

What is chromatin? How does prokaryotic and eukaryotic DNA differ in packaging? Differentiate between euchromatin and heterochromatin. Where are active genes located and why?

Chromatin is DNA associated with proteins, such as histones. Prokaryotic DNA is not associated with proteins and has a circular double-helix structure.

Euchromatin: less condensed, contains active genes because it is more accessible to transcription proteins.

Heterochromatin: more condensed, contains more proteins, and contains inactive genes.

List and describe the three levels of chromatin organization.

1. DNA wrapped around histones(nucleosomes)

2. Stacked/condensed Nucleosomes form a 30 nm fiber

3. Higher order packing such as chromosomes which are associated with more proteins

How many types of histone proteins are there? How does their composition allow for binding to DNA?

5 types

Amino acid side chains in histones are positively charged and interact with negative charge of DNA.

This electrostatic interaction helps stabilize the DNA-histone binding

What are scaffold proteins? what is their function?

proteins that bring together multiple other proteins to assemble them into functional complexes

They regulate signal transduction and help localize pathway components (organized in complexes) to specific areas of the cell

What is the orientation and directionality of DNA?

What is attached to the 5’ vs 3’ carbon of a deoxynucleotide?

Antiparallel. One strand runs 5’ to 3’ and the other 3’ to 5’.

The 3’ carbon has a hydroxyl group and the 5’ carbon has a phosphate group.

What is the origin of replication? what bases is it rich in and why?

Do eukaryotic cells have one origin of replication or multiple origins of replication? Why?

A region of DNA on a chromosome where initiator proteins bind and begin unwinding the double helix. It is rich in A-T base pairs because they are held together by 2 H-bonds instead of 3. This makes it easier to start unwinding the DNA.

Prokaryotic cells have 1 Ori. Eukaryotic cells have multiple origins since our chromosomes are much longer. Multiple origins increase the rate of DNA replication.

What are the functions of DNA polymerase I and III in replication? What end of a DNA strand can they add to?

Pol I → Proofreading, 5’ to 3’ DNA elongation, primer removal due to 5’ to 3’ exonuclease activity

Pol III → Proofreading, Faster 5’ to 3’ DNA elongation

They can only add to the 3’ end of an existing strand.

Describe replication initiation

1. Initiator proteins bind at the origin and start unwinding DNA.

2. Helicases work in both directions to continue unwinding DNA, forming a replicon with 2 replication forks. Helicases are ATP-dependent. SSB proteins keep ssDNA from reforming H bonds.

3. Primases place an RNA nucleotide primer on each of the template DNA strands 5’ to 3’.

Describe DNA replication elongation + how okazaki fragments are made.

Leading strand: DNA pol III uses dNTP precursors to add monophosphate deoxynucleotides to the 3’ end of the RNA primer, using a DNA template. It catalyzes the formation of phosphodiester bonds in condensation reactions.

Lagging Strand: The lagging strand is elongated discontinuously in fragments called “Okazaki fragments”. Each starts with a primer made by primase. DNA pol III elongates by adding deoxynucleotides to the 3’ end of the RNA primer until it reaches the next primer on the lagging strand. DNA pol I removes the primer 5’ to 3’ while catalyzing its replacement with deoxynucleotides. FEN1 trims “flaps” on the 5’ end of the DNA fragments that were displaced during primer replacement. DNA Ligase links the fragments of DNA.

What happens if a mistake occurs in replicating the daughter strand and what function does DNA polymerase have if errors occur? Does this fix all replication errors? Why is it important to have some room for error in replication?

If a wrong nucleotide is placed, DNA polymerases can catch the mistake by proofreading. This is accomplished by DNA pols catalyzing the hydrolysis of an incorrect nucleotide and replacing it the right one. DNA pols then continue elongation.

Describe replication termination in prokaryotes and eukaryotes

In prokaryotes, replication ends when the replication forks meet and fuse.

In Eukaryotes, replicons merge, creating one big replication bubble. The ends of lagging strands present a problem because once the last primer is removed, DNA polymerase cannot extend from the 5’ end of the fragment. To avoid chromosome shrinkage, telomerase binds to the DNA and uses its RNA sequence to extend the lagging strand template. This is thanks to telomerases’ reverse transcriptase activity. Multiple repeating telomere sequences are synthesized. Finally, a last Okazaki fragment is made using the recently extended template strand which completes DNA replication.

Define the role of the following proteins and enzymes in replication: initiator proteins, helicase, DNA clamp, single-stranded binding proteins, primase, DNA ligase, Flap endonuclease I (FEN 1), and topoisomerase.

initiator proteins: bind to the ori and begin unwinding DNA

helicase: unwinds DNA by breaking the hydrogen bonds between strands at the replication forks. ATP-dependent.

SSB proteins: bind to single-stranded DNA and prevent hydrogen bonds from reforming the double-helical structure.

DNA clamp: keeps DNA polymerase III from falling off the DNA

Primase: Places RNA nucleotides (a primer) on the parental template from 5’ to 3’, providing a starting point for DNA polymerases to elongate.

DNA ligase: links the 3’ and 5’ ends of okazaki fragments

FEN1: Removes 5’ flaps of nucleotides on the ends of Okazaki fragments that may have been displaced by DNA pol 1

topoisomerase: Relieves strain ahead of the replication fork created as helicase unwinds the double helix. It cuts DNA, unwinds it, and then re-ligates.

Why are Okazaki fragments required?

Because DNA polymerases can only synthesize DNA 5’ to 3’, and since DNA template strands are antiparallel, only one new strand can be made continuously 5’ to 3’. The other strand must be made away from the replication fork in fragments.

Because the dna is unwound in the opposite direction of lagging strand synthesis, it can only be made in short fragments.

What is a replication bubble? What is a replisome?

A region of unwound dna where replication is occuring and 2 new strands are being synthesized. It has 2 forks.

A replisome is a multi-protein complex that synthesizes 2 strands of DNA at the same time. It is composed of Helicase, SSBs, Primase, DNA polymerases, and clamp proteins.

What does the statement, “replication is processive” mean and how is this achieved?

processive means the both strands are synthesised in the same overall direction at the replication fork at the same time. This happens because the lagging strand template loops around so that it faces in the same direction as the leading strand (new strands 5’ to 3’)

What are the benefits and detriments of mutations? Define germline and somatic mutations.

Mutations create variation that Natural selection can act on, leading to evolution. However, mutations can also lead to poor fitness and death.

Germline = mutations that are heritable and cause diseases or make you more prone to illness

Somatic = mutations that result in benign cysts or malignant tumors. Not heritable.

What are some general types of DNA damage that can occur in a cell? Is most DNA damage repaired?

Physical from environmental factors, or chemical from intracellular reactions.

yes, about 99.9%

List and describe the 6 common types of DNA damage that are usually repaired. What are the consequences if some of these are not repaired?

1. Depurination - hydrolytic removal of a purine (A or G) from the 1’ carbon of deoxyribose.

2. Deamination - hydrolytic removal of an amine group from A, C, or G (NOT T)

3. Break in the DNA - single or double-stranded breaks

4. Oxidative Damage - the deoxyribose sugar is damaged

5. Pyrimidine Dimerization - two adjacent pyrimidines dimerize

6. Inappropriate methylation of any base

Consequences = mutations that are passed on to daughter cells after replication

What types of mistakes are recognized, removed, and repaired in base excision repair? What are the steps and the enzymes involved?

deamination is repaired

1. A DNA glycosylase removes the damaged nitrogenous base portion, leaving behind a sugar-phosphate residue.

2. AP endonuclease nick the phosphodiester bond on the 5’ side of the abasic nucleotide, and phosphodiesterase catalyzes the final removal of the sugar-phosphate residue.

3. DNA polymerase places the correct deoxynucleotide

4. ligase seals the break

What type of repair mechanism is employed when there are pyrimidine dimers in DNA? What are the four enzymes that carry out this type of repair?

Nucleotide excision repair.

1. Excision nuclease

2. Helicase

3. DNA polymerase

4. DNA Ligase

As we learned in the previous unit, errors in replication are often repaired by DNA polymerase. What are these errors called? Describe the mechanism of repair for these errors.

Mismatches

Mismatch repair:

1. MutS and MutL recognize and bind to DNA at the mismatch, and recruit MutH

2. MutH recognizes methylation sequences on the Parent/non-template strand and cleaves a phosphodiester bond several bases away from the mismatch on the damaged strand.

3. Starting at the nick, helicase unwinds a segment of DNA containing the mismatch.

4. DNA pol III replaces the nucleotides 5’ to 3’ and ligase seals the strand.

Describe transcription coupled repair.

During transcription, RNA polymerase may run into damaged DNA and stall. This gives time for coupling proteins to find the site of damage and deploy repair machinery. When repaired, RNA pol backs up then resumes transcription.

DNA replication as well as environmental factors such as ionizing radiation can cause double-stranded DNA breaks. How is the DNA damage repaired, and what is this repair mechanism called?

By non-homologous end repair.

An endonuclease trims back the 3’ and 5’ ends of the break. Ku proteins (+ others) bind to the ends of the dna and further hydrolyze the strands to create complementary overlapping ends that can form H-bonds. Ligase then links each strand back together, but this results in nucleotide deletions.

What kind of DNA damage can be repaired by homologous recombination? What is the role of Rad51 in eukaryotes (RecA in prokaryotes and RadA in archaea) in repairing DNA damage?

Does the homologous repair mechanism result in DNA deletion?

Single-strand breakage at replication fork, double-strand breakage.

Rad51 proteins facilitate crossing over in meiosis 1 and function in homologous DNA repair. Rad51 (RecA and RadA) proteins bind to single-stranded 3’ DNA overhangs and form a nucleoprotein filament. The nucleoprotein filament searches for a homologous sequence and promotes strand invasion of the homologous dna duplex. DNA polymerase can then extend from the 3’ end of the invading strand.

NO deletions

DNA gyrase is a type of topoisomerase that releives strain specifically from…?

Helicase unwinding DNA during replication.

What kind of DNA damage does UV light cause?

pyrimidine dimers