CHAPTER 7 - THE GENETIC MATERIAL

Watson, Crick, and Rosalind Franklin

additionally, x-ray diffraction data "obtained" from maurice wilkins and rosalind franklin suggested that: - DNA is a long a skinny molecule that has two similar parts which are parallele to one another along their lengths (long axis) - the molecule is helical (or spiral like) - The diameter of the molecule is consistent along its length with a pyrimidine+purine at its center, but not a purine and pyrimidine pair up.

Topoisomerase

corrects "overwinding" ahead of replication forks by breaking, swiveling, and rejoining DNA strands works at the region ahead of the replication fork to prevent supercoiling

how double helix is formed

each unit in the backbone is linked by 5' to 3' phosphodiester bonds The two helices(sugar phosphate backbones) are held together by *hydrogen bonds* between the two bases. each base pair consists of one purine and one pyrimidine: G pairs with C. A pairs with T. Complementary base pairing. Different number of hydrogen bonds for base pairs. The bases are stacked one on top of another, which adds greatly to the stability of the molecule by excluding water The two sugar phosphate backbones run in opposite direction, which is why they are anti parallel. due to more hydrogen bonds, DNA ritch with G and C bind more tightly and are more stable than A and T. each strand of DNA is polar as a result of the phosphate ends. These phosphate ends also determine the directionality of the strands.

end replication problem

eukaryotic chromosomes have a rod shaped end, posing a problem for DNA replication. The DNA at the very end of the chromosome cannot be fully copied in each round of replication, which results in a gradual shortening of the chromosome. WHY? in the lagging strand when the replication fork reaches the end of the chromosome, there is a short stretch of DNA that does not get covered by an Okazaki fragment (okazaki fragments can later be replaced by DNA after replication)- essentially there's no way to get the fragment started because the primer would fall beyond the end of the chromosome. Additionally, the primer of the last Okazaki fragment that does get made cannot be replaced with DNA like in other primers. thanks to these problems, part of the DNA at the end of a eukaryotic chromosome goes uncopied in each round of replication, leaving a single stranded over hand. Over multiple rounds of cell division the chromosome will get shorter and shorter as the process repeats itself. https://cdn.kastatic.org/ka-perseus-images/543664d6b0d7dde9627d6dc342d209291cf71133.png

Helicases

first replication enzyme to load on at the origin of replication enzymes that untwist the double helix at the replication forks it opens up the DNA at the replication fork moves the replication fork forward by unwinding the DNA, meaning it breaks the hydrogen bonds between nitrogenous base pairs.

Meselson and Stahl results

generation 0 - only heavy band in the center of the tube. (all N15) generation 1 - single band but intermediately between light and heavy (mix of N14 and N15). HYBRID. Reflects the dispersive and semi conservative models generation 2 - two bands. One as the same intermediate as before. the second was higher up (only N14) *each strand in a DNA molecule serves as a template for synthesis of a new, complementary strand.

semi-conservative replication

in each new DNA double helix, one strand is from the original molecule, and one strand is new Semi-conservative replication. In this model, the two strands of DNA unwind from each other, and each acts as a template for synthesis of a new, complementary strand. This results in two DNA molecules with one original strand and one new strand. First generation is the same as the dispersive model - a hybrid mix - which is why we need to observe the second generation to confirm. In the second generation there is a split between the two hybrid molecules in which 2 new pure light DNA strands appear at the top of the tube.

DNA polymerase I

removes the RNA primer and replaces it with DNA polymerase I at the end of replication This discontinuous synthesis results in the generation of fragments on the lagging strand called Okazaki fragments. DNA polymerase I recognizes a "nick" or break in the phosphate backbone, and then removes each RNA primer and fills the gaps with DNA. DNA ligase then covalently links the phosphate backbone.

dispersive replication

replication results in both original and new DNA dispersed among the two daughter strands Dispersive replication. In the dispersive model, DNA replication results in two DNA molecules that are mixtures, or "hybrids," of parental and daughter DNA. In this model, each individual strand is a patchwork of original and new DNA. since the strands are a mixture, there is a midpoint between the two of them until all of the heavy (old) DNA is completely gone.

DNA polymerase (III)

synthesizes new DNA only in the 5' to 3' direction extends the primers, adding on to the 3' end, to make the bulk of the new DNA. - always requires a pre-existing strand with a free 3' end in oder to extend. It cannot create a new strand on its own. - can only add nucleotides to the 3' end of a DNA strand OR 5' to 3' direction - require an RNA primer to begin - they proofread or check their work and remove a large majority of "wrong" nucleotide s that are accidentally added.

Telomere lengthening

telomeres need to be protected from a cell's DNA repair systems because they have single stranded over-hangs which can appear to look like damaged DNA. the overhang at the lagging strand end of the chromosome is due to incomplete replication Once the overhang is lengthened, primase the DNA polymerase can create complementary strands. these overhangs bind to complementary repeats in the nearby double stranded DNA, causing telomere ends to form protective loops.

lagging strand

the alternative new strand which runs in the 5' to 3' direction AWAY FROM THE FORK. this strand must be made in fragments because, as the fork moves forward, the DNA polymerase moving away from the fork has to come off and reattach on the newly exposed DNA. this strand is called the *lagging strand*

Leading Strand

the new strand which runs in the 5' to 3' direction towards the replication fork easy and continuous replication because it moves in the same direction as the replication fork. this continuously synthesized strand is the *leading strand*

conservative replication

the parental molecule serves as a template for the synthesis of an entirely new molecule Conservative replication. In this model, DNA replication results in one molecule that consists of both original DNA strands (identical to the original DNA molecule) and another molecule that consists of two new strands (with exactly the same sequences as the original molecule). lighter DNA new strand on the top. Older strand on the bottom of the testube.

Telomeres

the problem of loss of DNA at the end of chromosomes is solved with telomeres. The chromosomes are protected by long stretches of this repetitive DNA. To prevent the loss of genes as the chromosome ends wear down, the tips of eukaryotic chromosomes have specialized DNA "caps" called telomeres. telomeres consist of many repeats of the same short DNA sequence, which varies between organisms but is 5' TTAGGG 3' in humans and other mammals the repeats that make up a telomere are eaten away slowly over many division cycles, providing a buffer that protects the internal chromosomal region bearing the genes.

Okazaki fragments

the small fragments located on the lagging strand. while the leading strand can be extended from one prime alone, the lagging strand needs a new primer for each short okazaki fragment

discovering the genetic material

there was a controversy over what the genetic material really was, and it was shown differently by three independent experiments by three different researchers. 1. *The Griffith experiment (1928)* 2. *The Avery, MacLeod, and McCarthy experiment (1944)* 3. *The Hershey-Chase experiment (1952)*

Avery-MacLeod-McCarty experiment

they began with large cultures of heat-killed S cells and, through a long series of biochemical steps (determined by careful experimentation), progressively purified the transforming principle by washing away, separating out, or enzymatically destroying the other cellular components. By this method, they were able to obtain small amounts of highly purified transforming principle, which they could then analyze through other tests to determine its identity

what was known before DNA

*1.*we knew that discrete hereditary factors (genes) were associated with specific traits and linearly arrayed, but we did not know or understand their physical nature *2.* we knew that genes are carried on chromosomes, which are made of DNA and protein *3.* we knew that genes specify the structure of proteins (one gene - one protein hypothesis) *4.* we knew that there are three minimum requirements for genetic material:

Watson and Crick's structural model

- DNA is composed of four nucleotide: 1. dAMP (purine) 2. dGMP (purine) 3. dCMP (pyrimidine) 4. dTMP (pyrimidine)

three required properties of genetic material

1. *structure* must allow for faithful replication - since every cell carries copies of genetic material, it is copies up to trillions of times - mistakes in this process of copying should be rare. 2. *information content* - there must be a code that simplifies the complexity of the output or information. 3. *ability to change* - clearly each individual has different, yet still heritable traits. - new mutants appear regularly. - even though we don't want a lot of change, we know that it can change. Each gene is a little different between individuals.

why the transforming principle might be DNA?

1. The purified substance gave a negative result in chemical tests known to detect proteins, but a strongly positive result in a chemical test known to detect DNA. 2. The elemental composition of the purified transforming principle closely resembled DNA in its ratio of nitrogen and phosphorous. 3. Protein- and RNA-degrading enzymes had little effect on the transforming principle, but enzymes able to degrade DNA eliminated the transforming activity.

Mechanisms of DNA replications

1. helicases 2. single-strand binding proteins 3. topoisomerases 4. Primase 5. Polymerase (III) enzymes 6. Polymerase (I) 6. Okazaki Fragments 7. Ligase

primer

A short segment of DNA that acts as the starting point for a new strand

Telomerase

An enzyme that catalyzes the lengthening of telomeres in eukaryotic germ cells. it is an RNA dependent DNA polymerase, meaning an enzyme that can make DNA using RNA as a template.

Ligase

An enzyme that connects two fragments of DNA to make a single fragment the nicks that remain after the primers are replaced get sealed up by the enzyme DNA ligase.

Primase

An enzyme that joins RNA nucleotide to make the primer using the parental DNA strand as a template. synthesizes RNA primers that are complementary to the DNA strand DNA polymerases can only add nucleotide to the 3' end of an existing DNA strand, but they aren't able to add the first nucleotide alone. Instead, the primase enzyme makes a short stretch of nucleic acid complementary to the template that provides a 3' end of the DNA polymerase to start from. This is called the *RNA primer* The primase adds RNA primers to both strands of unzipped DNA.

Griffith Experiment Process

As part of his experiments, Griffith tried injecting mice with heat-killed S bacteria (that is, S bacteria that had been heated to high temperatures, causing the cells to die). Unsurprisingly, the heat-killed S bacteria did not cause disease in mice. The experiments took an unexpected turn, however, when harmless R bacteria were combined with harmless heat-killed S bacteria and injected into a mouse. Not only did the mouse develop pneumonia and die, but when Griffith took a blood sample from the dead mouse, he found that it contained living S bacteria!

replication forks

As the DNA unravels and opens up, 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 in both directions. at that point the helicases is then activated at the origin of replication and gets the process going. At the replication fork there are two molecules of DNA polymerase, each responsible for one of the two new DNA strands

Why was DNA importance a surprise?

BECAUSE 1. proteins do everything else in the cell 2. proteins make up 90% of the mass of the chromosome 3. DNA repetitive strings with simple (boring) structure 4. DNA seemed to have low information content (4 nucleotides vs. 20 amino acids) 5. It was only thought to be a structural component of a chromosome

Messelson-Stahl Experiment

Brainstormed 3 possible models (conservative, semiconservative, and dispersive) to explain DNA replication. Conducted an experiment to determine which one was correct. They used isotopes of nitrogen to tell old DNA and new DNA apart. new DNA was lighter bc it was prepared with lighter nitrogen and the old was prepared with heavy nitrogen. (Processes explained in specific flashcards)

Single Strand binding proteins

Coat the DNA around the replication fork to prevent rewinding of the DNA back into a double helix structure

Origins of Replication

How do DNA polymerases and other replication factors know wherer to begin? These specific locations on the DNA are called the *origins of replication* and are recognized by their unique sequence this unique seqeuence is recognized by specialized proteins that bind to the site and begin to open up and unravel the double stranded DNA.

The Griffith Experiment

In 1928, British bacteriologist Frederick Griffith conducted a series of experiments using Streptococcus pneumonia bacteria and mice. Griffith wasn't trying to identify the genetic material, but rather, trying to develop a vaccine against pneumonia. In his experiments, Griffith used two related strains of bacteria, known as R and S.

Avery, MacLeod, McCarty

In 1944, three Canadian and American researchers, Oswald Avery, Maclyn McCarty, and Colin MacLeod, set out to identify Griffith's "transforming principle."

R strain (rough)

R strain. When grown in a petri dish, the R bacteria formed colonies, or clumps of related bacteria, that had well-defined edges and a rough appearance (hence the abbreviation "R"). The R bacteria were nonvirulent, meaning that they did not cause sickness when injected into a mouse.

S strain (smooth)

S bacteria formed colonies that were rounded and smooth (hence the abbreviation "S"). The smooth appearance was due to a polysaccharide, or sugar-based, coat produced by the bacteria. This coat protected the S bacteria from the mouse immune system, making them virulent (capable of causing disease). Mice injected with live S bacteria developed pneumonia and died.

Griffith experiment conclusion

Some substance in the heat-killed virulent strain transformed the non-virulent form into the virulent form. Griffith concluded that the R-strain bacteria must have taken up what he called a "transforming principle" from the heat-killed S bacteria, which allowed them to "transform" into smooth-coated bacteria and become virulent.

How does telomerase work?

Telomerase binds to a special RNA molecule that contains a sequence that is complementary to the repetitive sequence on the telomere. it adds nucleotides to and extends the overhanging strand of the telomere DNA using this complementary RNA strand as a template. once the overhang is long enough, a matching strand can be made by the normal DNA replication machinery (using an RNA primer and DNA polymerase) producing a double stranded DNA.

Ways in which DNA can be replicated

There were three basic models for DNA replication that had been proposed by the scientific community after the discovery of DNA's structure. These models are illustrated in the diagram below: Semi Conservative. Dispersive, and Conservative

Avery, MacLeod, McCarty Conclusions

These results all pointed to DNA as the likely transforming principle. However, Avery was cautious in interpreting his results. He realized that it was still possible that some contaminating substance present in small amounts, not DNA, was the actual transforming principle Because of this possibility, debate over DNA's role continued until 1952, when Alfred Hershey and Martha Chase used a different approach to conclusively identify DNA as the genetic material

Messelson and Stahl procedure

They began by growing E. coli in medium, or nutrient broth, containing a "heavy" isotope of nitrogen15(An isotope is just a version of an element that differs from other versions by the number of neutrons in its nucleus.) When grown on medium containing heavy Nitrogen 15, the bacteria took up the nitrogen and used it to synthesize new biological molecules, including DNA. After many generations growing in the N15 medium, the nitrogenous bases of the bacteria's DNA were all labeled with heavy N15. Then, the bacteria were switched to medium containing a "light" N14 and allowed to grow for several generations. DNA made after the switch would have to be made up of N14 as this would have been the only nitrogen available for DNA synthesis. The researcher know how often Ecoli cells divided, so they were able to collect small samples in each generation and extract and purify the DNA. Then they measured the density of the DNA using a density gradient centrifugation and from the density could conclude the percent of either its N14 or N15 content.

Hershey-Chase Experiment

Used radioactive material to label DNA and protein; infected bacteria passed on DNA; helped prove that DNA is genetic material not proteins The Hershey-Chase experiment demonstrated that the genetic material of phages is DNA, not protein. The experiment uses two sets of T2 bacteriophage. In one set, the protein coat is labeled with radioactive sulfur (35S), not found in DNA. In the other set, the DNA is labeled with radioactive phosphorus (32P), not found in amino acids. Only the 32P is recovered from the E. coli, indicating that DNA is the agent necessary for the production of new phages

Watson and Crick - Chargoff

Watson and Crick based their model on Chargaff's principle and rule for double stranded DNA. - total number of pyrimidines (T+C) = total number of purines (A+G) - A pairs with T - G pairs with C While the numbers of pyrimidines and purines must equal one another, they can vary across different species