CH 16 MOLECULAR BASIS OF INHERITANCE DNA 1

CH. 16 – MOLECULAR BASIS OF INHERITANCE (DNA) 1

Griffith - Transformation Since Morgan showed that genes are found on chromosomes, the new issue was whether the genetic material was in the proteins of the chromosomes or the DNA. Most thought the PROTEINS because there are so many more possibilities and at that time, they didn’t know a lot about DNA. Fredrick Griffith He concluded that there must be something from the dead bacteria that gets incorporated into the live bacteria to convert it from harmless into harmful…thus killing the mice. He called this process transformation. Griffith did an experiment where he used streptococcus pneumoniae (which is a bacteria that causes pneumonia in mammals) and injected it into mice. He used harmless strains, and the mice were fine. He used harmful strains, and the mice died. Then he used heat to kill the harmful bacteria and injected that into the mice. The mice were fine. Lastly, he mixed the dead harmful bacteria with live harmless bacteria and injected the 2 mixture into the mice. The mice died.

Transformation = the process when a cell takes up DNA from the environment and assimilates it into its own genome. It can then start to express some of these “newly acquired” genes on its own. Transformation was first observed by Fredrick Griffith with his mice. 3

Avery – Transforming Agent = DNA Oswald Avery wanted to know what this “transforming agent” that Griffith found was. He focused on DNA, RNA and proteins. He purified each of the chemicals from the harmful bacteria and used each one, individually, to try to transform the harmless bacteria into the virulent strain. The only molecule that successfully transformed the bacteria was DNA. Thus, he found that DNA was the transforming agent. Even though Avery and his team (Mc. Carty and Mac. Leod) proved that DNA was the transforming agent, people were still skeptical that it was DNA that carried the genetic material. Because they really didn’t know about the structure of DNA, they didn’t understand how it could possibly carry all that information. 4

Hershey and Chase – DNA is genetic material Alfred Hershey and Martha Chase did more work in determining that DNA was the part of the chromosome that carried the genetic information. They worked with the T 2 phage (a phage or bacteriophage is a virus that infects the bacteria E. coli) and exposed it to radioactive sulfur (which was incorporated into the protein coat) and radioactive phosphorus (which was incorporated into the DNA). They let the different phages infect different batches of non-radioactive E. coli cells. Soon after the infection, the cells were whirled in a blender and then centrifuged. In the centrifuge, the cells formed a pellet at the bottom and the rest of the liquid formed a supernatant. They then checked for radioactivity. Here were their results: 35 S (proteins) – radioactivity in supernatant 32 P (DNA) – radioactivity in pellet (cells) SO…THE PART THAT GOT PUT INTO THE BACTERIA CELLS WAS THE DNA (not protein)!!! 5

Bacteriophage A bacteriophage is a virus that infects bacteria. A common one is T 2, and this infects E. coli. Phages are used a lot in scientific experiments (we will see more of this in the next few chapters). 6

Hershey and Chase Experiment - Specifics 7

Chargaff – Base Pairing Rules Erwin Chargaff did work with DNA from many different types of organisms. He found that “In the DNA of any one species, the amounts of the four nitrogenous bases are not all equal but are present in a characteristic ratio. ” He found that in humans, A = 30. 9% 19. 8% T = 29. 4% 19. 9% This information helped Watson and Crick come up with the structure of DNA C= G= These are now known as Chargaff’s Rules. 8

Franklin & Wilkins – X-ray crystallograph The race to find the correct structure of DNA was in full swing in the 1950’s. Linus Pauling came up with one idea that had 3 chains all linked together. This was incorrect. Jim Watson and Francis Crick visited Cambridge where they saw an Xray picture of DNA done by Rosalind Franklin (this was unpublished work). They knew she had concluded that the sugar-phosphate backbones were on the outside of the double helix. This was enough information to determine that the shape was a helix and Jim Watson was able to calculate important information from this image about DNA. Rosalind Franklin Maurice Wilkins X-ray crystallography is a technique where a picture is made as X-rays are deflected off of purified DNA samples. The patterns can be mathematically analyzed to figure out important dimensions of the molecules. 9

Watson and Crick – DNA Structure The nitrogenous bases are paired in specific combinations: adenine with thymine and guanine with cytosine. Only a pyrimidine-purine pair produces the diameter indicated by the Xray data. Based on details of their structure, adenine forms two hydrogen bonds only with thymine, and guanine forms three hydrogen bonds only with cytosine (Chargaff’s Rules. ) The base-pairing rules dictate the combinations of nitrogenous bases that form the “rungs” of DNA. The linear sequence of the four bases can be varied in countless ways, and each gene has a unique order of nitrogenous bases. Using information from Chargaff, Franklin, and Wilkins, Jim Watson and Francis Crick discovered the structure of DNA. They found that the bases match up on the inside of the chain, and the phosphates and sugars are on the outside of the chain. They also figured out that the strands are antiparallel, with the subunits running in the opposite direction. Along with Wilkins, they won the Nobel Prize for their work. With their idea, they also proposed the semi-conservative model of DNA Replication. While Jim Watson is still living, Francis Crick died 10 in 2004.

Meselson & Stahl – DNA Replication Watson and Crick proposed a method by which DNA replicates called the semi-conservative model. Meselson and Stahl came up with an experiment to support their hypothesis. The 3 models of DNA replication are: -Conservative – one whole piece of DNA has both of the strands from the parent DNA and the other one has two totally new strands - Dispersive – random pieces from the parent DNA show up in the new DNA in both strands - Semiconservative – Each new DNA molecule has one strand from the parent and one strand that’s new 11

Meselson & Stahl Experiment Proved the Semiconservative model of DNA replication They cultured E. coli for a few generations using a medium with “heavy nitrogen” (15 N). This way, the “parent” DNA would have this heavier type of nitrogen. They then put the bacteria into a medium with lighter nitrogen (14 N). This way, any NEW DNA they made would have the 14 N in it, rather than 15 N. They could then test the densities of the DNA by extracting it from the cells and centrifuging it. These are their results. 12

Models of DNA Replication Watson and Crick hypothesized that the semiconservative model was how DNA replicated. Meselson and Stahl confirmed this when they did their experiments using the heavy isotope of nitrogen. 13

Structure of DNA The structure of DNA is a double helix. It is two chains held together in the middle by hydrogen bonds between nitrogenous bases. The backbone of the molecule is made up of phosphates and sugars. The bond between the phosphates and sugars is called the phosphodiester linkage. A nucleotide is the basic unit of DNA. It is composed of a sugar, phosphate, and nitrogen base. Between A-T, there are 2 hydrogen bonds; Between C-G, there are 3 hydrogen bonds holding the bases together. 14

Double Helix 15

DNA Replication in PROKARYOTES The replication of a DNA molecule in PROKARYOTES begins at a special site called the origin of replication. � Enzymes separate the strands, forming a replication “bubble. ” � Replication proceeds in both directions until the entire molecule is copied. � 16

DNA Replication in Eukaryotes The Beginning There is a special site where replication starts. This sequence of DNA is recognized by proteins that come in and separate the 2 strands. Replication proceeds both ways. This is called the origin of replication; or the replication bubble and forms a replication fork. In eukaryotes, there a lots of origins, but in prokaryotes, there is only one origin. 17

� Several kinds of proteins participate in the unwinding of parental strands of DNA. � Helicases untwist the double helix and separate the template DNA strands at the replication fork. � Single-strand binding proteins bind to unpaired DNA strands, stabilizing them. � Unwound sections of parental DNA strands are now available to serve as templates for the synthesis of new complementary DNA strands. Unwinding DNA 18

Primers The enzymes that synthesize DNA cannot initiate synthesis of a polynucleotide. They can only add nucleotides to the end of an existing chain that is base-paired with the template strand. The initial nucleotide chain is a short stretch of RNA called a primer, synthesized by the enzyme primase. Primase starts a complementary RNA chain from a single RNA nucleotide, adding RNA nucleotides one at a time, using the parental DNA strand as a template. The RNA primers are eventually replaced with DNA nucleotides by DNA polymerase. 19

DNA Replication – Elongating the Strand Enzymes called DNA polymerases catalyze the synthesis of new DNA by adding nucleotides to a preexisting chain. (NOTE: there are many DNA polymerases in eukaryotes) The two strands of DNA in the double helix are antiparallel. 20

Antiparallel Strands of DNA The two strands of DNA run antiparallel – think 2 pencils going in the opposite directions. 5’ End = Free Phosphate 3’ End = Free Hydroxyl Group 21

Replication � � � DNA polymerases can add nucleotides only to the free 3 end of a growing DNA strand. A new DNA strand can elongate only in the 5 3 direction. Along one template strand, DNA polymerase can synthesize a complementary strand continuously by elongating the new DNA in the mandatory 5 3 direction. 22

Leading Strand vs. Lagging Strand The leading strand is a continuous strand is elongating TOWARDS the replication fork. It only needs ONE primer. The lagging strand is a continuous strand is elongating AWAY from the replication fork. Small Okazaki fragments are needed…each one using a NEW primer. Since DNA Polymerase can only add to the 3’ end, only the strand with the 3’ end going towards the replication fork can add continuously. 23

Okazaki fragments � � Unlike the leading strand, which elongates continuously, the lagging stand is synthesized as a series of short segments called Okazaki fragments. Although only one primer is required on the leading strand, each Okazaki fragment on the lagging strand must be primed separately. Another DNA polymerase, replaces the RNA nucleotides of the primers with DNA versions. DNA ligase joins the sugar-phosphate backbones of all the Okazaki fragments into a continuous DNA strand. 24

Proteins used for Replication Important Enzymes: - DNA Polymerase - Ligase - Primase - Helicase - Single Strand Binding Proteins DNA Polymerase 25

Proofreading in Replication Sometimes the wrong nucleotide is placed in a spot, or sometimes the nucleotides are changed by UV light or other radioactive substances. DNA polymerase proofreads each new nucleotide against the template nucleotide as soon as it is added If there is an error, the part with the mistake is cut out using the enzyme nuclease. Then DNA polymerase fills in the gap with the correct nucleotides. Ligase then attaches the backbones to make one strand. This is called nucleotide excision repair. (an example of mismatch repair) 26

� � Mutations can change the phenotype of an organism. If they occur in germ cells, which give rise to gametes, they can be passed on from generation to generation. The vast majority of such changes are harmful, but a very small percentage can be beneficial. Mutations are the source of the variation on which natural selection operates during evolution and are ultimately responsible for the appearance of new species. The balance between accuracy of DNA replication or repair and a low mutation rate has allowed the evolution of the rich diversity of species we see on Earth today. 27

Shortening of DNA Strands Because DNA Polymerase can only add to the 3’ end, the 5’ end can never be complete. Therefore, after many, many replications, the DNA strands become shorter. To prevent genes from eroding, chromosomes have telomeres. 28

Telomeres – NON CODING! Telomeres are segments of non-coding DNA that are found in eukaryotic chromosomes at the 5’ end. They usually consist of 1000’s of copies of a repetitive sequence. They act as buffers to protect the organisms genes. Because they become shorter with every round of replication, telomeres are lengthened by the enzyme telomerase. This enzyme is found in embryonic cells, and in adults, in germ – line cells (not somatic cells). Normal shortening of telomeres may protect organisms from cancer by limiting the number of divisions that somatic cells can undergo. 29

Prokaryotes � � Most bacteria have a single, circular, doublestranded DNA molecule associated with a small amount of protein. A bacterium has a dense region of DNA called the nucleoid. 30

Structure of Chromatin In the cell, eukaryotic DNA is packaged with protein to form chromatin. The packing of chromatin varies during the course of the cell cycle. Although interphase chromatin is generally much less condensed than the chromatin of mitotic chromosomes, it shows several of the same levels of higher-order packing. Levels of Chromatin Packing: 1. Nucleosome 2. 30 nm chromatin fibers 3. Looped Domains 4. Chromosomes 31

Heterochromatin vs. Euchromatin Heterochromatin – very tightly coiled; therefore it is NOT transcribed Euchromatin – “true chromatin”; it is less compact and therefore the RNA polymerase can attach and it can get transcribed; more loosely packed 32

Energy Source for DNA Replication The nucleotides that get added by DNA Polymerase are actually nucleoside triphosphates (NTP). These are nucleotides with 3 phosphates instead of 1. They are used as an energy source. The “extra” 2 phosphates break off to release energy (break bond = releases energy) NTP – nucleoside triphosphate The 2 phosphate molecule is called pyrophosphate. Pyrophosphate 33