THE MOLECULAR BASIS OF INHERITANCE

The search for genetic material led to DNA
_ Until the 1940s, the great variety of proteins seemed to indicate that proteins were the genetic material.
_ The discovery of the genetic role of DNA began with research by Griffith in 1928.
_ He studied Streptococcus pneumoniae, a bacterium that causes pneumonia in mammals. Fig 16.1.

_ the R strain, was harmless.

_ the S strain, was pathogenic.

_ Griffith mixed heat-killed S strain with live R strain bacteria and injected this into a mouse.
_ The mouse died and he recovered the pathogenic strain from the mouse's blood.
_ Griffith called this phenomenon transformation, a change in genotype and phenotype due to the assimilation of a foreign substance (now known to be DNA) by a cell.

_ In 1944, Avery, McCarty and MacLeod, after years of experiments with bacteria, announced that the transforming substance was DNA. Many were still skeptical.

_ Further evidence that DNA was the genetic material was derived from studies that tracked the infection of bacteria by viruses.
_ Viruses consist of a DNA molecule (sometimes RNA) enclosed by a protective coat of protein.
_ To replicate, a virus infects a host cell and takes over the cell's metabolic machinery. Movie!
_ Viruses that specifically attack bacteria are called bacteriophages or just phages. Fig 16.2a

_ In 1952, Hershey and Chase showed that DNA was the genetic material of the phage T2. Fig 16.2b
_ To determine the source of genetic material in the phage, Hershey and Chase designed an experiment where they could label protein or DNA and then track which entered the E. coli cell during infection.

_ They grew one batch of T2 phage in the presence of radioactive sulfur, marking the proteins but not DNA.

_ They grew another batch in the presence of radioactive phosphorus, marking the DNA but not proteins.

_ They allowed each batch to infect separate E. coli cultures.

_ They spun the cultured infected cells in a blender, shaking loose any parts of the phage that remained outside the bacteria.

_ The mixtures were spun in a centrifuge which separated the heavier bacterial cells in the pellet from lighter free phages and parts of phage in the liquid supernatant.

_ Hershey and Chase found that when the bacteria had been infected with T2 phages that contained radio-labeled proteins, most of the radioactivity was in the supernatant, not in the pellet.

_ When they examined the bacterial cultures with T2 phage that had radio-labeled DNA, most of the radioactivity was in the pellet with the bacteria.

_ Hershey and Chase concluded that the injected DNA of the phage provides the genetic information that makes the infected cells produce new viral DNA and proteins, which assemble into new viruses.

_ Other evidence - cells double the amount of DNA in a cell prior to mitosis and then distribute the DNA equally to each daughter cell.
_ Diploid sets of chromosomes have twice as much DNA as the haploid sets in gametes of the same organism.

_ By 1947, Chargaff had developed a series of rules based on a survey of DNA composition in organisms.

_ He already knew that DNA was a polymer of nucleotides consisting of a nitrogenous base, deoxyribose, and a phosphate group. Fig 16.3

_ The bases could be adenine (A), thymine (T), guanine (G), or cytosine (C).

_ Chargaff noted that the DNA composition varies from species to species.

_ In any one species, the four bases are found in characteristic, but not necessarily equal, ratios.

_ He also found a regularity in the ratios of nucleotide bases which are known as Chargaff's rules.

_ The number of adenines was approximately equal to the number of thymines (%T = %A).

_ The number of guanines was approximately equal to the number of cytosines (%G = %C).

_ Human DNA is 30.9% adenine, 29.4% thymine, 19.9% guanine and 19.8% cytosine.

_ Wilkins and Franklin used X-ray crystallography to study the structure of DNA. Fig 16.4

_ Watson and Crick, building on the work of Wilkins and Franklin, began to work on a model of DNA with two strands, the double helix. Fig 16.5.
_ The key breakthrough came when Watson put the sugar-phosphate chain on the outside and the nitrogen bases on the inside of the double helix.

_ The sugar-phosphate chains of each strand are like the side ropes of a rope ladder.

_ Pairs of nitrogen bases, one from each strand, form rungs.

_ The ladder forms a twist every ten bases.

_ The nitrogenous bases are paired in specific combinations: adenine with thymine and guanine with cytosine.

_ Pairing like nucleotides did not fit the uniform diameter indicated by the X-ray data.

_ A purine-purine pair would be too wide and a pyrimidine-pyrimidine pairing would be too short.

_ Only a pyrimidine-purine pairing would produce the 2-nm diameter indicated by the X-ray data.

_ In addition, Watson and Crick determined that chemical side groups of the nitrogen bases would form hydrogen bonds, connecting the two strands. Fig 16.6.

_ Based on details of their structure, adenine would form two hydrogen bonds only with thymine and guanine would form three hydrogen bonds only with cytosine.

_ This finding explained Chargaff's rules.

_ The linear sequence of the four bases can be varied in countless ways.

_ Each gene has a unique order of nitrogen bases.

DNA replication
_ In a second paper Watson and Crick published their hypothesis for how DNA replicates.

_ When a cell copies a DNA molecule, each strand serves as a template for ordering nucleotides into a new complementary strand. Fig 16.7.
_ Watson and Crick's model, semiconservative replication, predicts that when a double helix replicates, each of the daughter molecules will have one old strand and one newly made strand. Fig 16.8.
_ Other competing models, the conservative model and the dispersive model, were also proposed.
_ Experiments in the late 1950s by Meselson and Stahl supported the semiconservative model, proposed by Watson and Crick, over the other two models.

A large variety of enzymes and other proteins carries out DNA replication

_ It takes E. coli less than an hour to copy each of the 5 million base pairs in its single chromosome and divide to form two identical daughter cells.
_ A human cell can copy its 6 billion base pairs and divide into daughter cells in only a few hours, with only one error per billion nucleotides.

_ More than a dozen enzymes and other proteins participate in DNA replication.

_ The replication of a DNA molecule begins at special sites, origins of replication. Fig 16.10. Movie!

_ In bacteria, this is a single specific sequence of nucleotides that is recognized by the replication enzymes.

_ These enzymes separate the strands, forming a replication "bubble".

_ Replication proceeds in both directions until the entire molecule is copied.

_ In eukaryotes, there may be hundreds or thousands of origin sites per chromosome.

_ At the origin sites, the DNA strands separate forming a replication "bubble" with replication forks at each end.

_ The replication bubbles elongate as the DNA is replicated and eventually fuse.

_ DNA polymerases catalyze the elongation of new DNA at a replication fork.

_ Nucleotides are added to the growing end of the new strand by the polymerase.

_ The raw nucleotides are nucleoside triphosphates.

_ Each has a nitrogen base, deoxyribose, and a triphosphate tail.

 

_ As each nucleotide is added, the last two phosphate groups are hydrolyzed to form pyrophosphate. Fig 16.11.

 

_ The exergonic hydrolysis of pyrophosphate to two inorganic phosphate molecules drives the polymerization of the nucleotide to the new strand.

_ The strands in the double helix are antiparallel. Fig 16.12.

_ The sugar-phosphate backbones run in opposite directions.

_ Each DNA strand has a 3' end with a free hydroxyl group attached to deoxyribose and a 5' end with a free phosphate group attached to deoxyribose.

_ DNA polymerases can only add nucleotides to the free 3' end of a growing DNA strand. Fig 16.13.

_ A new DNA strand can only elongate in the 5'->3' direction.

_ At the replication fork, one parental strand (3'-> 5' into the fork), the leading strand, can be used by polymerases as a template for a continuous complementary strand. Movie!

_ The other parental strand (5'->3' into the fork), the lagging strand, is copied away from the fork in short segments (Okazaki fragments). Movie!

_ Okazaki fragments, each about 100-200 nucleotides, are joined by DNA ligase to form the sugar-phosphate backbone of a single DNA strand.

To start a new chain requires a primer, a short segment of RNA. Fig 16.14.

_ The primer is about 10 nucleotides long in eukaryotes.

_ Primase, an RNA polymerase, links ribonucleotides into the primer.

_ Another DNA polymerase later replaces the primer ribonucleotides.

_ The leading strand requires the formation of only a single primer.

_ The lagging strand requires formation of a new primer as the replication fork progresses.

_ After the primer is formed, DNA polymerase can add new nucleotides away from the fork until it runs into the previous Okazaki fragment.

_ The primers are converted to DNA before DNA ligase joins the fragments together.

_ The lagging strand is copied away from the fork in short segments, each requiring a new primer.

Summary of replication. Fig 16.16.    Movie!

The main proteins of replication. Fig 16.15.

In addition to primase, DNA polymerases, and DNA ligases, several other proteins have prominent roles in DNA synthesis.

_ A helicase untwists and separates the template DNA strands at the replication fork.
_ Single-strand binding proteins keep the unpaired template strands apart during replication.

DNA polymerase proofreads each new nucleotide against the template nucleotide as soon as it is added.

_ If there is an incorrect pairing, the enzyme removes the wrong nucleotide and then resumes synthesis.

_ The final error rate is only one per billion nucleotides.

_ DNA molecules are constantly subject to potentially harmful chemical and physical agents.

_ Mismatched nucleotides that are missed by DNA polymerase or mutations that occur after DNA synthesis is completed can often be repaired.

_ Each cell continually monitors and repairs its genetic material, with over 130 repair enzymes identified in humans.

_ In mismatch repair, special enzymes fix incorrectly paired nucleotides.

_ A hereditary defect in one of these enzymes is associated with a form of colon cancer.

_ In nucleotide excision repair, a nuclease cuts out a segment of a damaged strand. Fig 16.17.

_ The gap is filled in by DNA polymerase and ligase.

_ The importance of the proper functioning of repair enzymes is clear from the inherited disorder xeroderma pigmentosum.

_ In individuals with this disorder, mutations in their skin cells are left uncorrected and cause skin cancer.

The ends of DNA molecules are replicated by a special mechanism. Fig 16.18.

_ The usual replication machinery provides no way to complete the 5' ends of daughter DNA strands.

_ Repeated rounds of replication produce shorter and shorter DNA molecules.

_ The ends of eukaryotic chromosomal DNA molecules, the telomeres, have special nucleotide sequences. Fig 16.19b.

_ In human telomeres, this sequence is typically TTAGGG, repeated between 100 and 1,000 times.

_ Telomeres protect genes from being eroded through multiple rounds of DNA replication, providing a mechanism to restore shortened telomeres.

_ Telomerase uses a short molecule of RNA as a template to extend the 3' end of the telomere.

_ There is now room for primase and DNA polymerase to extend the 5' end.

_ Telomerase is present in germ cells, ensuring that zygotes have long telomeres.

_ Active telomerase is also found in cancerous somatic cells.

_ This overcomes the progressive shortening that would eventually lead to self-destruction of the cancer.

_ Telomerase is not present in most cells of multicellular organisms.

_ Therefore, the DNA of dividing somatic cells and cultured cells does tend to become shorter.

_ Telomere length may be a limiting factor in the life span of certain tissues and of the organism.