Chapter 10

The Structure, Replication and Repair

of DNA

Key Questions

• How did biologists discover what genes were made of and what they did?

• What is the structure of DNA?

• How does DNA’s structure allow it to act as a template for its own replications?

• What is a mutation and why are mutations important?

Structure of DNA-Overview• Each nucleotide of DNA

consists of:– A sugar —

deoxyribose– A phosphate– A base — there are 4

bases; • 2 of the 4 bases are

pyrimidines: cytosine (C) and thymine (T);

• The other 2 bases are purines: denine(A) and guanine (G)

Structure of DNA-A Historic Story (1)• Late 1800s scientists postulated a biochemical basis

– Friedrich Miescher (1869): isolated DNA (called it nucleic acid)– Researchers became convinced chromosomes carry genetic information

• 1920s to 1940s expected the protein portion of chromosomes to be the genetic material

• Late 1920s Frederick Griffith was working with Streptococcus pneumoniae肺炎鏈球菌

– Strains that secrete capsules look smooth and can cause fatal infections in mice

– Strains that do not secrete capsules look rough and infections are not fatal in mice

• Griffith’s experiments (next page) showed that:– Genetic material from the heat-killed type S bacteria had been

transferred to the living type R bacteria– This trait gave them the capsule and was passed on to their

offspring• Griffith did not know the biochemical basis of his transforming

principle

• Rough strains (R) without capsule are not fatal– No living bacteria found

in blood• Smooth strains (S) with

capsule are fatal– Capsule prevents

immune system from killing bacteria

– Living bacteria found in blood

• If mice are injected with heat-killed type S, they survive

• Mixing live R with heat-killed S kills the mouse– Blood contains living

S bacteria– Transformation

Griffith’s Bacterial Transformations

Structure of DNA-A Historic Story (2)• Avery, MacLeod, and McCarty used purification methods to reveal that

DNA is the genetic material– 1940s interested in bacterial transformation– Only purified DNA from type S could transform type R– Purified DNA might still contain traces of contamination that may be

the transforming principle– Added DNase, RNase and proteases– RNase and protease had no effect– With DNase no transformation– DNA is the genetic material

• 1952, Hershey and Chase studied T2 virus infecting Escherichia coli– Bacteriophage or phage– Phage coat made entirely of protein

• DNA found inside capsid

Structure of DNA-A Historic Story (3)• 1952, Hershey and Chase studied T2 virus infecting Escherichia coli

– Bacteriophage or phage– Phage coat made entirely of protein

• Bacteriophage– A virus that infects bacteria– Viruses are composed of protein and DNA (or RNA)– Viruses are capable of forcing host cells to make more viruses– Viruses are not living organisms

Structure of DNA-A Historic Story (3)• Shearing force from a blender will separate the phage coat from the

bacteria• 35S will label proteins only; 32P will label DNA only• Experiment to find what is injected into bacteria- DNA or protein?• DNA found inside capsid: Results support DNA as the genetic material

Hershey and Chase — The Blender Guy/Gal

Structure of DNA-A Historic Story (3)

Structure of DNA-A Historic Story (4)• Chagaff’s Rules:• Found the proportions of

the bases in many different species

• The amount of A is equal to T

• The amount of G is equal to C

Structure of DNA-A Historic Story (5)• Solving DNA structure:• 1953, James Watson and Francis Crick, with Maurice Wilkins, proposed

the structure of the DNA double helix• Watson and Crick used Linus Pauling’s method of working out protein

structures using simple ball-and-stick models• Rosalind Franklin’s X-ray diffraction results provided crucial

information • Erwin Chargoff analyzed base composition of DNA that also provided

important information

Rosalind Franklin’s Contribution• Crystallographer• Accurately measured the

density of DNA, the number of water molecules

• Discovered that DNA had 2 slightly different structures

• Used Franklin’s data and work extensively, but did not cite it

• Applied Chargaff’s Rules• Built several models of DNA• Found ball-and-stick model

consistent with data• Watson and Crick awarded

Nobel Prize in 1962• Rosalind Franklin had died

and the Nobel is not awarded posthumously

Structure of DNA-A Historic Story (5)

DNA Structure — Linkages• DNA is

– Double stranded– Helical– Sugar-phosphate backbone– Bases on the inside– Stabilized by hydrogen bonding– Base pairs with specific pairing– AT/GC or Chargoff’s rule– A pairs with T– G pairs with C– Keeps with consistent– 10 base pairs per turn– 2 DNA strands are complementary– 5’ – GCGGATTT – 3’– 3’ – CGCCTAAA – 5’– 2 strands are antiparallel– One strand 5’ to 3’– Other stand 3’ to 5’

Computer Generated Model of DNA

Figure 10-9

What Is a Gene?• To Mendel, in 1865, it was just an abstraction

– Places on chromosomes– Pure information– Important to understand genes in chemical terms

• 1908, Archbold Garrod proposed relationship between genes and the production of enzymes

• Studied patients with metabolic defects• Alkaptonuria- patient’s body accumulates abnormal levels of

homogentisic acid (alkapton)• Hypothesized disease due to missing enzyme• Knew it had a recessive pattern of inheritance• Inborn error of metabolism

What Causes Alkaptonuria?

Biochemical Importance of Genes

• Alkaptonuria — black urine stains• Garrod suspected that it might be inherited• Caused by a recessive allele• A crucial enzyme is missing; HA accumulates in

the body and is excreted in the urine• Suggested that genes might work by specifying

enzymes

1 Gene — 1 Enzyme• In the early 1940s, George

Beadle and Edward Tatum rediscovered Garrod’s work, using Neurospora crassa(common bread mold), showed that 1 gene could specify 1 enzyme

• Minimum requirements for growth are carbon source (sugar), inorganic salts, and biotin

• Mutant strains would be unable to grow unless supplemented

• Compare to wild-type or normal

• A single mutation resulted in the requirement for a single type of vitamin

• Stimulated research into other substances including arginine, an amino acid

• Isolated several mutants requiring arginine for growth• Examined for ability to grow in the presence of precursors• 3 groups based on requirements• Beadle and Tatum conclude that single gene controls the

synthesis of a single enzyme– One gene – one enzyme hypothesis

1 Gene — 1 Enzyme

1 Gene — 1 Polypeptide• Sickle Cell Anemia• Differences in gene has caused

differences in the hemoglobin protein, not in the enzyme

• One gene – one enzyme hypothesis has been modified

• Enzymes are only one category of cellular proteins

• More accurate to say one gene encodes a polypeptide– Hemoglobin composed of 4

polypeptides required for function

• One gene – one polypeptide theory• Genes influence phenotype by

specifying polypeptides

DNA Replication

• 3 different models for DNA replication proposed in late 1950s– Semiconservative– Conservative– Dispersive

• Newly made strands are daughter strands

• Original strands are parental strands

• In 1958, Matthew Meselsonand Franklin Stahl devised experiment to differentiate among 3 proposed mechanisms

• Nitrogen comes in a common light form (14N) and a rare heavy form (15N)

• Grew E.coli in medium with only 15N

• Then switched to medium with only 14N

• Collected sample after each generation

• Original parental strands would be 15N while newly made strands would be 14N

• Results consistent with semiconservativemechanism

DNA Replication

Simultaneously Copying DNA Strands• DNA replication occurs in replication forks• These are Y-shaped regions of DNA where the 2 strands of the helix

have come apart• Nucleotides add directly to the 3’ end of an RNA primer; the other

strand is produced in short fragments (Okazaki Fragments) that are joined together by DNA ligase

• In the leading strand– DNA primase makes one RNA primer– DNA polymerase attaches nucleotides in a 5’ to 3’ direction as it

slides forward• In the lagging strand

– DNA synthesized 5’ to 3’ but in a direction away from the fork– Okazaki fragments made as a short RNA primer made by DNA

primase at the 5’ end and then DNA laid down by DNA polymerase• RNA primers will be removed by DNA polymerase and filled in with

DNA• DNA ligase will join adjacent DNA fragments

DNA Polymerization• DNA polymerase — enzyme

that strings together the nucleotides

• During replication 2 parental strands separate and serve as template strands

• New nucleotides must obey the AT/GC rule

• End result 2 new double helices with same base sequence as original

DNA Polymerization

RNA Polymerase

• An RNA polymerase makes an RNA primer that provides a 3’ end for DNA polymerase

• Then DNA polymerase links together the nucleotides that assemble opposite the template into a new strand of DNA

Sources of Genetic Diversity• Recombination• Crossing over• Mixing of gametes• Mutations — permanent changes in DNA — probably the ultimate

source• Kinds of Mutations:

– Point mutations — change 1 or several nucleotide pairs• Base substitution – replacement of 1 base by another• Insertion — addition of 1 or more nucleotides• Deletion — the removal of 1 or more nucleotides

– Chromosomal mutations — large regions of chromosome changes• Deficiencies• Translocations• Inversion• Duplications

Mutation Rate

• Measure of how often mutations occur• Depends on how often a sequence mutates, and

on how efficiently cells repair these mutations• Mutation hot spots exist that are more likely to

mutate• Mutation rates are usually low

Sunburn Damages DNA

Correcting Mistakes

• An enzyme detects something wrong in 1 strand of the DNA and removes it

• Then DNA polymerase copies the information in the intact second strand and creates a new stretch of DNA

• DNA ligase seals the gap