The Molecular Basis of Inheritance

Griffith’s Experiments (1928) using Streptococcus pneumoniae and mice

1. Mice injected with live, infective S cells (control to show effect of S cells)

RESULT: Mice die. Live, infective S cells in their blood; shows that S cells are virulent.

2. Mice injected with live, noninfective R cells (control to show effect of R cells)

RESULT: Mice live. No live R cells in their blood; shows that R cells are nonvirulent.

Griffith’s Experiments, cont. 3. Mice injected with heat-killed S cells (control to show effect of dead S cells)

RESULT: Mice live. No live S cells in their blood; shows that live S cells are necessary to be virulent to mice.

4. Mice injected with heat-killed S cells plus live R cells

RESULT: Mice die. Live S cells in their blood; shows that living R cells can be converted to virulent S cells with some factor from dead S cells.

Hershey and Chase’s Experiments (1952)

Watson and Crick (1953)

!  Deoxyribonucleic acid (DNA) molecule forms the genetic material of all living organisms

6

Rosalind Franklin: Discovered DNA Structure through X-ray Diffraction

!  Performed X-ray diffraction studies to identify the 3-D structure

•  Discovered that DNA is helical •  Using Maurice Wilkins’ DNA fibers,

discovered that the molecule has a diameter of 2 nm and makes a complete turn of the helix every 3.4 nm

Chargaff’s Rules

!  Erwin Chargaff determined that •  Amount of adenine = amount of thymine •  Amount of cytosine = amount of guanine •  Always an equal proportion of purines (A and G)

and pyrimidines (C and T)

7

Nucleotide Subunits of DNA

!  DNA is made of subunits called nucleotides

!  Each nucleotide consists of a phosphate group, a deoxyribose and a nitrogenous base

!  The nucleotides are covalently linked via the sugar-phosphate backbone

!  Each strand of DNA has a 5’ and a 3’ end

Purin

es

Pyrim

idin

es

Adenine Guanine NH2 C C N N

N C

H N C

C H O

H

H O C N C

H N C NH2

H C H O

O C N C

H N C H3C

C H H O

O C N C

H N C H

C H

NH2 C C N N

N C

H N C

C H H

Nitrogenous Base

4´ 5´

1´ 3´ 2´

2 8 7 6

3 9 4 5

1 O P O–

–O

Phosphate group

Sugar

Nitrogenous base

O CH2

N N

O

N

NH2

OH in RNA Cytosine

(both DNA and RNA) Thymine

(DNA only) Uracil

(RNA only)

OH H in DNA

DNA Structure

!  Phosphodiester bond •  Bond between

adjacent nucleotides •  Formed between the

phosphate group of one nucleotide and the 3! —OH of the next nucleotide

!  The chain of nucleotides has a 5!-to-3! orientation

Base CH2

O

5´

3´

O P O

OH

CH2

–O O

C

Base O

PO4

Phosphodiester bond

DNA Structure, cont.

!  Complementarity of bases

!  A forms 2 hydrogen bonds with T

!  G forms 3 hydrogen bonds with C

!  Gives consistent diameter

A

H

Sugar

Sugar Sugar

Sugar

T

G C

N H

N O

H

CH3

H H N

N N H N N

N

H

H H

N O H H

H N

N H N

N H N N

Hydrogen bond

Hydrogen bond

DNA: Complimentary Base Pairing DNA Double Helix 2 nm

5' end

Distance between each pair of bases =0.34 nm

Each full twist of the DNA double helix =3.4 nm

Phosphate group

5-carbon sugar deoxyribose)

Nitrogenous base (guanine)

Hydrogen bond 3' end

!  Two molecules of DNA are connected through hydrogen bonds between the nitrogenous bases.

!  [A] = [T] and [G] = [C]

DNA Replication

!  3 possible models 1.  Conservative model 2.  Semiconservative model 3.  Dispersive model

Meselson and Stahl Experiment, 1957

3 Replication Models

2nd replication

Parental DNA Replicated DNA

KEY

a. Semiconservative replication

1st replication

b. Conservative replication c. Dispersive replication

Meselson-Stahl Experiment (cont.)

1st replication

15N medium 14N medium

2nd replication

Meselson-Stahl Experiment (cont.)

15N–14N hybrid DNA

14N–14N (light) DNA

15N–15N (heavy) DNA

CsCl forms a density gradient during centrifugation, with the highest density at the bottom of the tube.

DNA molecules move to positions where their density equals that of the CsCl solution and form bands. Shown are the positions of differently labeled DNA molecules. Experimentally the bonds are detected by absorbance of UV light.

Meselson-Stahl Experiment (cont.)

15N–14N hybrid DNA

15N–15N (heavy) DNA

DNA from 15N medium

DNA after one replication in 14N

15N–14N hybrid DNA

DNA after two replications in 14N

14N–14N (light) DNA

Meselson-Stahl Experiment (cont.)

Semiconservative

Conservative

Dispersive

The results support the semiconservative model.

Matches results

Does not match results

Does not match results

Two replications in 14N 15N medium One replication in 14N

Model for semiconservative Replication

Direction of replication

Old

New

Complementary base pairing in the DNA double helix: A pairs with T, G pairs with C.

The two chains unwind and separate.

Each “old” strand is a template for the addition of bases according to the base-pairing rules.

The result is two DNA helices that are exact copies of the parental DNA molecule with one “old” strand and one “new” strand

Enzymes of DNA Replication

!  Helicase unwinds the DNA

!  Primase synthesizes RNA primer (starting point for nucleotide assembly by DNA polymerases)

!  DNA polymerases assemble nucleotides into a chain, remove primers, and fill resulting gaps

!  DNA ligase closes remaining single-chain nicks

Assembling a Complementary Chain DNA Replication: Leading and Lagging Strand Assembly

DNA Replication: Enzyme Activities

Unwinding enzyme (helicase)

Primase RNA primers

Replication fork

Overall direction of replication

1 Helicase unwinds the DNA, and primases synthesize short RNA primers.

RNA

Leading strand

Lagging strand DNA polymerase

DNA polymerase

RNA DNA

2 RNA primers are used as starting points for the addition of DNA nucleotides by DNA polymerases.

DNA Replication: The Lagging Strand

Primer being extended by DNA polymerase

DNA polymerase

Newly synthesized primer

DNA unwinds further, and leading strand synthesis proceeds continuously, while a new primer is synthesized on the lagging strand template and extended by DNA polymerase III.

3

DNA polymerase

Nick Another type of DNA polymerase (I) removes the RNA primer, replacing it with DNA, leaving a nick between the newly synthesized segments.

4

DNA Replication: The Lagging Strand (Okazaki Fragments)

DNA ligase Nick is closed by DNA ligase. 5

Lagging strand Leading strand

Newly synthesized primer

DNA polymerase

Primer being extended by DNA polymerase III

6 DNA continues to unwind, and the synthesis cycle repeats as before: continuous synthesis of leading strand and synthesis of a new segment to be added to the lagging strand.

Telomers shorten with each Replication

Telomerase adds Telomere Repeats Replication From Multiple Origins

Proofreading By DNA Polymerase Photorepair: A Specific Repair Mechanism

!  For one particular form of damage caused by UV light

!  Thymine dimers •  Covalent link of adjacent

thymine bases in DNA

!  Photolyase •  Absorbs light in visible range •  Uses this energy to cleave

thymine dimer

Excision Repair: A Non-Specific Repair Mechanism

!  Damaged region is removed and replaced by DNA synthesis

!  3 steps 1.  Recognition of damage 2.  Removal of the

damaged region 3.  Resynthesis using the

information on the undamaged strand as a template 32

!  E. coli has 3 DNA polymerases •  DNA polymerase I (pol I)

•  Acts on lagging strand to remove primers and replace them with DNA

•  DNA polymerase II (pol II) •  Involved in DNA repair processes

•  DNA polymerase III (pol III) •  Main replication enzyme

•  All 3 have 3!-to-5! exonuclease activity – proofreading •  DNA pol I has 5!-to-3! exonuclase activity

Different DNA Polymerase Have Different Functions

Nucleosomes and Chromatin Fiber

Chromosome in

metaphase

DNA

2 nm

Nucleosome and DNA wound around core of 2 molecules each of H2A, H2B, H3, H4

Linker

10-nm chromatin fiber

Nucleosomes Linkers

H1

30-nm chromatin fiber

Chromatin fiber

Solenoid

Prokaryotic DNA Replication