Chapter 16 BIOL 102

The Molecular Basis of Inheritance

Rob Swatski

Assoc. Prof. Biology

HACC – York Campus

Overview: Life’s Operating Instructions

1953: James Watson & Francis Crick

- double-helix model

- structure of deoxyribonucleic acid (DNA)

DNA directs development of traits:

- biochemical

- anatomical

- physiological

- behavioral

The Search for the Genetic Material

After Morgan’s research on genes & chromosomes, DNA & protein became likely candidates for

the genetic material

The key factor was choosing appropriate experimental organisms

The role of DNA in heredity was first discovered by studying bacteria and the viruses that infect

them

1928: Frederick Griffith worked with 2 bacterial strains:

one pathogenic & one harmless

When he mixed heat-killed remains of the pathogenic strain with living cells of the harmless strain:

some living cells became pathogenic

He referred to this as transformation

- we now define it as a change in genotype & phenotype due to assimilation of foreign DNA

Living S cells (control)

Living R cells (control)

Heat-killed S cells (control)

Mixture of heat-killed S cells & living R cells

Mouse dies Mouse dies Mouse healthy Mouse healthy

Living S cells

RESULTS

EXPERIMENT

1944: Avery, McCarty, & MacLeod announced that DNA was the transforming substance

- based on experimental evidence showing only DNA helped transform harmless bacteria into

pathogens

- many biologists remained skeptical, mainly because little was known about DNA

Bacterial cell

Phage capsid

Tail sheath

Tail fiber

DNA

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More Evidence: Bacteriophages (Phages)

1952: Alfred Hershey & Barbara Chase experiments:

- showed that DNA is the genetic material of T2 phage

- Results: only 1 of the 2 components of T2 (DNA or protein) enters an E. coli cell during infection

Concluded that the phage’s injected DNA provides the genetic information

EXPERIMENT

Phage

DNA

Bacterial cell

Radioactive

protein

Radioactive

DNA

Batch 1: radioactive sulfur (35S)

Batch 2: radioactive phosphorus (32P)

EXPERIMENT

Phage

DNA

Bacterial cell

Radioactive

protein

Radioactive

DNA

Batch 1: radioactive sulfur (35S)

Batch 2: radioactive phosphorus (32P)

Empty protein shell

Phage DNA

EXPERIMENT

Phage

DNA

Bacterial cell

Radioactive protein

Radioactive DNA

Batch 1: radioactive sulfur (35S)

Batch 2: radioactive phosphorus (32P)

Empty protein shell

Phage DNA

Centrifuge

Centrifuge

Pellet

Pellet (bacterial cells and contents)

Radioactivity (phage

protein) in liquid

Radioactivity (phage DNA)

in pellet

Additional Evidence

It was known that DNA is a polymer of nucleotides:

- nitrogenous base, a sugar, & a phosphate group

1950: Erwin Chargaff showed DNA composition varies between species

- this evidence of diversity made DNA a more credible candidate for the genetic material

Sugar–phosphate backbone

5 end

Nitrogenous bases

Thymine (T)

Adenine (A)

Cytosine (C)

Guanine (G)

DNA nucleotide

Sugar (deoxyribose) 3 end

Phosphate

Chargaff’s Rules

In any species there is an equal number of:

A and T

&

G and C

Building a Structural Model of DNA

The next challenge was to relate DNA structure with function

Maurice Wilkins & Rosalind Franklin: used X-ray crystallography to study molecular structure

- took pictures of DNA

Rosalind Franklin Franklin’s X-ray diffraction photograph of DNA

Franklin’s images of DNA enabled Watson to deduce:

- shape: double helix

- width (double-stranded)

- spacing of N-bases

(c) Space-filling model

Hydrogen bond 3 end

5 end

3.4 nm

0.34 nm

3 end

5 end

(b) Partial chemical structure

(a) Key features of DNA structure

1 nm

Watson & Crick built double helix models to match the x-ray & chemical evidence

Franklin’s DNA structure hypothesis:

- 2 sugar-phosphate backbones

- paired nitrogenous bases in-between

Watson built a model in which the backbones were antiparallel (their subunits run in opposite

directions)

Watson & Crick first thought bases paired “like with like” (A-A, etc.)

- but this does not result in a uniform width

Purine + purine: too wide

Pyrimidine + pyrimidine: too narrow

Watson & Crick concluded that:

- Adenine (A) paired only with Thymine (T)

- Guanine (G) paired only with Cytosine (C)

This model explains Chargaff’s rules:

“in any organism the amount of

A = T and the amount of G = C”

Cytosine (C)

Adenine (A) Thymine (T)

Guanine (G)

DNA Replication and Repair

Watson & Crick noted that the specific base-pairing suggested a possible DNA copying mechanism

The Basic Principle: Base Pairing to a Template Strand

• Since the 2 strands of DNA are complementary, each strand acts as a template for building a new strand in replication

• In DNA replication, the parent molecule unwinds & 2 new daughter strands are built based on base-pairing rules

A T

G C

T A

T A

G C

(a) Parent molecule

A T

G C

T A

T A

G C

(c) “Daughter” DNA molecules, each consisting of one parental strand & one new strand

(b) Separation of strands

A T

G C

T A

T A

G C

A T

G C

T A

T A

G C

Semiconservative Model of Replication

• Predicts that when a double helix replicates, each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) & one newly made strand

Competing models:

- Conservative model: the 2 parent strands rejoin

- Dispersive model: each strand is a mix of old & new

Parent cell First

replication Second

replication

(a) Conservative model

(b) Semiconservative model

(c) Dispersive model

• Experiments by Matthew Meselson & Franklin Stahl supported the semiconservative model

• They labeled the nucleotides of the old strands with a heavy isotope of N, while any new nucleotides were labeled with a lighter isotope

EXPERIMENT

RESULTS

1

3

2

4

Bacteria cultured in medium containing 15N

Bacteria transferred to medium containing 14N

DNA sample centrifuged after 20 min (after 1st application)

DNA sample centrifuged after 20 min (after 2nd replication)

Less dense

More dense

• The 1st replication produced a band of hybrid DNA, eliminating the conservative model

• A 2nd replication produced both light & hybrid DNA, eliminating the dispersive model & supporting the semiconservative model

CONCLUSION

First replication Second replication

Conservative model

Semiconservative model

Dispersive model

• DNA replication is remarkable in its speed & accuracy

• More than a dozen enzymes & other proteins participate in DNA replication

Getting Started

• Replication begins at special sites called origins of replication, where the 2 DNA strands are separated, opening up a replication “bubble”

• A eukaryotic chromosome may have 100’s or 1000’s of origins of replication

• Replication proceeds in both directions from each origin, until the entire molecule is copied

Origin of replication Parental (template) strand

Daughter (new) strand

Replication fork

Replication bubble

Double-stranded DNA molecule

Two daughter DNA molecules

(a) Origins of replication in prokaryotes

0.5 µm

0.25 µm

Origin of replication Double-stranded DNA molecule

Parental (template) strand Daughter (new) strand

Bubble Replication fork

Two daughter DNA molecules

(b) Origins of replication in eukaryotes

• At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating

• Helicases are enzymes that untwist the double helix at the replication forks

• Single-strand binding proteins bind to & stabilize single-stranded DNA until it can be used as a template

• Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, & rejoining DNA strands

Topoisomerase

Primase

RNA primer

Helicase

Single-strand binding proteins

5

3

5

5 3

3

• DNA polymerases cannot initiate synthesis of a polynucleotide

- they can only add nucleotides to the 3 end

• The initial nucleotide strand is a short RNA primer

(5–10 nucleotides long)

• The 3 end serves as the starting point for the new DNA strand

• Primase: can start an RNA chain from scratch & adds RNA nucleotides one at a time using the parental DNA as a template

Topoisomerase

Primase

RNA primer

Helicase

Single-strand binding proteins

5

3

5

5 3

3

Synthesizing a New DNA Strand

• DNA polymerases catalyze the elongation of new DNA at a replication fork

- most require a primer & a DNA template strand

• The rate of elongation is approx. 500 nucleotides per second in bacteria & 50 per second in human cells

• Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate

• dATP supplies adenine to DNA & is similar to the ATP of energy metabolism

- the difference is in their sugars: dATP has deoxyribose while ATP has ribose

• As each dATP joins the DNA strand, it loses 2 phosphate groups as a molecule of pyrophosphate

A

C

T

G

G

G

G C

C C

C

C

A

A

A

T

T

New strand 5 end

Template strand 3 end 5 end 3 end

3 end

5 end 5 end

3 end

Base

Sugar

Phosphate

Nucleoside triphosphate

Pyrophosphate

DNA polymerase

Antiparallel Elongation

• The double helix has an antiparallel structure

- the 2 strands are oriented in opposite directions

- this affects replication

• DNA polymerases add nucleotides only to the free 3end of a growing strand

- therefore, a new DNA strand can elongate only in the 5to3direction

• Along one template strand of DNA, the DNA polymerase continuously synthesizes a leading strand, moving toward the replication fork

Leading strand

Leading strand Lagging strand

Lagging strand

Origin of replication

Primer

Overall directions of replication

Origin of replication

RNA primer

Sliding clamp

DNA pol III Parental DNA

3

5

5

5

5

5

5

3

3

3

Helicase

Single-strand binding proteins

• To elongate the other new strand (the lagging strand), DNA polymerase must work in the direction away from the replication fork

• The lagging strand is synthesized as a series of segments called Okazaki fragments, which are joined together by DNA ligase

Origin of replication

Leading strand

Leading strand

Lagging strand

Lagging strand

Overall directions of replication

1 2

Template strand

RNA primer for fragment 1

Okazaki fragment 1

RNA primer for fragment 2

Okazaki fragment 2

Overall direction of replication

3

3

3

3

3

3

3

3

3

3

3

3

5

5

5

5

5

5 5

5 5

5 5

5

2

2

2 1

1

1

1

1

The DNA Replication Complex

• The proteins that participate in DNA replication form a large complex called a “DNA replication machine”

• Recent studies support a model in which DNA polymerase molecules “reel in” parental DNA & “extrude” newly made daughter DNA molecules

Parental DNA

DNA pol III

Leading strand

Connecting protein

Helicase

Lagging strand DNA pol III

Lagging strand template

5

5

5

5

5

5

3 3

3 3

3

3

Proofreading & Repairing DNA

• DNA polymerases proofread newly made DNA & replace any incorrect nucleotides

• DNA can be damaged by chemicals, radioactive emissions, X-rays, UV light, & certain molecules (in cigarette smoke for example); it can also undergo spontaneous changes

• Mismatch repair: repair enzymes correct errors in base pairing

• Nucleotide excision repair: a nuclease cuts out & replaces damaged stretches of DNA

Nuclease

DNA polymerase

DNA ligase

Nucleotide Excision Repair

Nuclease

Evolutionary Significance of Altered DNA Nucleotides

• Error rate after proofreading repair is low but not zero

• Sequence changes may become permanent & can be passed on to the next generation

• These changes (mutations) are the source of the genetic variation upon which natural selection operates

Replicating the Ends of DNA Molecules

• Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes

• The usual replication machinery provides no way to complete the 5 ends

- repeated rounds of replication produce shorter DNA molecules with uneven ends

Ends of parental DNA strands

Leading strand

Lagging strand

Last fragment Next-to-last fragment

Lagging strand RNA primer

Parental strand Removal of primers and replacement with DNA where a 3 end is available

Second round of replication

Further rounds of replication

New leading strand

New lagging strand

Shorter and shorter daughter molecules

3

3

3

3

3

5

5

5

5

5

Telomeres

• Nucleotide sequences at the ends of eukaryotic chromosomal DNA molecules

• Telomeres do not prevent the shortening of DNA molecules, but they do postpone the erosion of genes near the ends of DNA molecules

- the shortening of telomeres is thought to be connected to aging

1 µm

• If chromosomes of germ cells became shorter in every cell cycle, essential genes would eventually be missing from the gametes they produce

• Telomerase: catalyzes the lengthening of telomeres in germ cells

• The shortening of telomeres might protect cells from cancerous growth by limiting the number of cell divisions

• There is evidence of telomerase activity in cancer cells, which may allow cancer cells to persist

• The prokaryotic chromosome is a double-stranded, circular DNA molecule associated with a small amount of protein

- the DNA is “supercoiled” & found in the nucleoid region of the cell

• Eukaryotic chromosomes have linear DNA molecules associated with a large amount of protein

• Chromatin: a complex of DNA & protein found in the nucleus of eukaryotic cells

• Chromosomes fit into the nucleus through an elaborate, multilevel system of packing

• Histones: proteins responsible for the 1st level of DNA packing in chromatin

• 10-nm fiber (diameter) – “thin” fiber

– DNA winds around histones to form strings of nucleosome “beads”

• 30-nm fiber (diameter) – “thick” fiber

– interactions between nucleosomes cause the thin fiber to coil or fold into this thicker fiber

DNA double helix (2 nm diameter)

Nucleosome (10 nm “thin” fiber)

Histones Histone tail

H1

Nucleosomes, or “beads on a string” (10 nm fiber)

30 nm fiber

Chromatid (700 nm)

Loops Scaffold

300 nm fiber

Replicated chromosome (1,400 nm) 30 nm “thick”

fiber Looped domains (300 nm fiber)

Metaphase chromosome

• Most chromatin is loosely packed euchromatin in the nucleus during interphase & condenses prior to mitosis

• During interphase a few regions of chromatin (centromeres & telomeres) are highly condensed into heterochromatin

- this dense packing of chromatin makes it difficult for the cell to express genetic information coded in these regions

• Though interphase chromosomes are not highly condensed, they still occupy specific restricted regions in the nucleus

5

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