5.DNA

Chapter 16

Central Dogma

The Search for the Genetic Material

• _{Two components of chromosomes— DNA and}

protein —became candidates for the genetic material

• _{Frederick Griffith in 1928}

– _{2 kinds of bacteria: a pathogenic “S” strain and a}

harmless “R” strain

– _{Coined term transformation, now defined as a}

LE 16-2

Living S cells (control)

Living R cells (control)

Heat-killed S cells (control)

Mixture of heat-killed S cells and living R cells

Mouse dies

Living S cells are found in blood sample Mouse healthy Mouse healthy Mouse dies

The Search for the Genetic Material

• _{1944, Avery, McCarty, and MacLeod: DNA is}

“transforming agent”

– Experimental evidence that only DNA worked in

transforming harmless bacteria into pathogenic bacteria

• _{In 1952, Hershey and Chase performed experiments}

showing that DNA is the genetic material of a bacteriophage known as T2

– _{Experiment to determine if DNA or protein enters an E.}

coli cell during infection

osulibrary.orst.edu/ specialcollections/coll/p...

Additional Evidence That DNA Is the Genetic Material

• _{In 1947, Chargaff reported that DNA composition}

varies from one species to the next

• _{By the 1950s, known that DNA is a polymer of}

Building a Structural Model of DNA

• _{Wilkins and Franklin used a}

technique called X-ray crystallography to study molecular structure

– _{Franklin produced a picture of}

the DNA molecule

– _{It enabled Watson to deduce}

• _{Franklin : two antiparallel sugar-phosphate}

backbones, nitrogenous bases paired in the molecule’s interior

Building a Structural Model of DNA

5 end

3 end

5 end 3 end

Space-filling model Partial chemical structure

Hydrogen bond

Key features of DNA structure 0.34 nm

A and G

C, T and U

• _{Watson and Crick : bases paired like with like; this}

gave uneven width

• _{Pairing a purine with a pyrimidine resulted in a}

uniform width

Building a Structural Model of DNA

Purine + purine: too wide

Pyrimidine + pyrimidine: too narrow

Purine + pyrimidine: width consistent with X-ray data

A, G

• _{Watson and Crick:}

adenine paired only with thymine, and guanine

paired only with cytosine

Adenine (A) Thymine (T)

Guanine (G) Cytosine (C) Sugar

Sugar

Sugar Sugar

The Basic Principle: Base Pairing to a Template Strand

• _{Watson and Crick first to reason: Since the two}

strands of DNA are complementary (what does this mean?) each strand acts as a template for building

a new strand in replication

– _{In DNA replication, the parent molecule unwinds,}

Semiconservative Replication

• _{Watson and Crick’s semiconservative model}

of replication:

– _{each daughter molecule will have one old strand}

• _{Meselson and Stahl}

– _{Labeled nucleotides of DNA with a heavy isotope of}

nitrogen

– _{DNA replication occurs in presence of nucleotides}

labeled with a lighter isotope of N

• _{The first replication produced a band of hybrid DNA}

• _{A second replication produced both light and hybrid DNA}

Origins of Replication

• _{DNA Replication begins at origins of replication}

– _{the two DNA strands separate, opening up a replication}

“bubble”

– _{At the end of the bubble is a replication fork}

– _{Hundreds (even thousands) of origins of replication on a}

eukaryotic chromosome

– _{Replication proceeds in both directions from each origin}

Two daughter DNA molecules

Parental (template) strand

Daughter (new) strand 0.25 µm

Replication fork Origin of replication

Bubble

Elongating a New DNA Strand

• _{Enzymes called DNA polymerases catalyze the}

elongation of new DNA

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

New strand

5 end

Phosphate Base Sugar

Template strand

3 end 5 end 3 end

5 end

3 end

5 end

3 end

Nucleoside triphosphate

DNA polymerase

Antiparallel Elongation • _{DNA Double helix}

is antiparallel

• _{DNA polymerases}

add nucleotides only to the 3end of a growing

strand

• _{a new DNA strand}

can elongate only in the 5to

3direction

• _{The leading strand:}

DNA polymerase

synthesizes the new strand continuously

• _{The lagging strand: DNA}

polymerase must work away from the

replication fork

– _{Synthesized as Okazaki}

fragments, which are joined together by DNA ligase

Antiparallel Elongation

DNA pol III

Template strand Leading strand Lagging strand DNA ligase Template strand

• _{http://www.pbs.org/wgbh/aso/tryit/dna/shoc}

Priming DNA Synthesis

• _{DNA polymerases cannot}

initiate synthesis of a new strand

• _{The initial nucleotide strand}

is a short RNA or DNA primer

– _{An enzyme called primase can}

start an RNA chain from scratch

• _{One primer for leading}

Other Proteins That Assist DNA Replication

• Helicase untwists the double helix and separates the DNA strands at the replication fork

• Single-strand binding protein binds to and stabilizes single-stranded DNA

• _{Topoisomerase corrects “overwinding” ahead of replication} forks by breaking, swiveling, and rejoining DNA strands

• _{Primase synthesizes an RNA primer at the start of the leading} strand and the Okazaki fragments

• _{DNA pol III synthesizes the leading strand and Okazaki} fragments

• _{DNA pol I removes primer from the 5} ends of the leading strand and Okazaki fragments, replacing primer with DNA • _{DNA ligase joins the 3} end of the DNA that replaces the

LE 16-16 5 3 Parental DNA 3 5

Overall direction of replication

DNA pol III

Replication fork Leading strand DNA ligase Primase OVERVIEW

PrimerDNA pol III

DNA pol I Lagging strand Lagging strand Leading strand Leading strand Lagging strand Origin of replication

The DNA Replication Machine as a Stationary Complex

• _{The proteins involved in DNA replication form a}

large complex

• _{This DNA replication machine is probably}

stationary during replication, reeling in the DNA strand to be copied

• _{http://www.youtube.com/watch?v=-mtLXpgjHL0&N}

Proofreading and Repairing DNA

1. DNA polymerases proofread newly made DNA

2. In mismatch repair of DNA, repair enzymes correct errors in base pairing

3. In nucleotide excision repair,

enzymes cut out and replace damaged

stretches of DNA

DNA ligase DNA

polymerase

DNA ligase seals the free end of the new DNA to the old DNA, making the strand complete. Repair synthesis by

a DNA polymerase fills in the missing nucleotides. A nuclease enzyme cuts

the damaged DNA strand at two points and the damaged section is removed.

Nuclease

Replicating the Ends of DNA Molecules

• _{The usual replication machinery provides no way}

to complete the 5 ends,

– repeated rounds of replication produce shorter DNA molecules

• _{Ends of eukaryotic chromosomal DNA molecules:}

nucleotide sequences called telomeres

– _{Telomeres postpone the erosion of genes near the}

LE 16-18

End of parental DNA strands

5

3

Lagging strand 5

3 Last fragment RNA primer Leading strand Lagging strand Previous fragment

Primer removed but cannot be replaced with DNA because no 3 end available for DNA polymerase

5

3

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

Second round of replication 5 3 5 3 Further rounds of replication New leading strand

New leading strand

Telomerase

• _{Essential genes would be missing from the}

gametes if germ cells shortened every generation

• _{An enzyme called telomerase catalyzes the}

Race to be right!

– _{What makes up a nucleotide?}

– _{What is the difference between a purine and a}

pyrmidine?

– _{What are the base pair rules?}

– _{Name as many enzymes involved in DNA replication}

that you can. Name the function of each.