DNA Structure and Replication

Ch. 14

DNA and Heredity • Mendel set the stage for

inheritance patterns, but it was not yet known that this is through DNA

• Proteins were considered the better source of variation, why?– More possible variation; 20 AA

vs. 4 Base pairs • A series of researches over

decades showed it really DNA:– Griffith– Avery– Hershey and Chase

Frederick Griffith Kills Mice• Worked with Streptococcus pneumoniae

– S-Type (smooth) virulent; deadly– R-Type (rough) non-virulent; pretty okay

• Ran for tests on mice:1) S-type injected into mice

– Mice die2) R-type injected into mice

– Mice live3) Heat-killed S-type injected into mice

– Mice live4) Heat-killed S-type and R-type injected

into mice– Mice die

Conclusion:• Some material from dead S-type changed

R-type into S-type (transformed)

Avery Doesn’t Kill Mice• Works with Streptococcus

pneumoniae too, but only in test tubes

• Used enzymes to destroy either the proteins, DNA, or RNA of the cells and then tried to transform them

1) R-type + S-type with destroyed proteins transformation

2) R-type + S-type with destroyed RNA transformation

3) R-type + S-type with destroyed DNA no transformation

Conclusion:• DNA must be needed to

transform bacteria cells, so it must be the key to heredity

Hershey and Chase End the Debate• Worked with E. coli and a

bacteriophage called T2– Virus that infects only bacteria; made

of just DNA and a protein coat• Labeled DNA and protein coat of

virus with radioactive P and S isotopes and traced them through the virus life cycle

1) E.coli + Virus with labeled protein coat no radioactivity in offspring

2) E.coli + Virus with labeled DNA radioactivity in offspring

Conclusion:• DNA, not proteins, are passed on to

offspring

Structure of DNA• Discovered by Watson and Crick with

help from Franklin and Wilkins• X-Ray diffraction image of DNA

– X-rays shot through crystal containing molecule

– Photograph film catches areas exposed when x-rays deflect

• What is DNA’s shape?– Double helix

• What is it made of?– Nucleotides : Adenine (A), Guanine (G),

Thymine (T), and Cytosine (C)• How are the nucleotides connected?

– Phosphodiester bonds in a sugar-phosphate backbone; creates 5’ end and a 3’ end

– Strands held together by H-bonds

Structure of DNA• How do nucleotides match up?

– Purines (two rings) with Pyrimidines (one ring)

– A-T; G-C• What is the vocab word for this type of

pairing?– Complementary base pairing

• In order for pairing to happen, the two strands in DNA must run opposite directions. What is this called?– Antiparallel

• Other dementions:– DNA is 2n wide (only Purine-Pyrimidine

combination makes this length)– Full twist is 3.4 nm long– Distance between each nucleotide is 0.34nm– SO…there are 10 bases/turn

Semiconservative Replication?• In replication, one strand is used

as a template (guide) to build a new strand

• Unzipping DNA allows both strands to be copied at the same time, thus producing copies that each have one full strand from the starting DNA

• Other options existed…1) Conservative model DNA is

template but original DNA reforms and new DNA has no original strands

2) Dispersive Replication model old and new DNA strands mix as they form

Meselson and Stahl• Worked with DNA made of

“heavy” 15N isotope• Mixed “heavy” DNA with 14N, did

one replication, and then separated DNA types by centrifuge (heavy ones sink more)

• Allowed DNA to replicate again and centrifuged again

1) Semiconservative 1st; lighter DNA (half 15N and half 14N), 2nd; lightest appears (all 14N)

2) Conservative 1st; heavy DNA (all 15N) and lightest (all 14N), 2nd; same

3) Dispersive all DNA gets lighter as more 14N is used

Vocabulary Explosion!• Deoxyribosenucleoside

triphosphates building blocks of DNA (dATP, dGTP, dCTP, dTTP)

• DNA Helicase breaks H-bonds and unwinds DNA

• Topoisomerse untwists downstream DNA; DNA twists as it is unzipped

• SSBs (Single-stranded binding proteins) hold unzipped DNA strands so they don’t adhere

• DNA polymerase III main enzyme used to copy DNA

• Sliding DNA Clamp helps DNA polymerase stay attached to DNA

Vocabulary Explosion 2! The Sequel • DNA polymerase I

removes RNA primers at 5’ end

• Primase makes primers; RNA nucleotides that mark the start of replication

• DNA ligase fixes breaks in sugar-phosphate backbone

• Leading strand DNA strand continuously replicated

• Lagging strand DNA strand replicated in fragments (Okazaki fragments)

DNA Replication: Getting Started• DNA strands have a 5’ and 3’ end,

but nucleotides can only be added to the 3’ (free –OH ready for dehydration reaction)

• New DNA is built 5’3’, so the template is “read” 3’5’

• Replication starts at ori region of the DNA (origin of replication)– Eukaryotic DNA is too long to

replicate from end to end– Hundreds ori sights exist and

replication goes in both directions (replication bubbles)

• DNA helicase unwinds the strands creating a replication fork (Y-structure)

• SSBs hold stands apart

DNA Replication: Primers• Pulling apart strands causes

the DNA to twist and bundle up

• Topoisomerase cuts DNA ahead of the replication fork, untwists it, and rebinds it

• DNA Poly III needs a 3’ end to start replication

• Primers (RNA) are base paired at ori and provide a 3’ for Poly III

• Primers are built by Primase • Primers are removed by DNA

Polymerase I (exonuclease) and replaced with DNA

DNA Replication: Two Types of Synthesis• DNA is antiparallel, so one

strand is read 3’5’ (leading strand) while the other runs 5’3’ (lagging strand)

• DNA cannot be added in the 5’3’ direction

• Leading stand as continuous replication

• Lagging strand is replicated in sections (Okazaki Fragments)– Leaves gaps which are filled

in by DNA Poly I and Ligase

DNA Replication: Finishing Up• When bubbles meet, the enzymes detach

and DNA re-adheres• If DNA is liner, what happens to the

starting stands on either end?– They are not be replicated; short section is

lost after each replication DNA gets shorts with time

• Telomere noncoding area at the end of DNA (5’-TTAGGG-3’) that protect against this– Shortening is believed to be the main cause

of aging and death• Telomerase enzyme that adds more

telomeres; only active as an embryo• What type of cell is telomerase also active

in?– Cancer; If we can turn these off cancer will

divide itself to death

DNA Replication: Opps…Mistake…• Polymerase is not perfect; makes

a base-pair mismatch 1: 1,000 nucleotides

• Proofreading mechanism Poly III can backup and use exonuclease to replace mistakes

• Lowers mutation rates to 1:1 million nucleotides

• What if Poly III miss the mistake?– DNA repair mechanisms run along

the DNA double checking it– Any area wider or narrower than

2nm must have a mistake and replaces the nucleotide

DNA Compaction• DNA is around 2 meter long and must fit

in a 10mm nucleus– Most is compacted and only opened for

making proteins• Chromatin DNA and Chromosomal

proteins• Histone small, positively charged

proteins; bind negative backbone of DNA

• Histones join together and wrap DNA around them to make nucleosomes

• Strings of nucleosomes connected by linkers string of beads

• Decreases DNA size by a factor of 7!• Nonhistone proteins effect histone

binding so regions of DNA become accessible

DNA Compaction• Nucleosome strings (10-nm

chromatin fibers) can wrap around a H1 histone to make 30-nm chromatin fiber– Solenoid model helix of

nucleosomes• This level of condensing

protects against damage• Euchromatin loosely packed

regions (light color band on chromosome); often expressed

• Heterchromatin densely packed regions (dark color band on chromosome); often deactivated genes

Bacterial DNA• Prokaryotes have no need for

histones– DNA is one circular ring (bacterial

chromosome) that is short enough

– Kept compacted in a mass called the nucleoid

• Addition al DNA can be absorbed by bacteria– Plasmids short DNA rings – Can be copied and exchanged

with other bacteria of the same species or genus

– Can help form drug resistant bacteria

Homework• Suggested Homework:– Test Your Knowledge Ch. 14

• Actual Homework:– Interpret the Data Ch. 14– Discuss the Concepts #1

and #4• Lab Reports due 12/11• Papers due. 12/13