Lecture #18 – 10/17/01 – Dr. Wormington
DNA Replication – The Story So Far
•Semiconservative
•Hydrolysis of 5' dNTP • 3'HON4pN3pN2pN1p5'... + PPi • 2Pi
Provides Energy for Phosphodiester Bond Formation
•Occurs Only 5'• 3'
•Initiates at Discrete Origins
•Bidirectional
•Requires a Template and a Primer (usually RNA)
•So where are we?
Everything You Need to Know About Life At The Replication Fork
Hint – The Players’ Names Tell You What They Do!
Leading Strand Synthesis
Proceeds in Same Direction
As Replication Fork
DNA Helicase unwinds
the 2 strands
Single-stranded DNA
binding proteins hold the 2
RNA Primase strands apart
makes primer
Lagging Strand Synthesis
Proceeds in Opposite Direction
As Replication Fork – Synthesized in Short pieces termed Okazaki Fragments
Continuing the Story
DNA Synthesis is Semi-Discontinuous
Leading strand
is synthesized
as a single
continuous
strand
Lagging strand
is synthesized
as a series
of short, discontinuous
Okazaki fragments
which are
subsequently
Ligated together into
a single continuous
strand.
Its synthesis lags
behind the leading
strand as more
steps are required
Remember! DNA Replication is Bidirectional – So
Are Reversed for the
Replication Fork
Going this a way
Lagging
Leading
The Leading & Lagging Strands
For the Replication Fork
Going this a way
Leading
Lagging
The End of the Story
DNA Polymerase I
removes RNA primers
& replaces them with DNA
DNA Ligase links
Okazaki Fragments into
a single continuous strand
The 3 Major DNA Repair Mechanisms in Cells
Replication Errors Occur Only 10-4 base pairs
Repair Activities Reduce Overall Errors to 10-9 base pairs
Removes incorrectly inserted nucleotides during replication
Removes incorrectly inserted nucleotides after replication
e.g., Recognizes an AC base pair - removes the C & replaces it with T or vice versa
Note - The repair system does know if it's the A or the C which should be removed - don't ask
Recognizes damaged DNA, e.g.,
UV photocrosslinked bases
DNA Replication – The Take Home
•Semiconservative
•Hydrolysis of 5' dNTP • 3'HON4pN3pN2pN1p5'... + PPi • 2Pi
Provides Energy for Phosphodiester Bond Formation
•Occurs Only 5'• 3'
•Initiates at Discrete Origins
•Bidirectional
•Requires a Template and a Primer (usually RNA)
•Semi-Discontinuous
Leading Strand is Continuous
Lagging Strand is Discontinuous Okazaki Fragments
•3 DNA Repair Mechanisms
•Proofreading During DNA Replication
•Mismatch Repair (Post-Replication)
•Excision Repair (Post-Replication)
G2 DNA Damage
Checkpoint
What's a Gene? Beadle & Tatum 1 Gene = 1 Enzyme (& Usually 1 Polypeptide)
Prototroph =
wild-type for
synthesis of a
given product
e.g., amino acid
Mutant in C
Mutant in B
Mutant in A
Auxotroph =
mutant which
cannot synthesize
a given product
Therefore, the
missing product
must be provided
in nutrient media
or cells fail to
grow
The Central Dogma or Information Storage & Transfer in Biological Systems
Information Transfer from Nucleic Acids to Protein is Unidirectional
Replication
Transcription
Translation
Replication e.g., Picornaviruses
Hepatitis C, Polio
Transcription
Reverse-Transcription
e.g., Retroviruses, HIV
Translation
Gene Expression – The Big Picture
In prokaryotes,
transcription
and translation
Both occur
in the same
compartment
In eukaryotes
transcription occurs
in the nucleus but
translation occurs
in the cytoplasm
mRNAs
must be
exported
For a given gene
Only 1 strand is read
Which one?
Where to Start?
Where to Stop?
What is the "code" to
"translate" the bases
in mRNA into
amino acids
Where to Start?
Where to Stop?
The Big Picture cont'd – 3 Essential Roles for RNA in Translation
Catalyst = ribosomal RNA
Adaptor = transfer RNA
Template = messenger RNA
Transcription Initiation & Elongation – The Big Picture
Transcription Occurs only 5' • 3' – Initiation Occurs at a Promoter
•Hydrolysis of 5'rNTP • 3'OHN4pN3pN2pN1p5'...+ PPi
Provides Energy for Phosphodiester Bond Formation in RNA
Unlike DNA synthesis, PPi is not hydrolyzed to 2 Pi
Transcription Elongation & Termination – The Big Picture
Transcription stops at a Terminator
Termination Releases the mRNA
Unlike DNA polymerase, RNA polymerase does not proofread
Error rate is extremely low 10-4 – 10-5
Breaking the Genetic Code – Nirenberg & Matthaei, 1961
Synthetic mRNA
Templates
Protein Product
Cell-free
Translation
reaction
Why a triplet code? Consider 4 bases (A,C,G,U); 20 amino acids
1 base = 4 codons (41) – Not enough
2 bases = 16 codons (42) – Still not enough
3 bases = 64 codons (44) – More than enough! Why have 44 "extra" codons?
The Genetic Code Is Almost Universal, Degenerate (Redundant)
& Has 1 Start (AUG) and 3 Stops (UAA, UAG, UGA)
A sequence of codons starting with an AUG
and terminating with UAA or UAG or UGA defines an Open Reading Frame
Consider the following hypothetical mRNA Sequence:
5' AGAGGCCCUGUGCAUCUAUGCCGUUGCGAUA 3'
Could be translated each of 3 ways:
AGA GGC CCU GUG CAU CUA AUG GCC GUU UGA AUA
GAG GCC CUG UGC AUC UAA UGG CCG UUU GAA UA
AGG CCC UGU GCA UCU AAU GGC CGU UUG AAU
Note: Translation Only Proceeds 5' • 3'
AGAGGCCCUGUGCAUCUAAUG GCC GUU UGA AUA
The actual reading frame starts
and stops
This would generate a tripeptide
methionine-alanine-valine = met-ala-val = MAV