Rea Lec 9 DNA Replication

8/13/2019 Rea Lec 9 DNA Replication

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On the lagging strand, DNA is made in fragments

On the leading strand, only one initialprimer is needed. On the lagging strand, many primers

are needed. The RNA primers are removed by a

nucleasethat recognizes the RNA/DNA

heterodimer. ADNA repair polymerase with

proofreading then fills in the gap (end of

Okazaki is primer). The completed fragments are finally

joined/sealed byDNA ligase.

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DNA replication requires the coordination of

many proteins to form the replication machine

1. Need to unzip DNA-helicase (uses ATP)2. DNA polymerase3. Sliding clamp-keeps DNA pol on DNA.

Putting this on requires another protein-theclamp loader (uses ATP).

4. Need to stabilize ssDNA so it doesntrehybridize and keep it elongated-single-

strand binding protein (SSBPs)5. Primase, a nuclease (not shown here), DNA

repair pol, DNA ligase{For Lagging Strand

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many proteins to form the replication machine *


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Telomerase replicates the ends of

eukaryotic chromosomes

Problem: DNA polymerase cannotsynthesize DNA in the 3-to-5direction.And, at the ends of chromosomes there is no place

to lay down an RNA primer. How are telomeres replicated? Solution: Eukaryotes have special

repetitive DNA sequences in theirtelomeres that recruit telomerase. Telomerase is part protein and part

RNA. It recognizes the repeats and

adds more repeats every time acell replicates its DNA.

Telomeres also identify ends ofchromosomes rather than dsDNA

breaksTelomerase linked to both

cancer and aging.

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Lagging strand cannot be completed

Must have a base-paired residue

with a 3hydroxyl to be

synthesized by the DNA

polymerasePrimase requires ~ 20 base pairs to

generate a 10 base pair primer

At some point, there is not enough

room left on the template strand

for the primase

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A DNA mismatch repair system removes

replication errors that escape DNA

polymerase proofreading DNA mismatch repairthe backup system

Fixes DNA mismatchesleft behind by replication machine. Pretty effective (>99%), but not perfect!


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Because germline mutations result in an entire

organism having mutation, protecting the

germ cells from mutations is critical Germ cellsthe reproduction cells = sperm and egg (ex. genetic diseases like SCA) Somatic cellsevery other cell in

your body (ex. cancer*)Due largely to the accumulation of

mutations over time. Anything that

speeds up this process could be

disastrous (ex. Mutation or deletion of

DNA repair enzyme).

colon

cancer in

women

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Mismatches must be repaired properly to

avoid mutations

bad worse good

In eukaryotes, still not known how DNA repair machinery tellsthe difference between the 2 strands. New DNA might be nicked

(ss breaks).

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Figure 6-21a Essential Cell Biology ( Garland Science 2010)

BAD*


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Figure 6-21c Essential Cell Biology ( Garland Science 2010)

GOOD

GOOD

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Figure 6-21b Essential Cell Biology ( Garland Science 2010)

BAD

WORSE

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Mismatches must be repaired properly to

avoid mutations

bad worse good

In eukaryotes, still not known how DNA repair machinery tellsthe difference between the 2 strands. New DNA might be nicked

(ss breaks).


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DNA mismatch repair

Distorts dsDNA; hencecan be recognized as

different

*Nick identifies

the newly

synthesized

strand


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Spontaneous events that compromise DNA integrity*


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Spontaneous events that compromise DNA integrity*


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If not fixed, can lead to mutations


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Figure 6-24 Essential Cell Biology ( Garland Science 2010)

Thymine Dimers can form as consequence of UV radiation

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Damaged DNA can repair itself

using its backupcopy

Can use complementary strand as template. Since most DNA damage creates strange

looking structures, easy to differentiate thetwo strands.

Proteins (nucleases) involved in Step 1vary with different types of DNA damage.

Base Excision Repair (BER) System

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What happens when both strands

of DNA are damaged?

Can happen from ionizingradiation, replication fork errors,

various chemicals and

metabolites, etc.

Nonhomologous end-joining(NHEJ) is the most common

mechanism to repair dsDNA

breaks in somatic cells. Usually OK since most ofgenome non-coding.

Quick and dirty.

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Homologous Recombination

Can produce error-free repairs. Involves using entirely separate DNA

duplex(ex sister chromatids) to fix

dsDNA break. Also used extensively to produce genetic

diversity during meiosis (swapping between

maternal and paternal chromosomes).

Requires extensive stretches of sequencesimilarity (homology). But doesnt have to

be absolutely perfect homology.

Donor DNA needs to be in closeproximity following dsDNA break.

Versatile DNA repair mechanism. Highlyconserved.

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Homologous Recombination*


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Homologous recombinationduring meiosis

more common less common

If heteroduplex has anymismatches, DNA can

undergo mismatch repair.

Can lead to 2 differenttypes of recombination.

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Gene Conversion

Crossover

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Barbara McClintock(1902 1992)

Discovery of Genetic TranspositionJumping GenesTransposons

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Mobile Genetic Elements (Transposons)

jumping genes (molecular parasites ?) Short specialized sequences of DNA that can move throughout a

cells genome. Can carry other genes. Responsible for much more rapid evolutionary genetic changes. Typically affect only that cell and its descendants.

Can be major cause ofantibiotic resistant bacteria.

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Mobile Genetic Elements (Transposons)

inverted repeats5---GACTGCGCAGTC---3

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Mobile Genetic Elements encode the

components they need for movement

Unlike HR, dont require sequence homology. Contains

(1) Gene for transposase(catalyzes the movement of that element

via specialized recombination)(2) DNA sequences that are recognized by its transposase.

Nearly half of human genomeis occupied by millions of

copies of various mobile

genetic elements!!

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Human genome contains 2 major

families of transposable elements

1. DNA-only transposons2. Retrotransposons

Uses RNA intermediate Unique to eukaryotes Most common type

L1 element (LINE-1); 15% human genome Alu sequence; ~1 million copies in our

genome; dont encode their own reverse

transcriptase Both proliferated in primates relatively

recently.

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AluSequence Distribution

Arthrobacter luteus restriction endonuclease~ 300 bps

Formed from the 7SL RNA component

of the Signal Recognition Particle

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Viruses: the ultimate mobile DNA


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Viruses: the ultimate mobile DNA

Essentially strings of genes wrapped in a protein coat. Very small. Parasitesthey need to use cells machinery to replicate. Often lethal (ex lytic) to cell.

Retroviruses are found only in eukaryotic cells.


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Retroviruses make DNA from an RNA template

using reverse transcriptase *


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Retroviruses make DNA from an RNA template

using reverse transcriptase

Latent phase;

virus can hide

for a long time

Lytic phase

Major drug target for AIDSsince unique to virus

e.g., HIV*


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End


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Chapter 7

From DNA to Protein: How Cells

Read the Genome

EssentialCell Biology

Third Edition

Copyright Garland Science 2010


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Charles Robert Darwin Alfred Russell Wallace


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Francis Harry C. Crick James Dewey Watson


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The Central Dogma of Molecular Biology

Occurs in all cells frombacteria to humans.

One of the definingcharacteristics of living cells. Allows massive amplification

of signals from a single gene.

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Caught in the Act

Actively Transcribing Vertebrate DNA


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The efficiency of gene expression

is quite variable

Translation efficiency, as well asRNA and protein stability vary

greatly among genes


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Structure of RNA

Differs from DNA in 2 significant ways:1. ribonucleotides vs deoxyribnucleotides2. uracil vs thymine


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RNA is typically single stranded

Because RNA is single stranded, intramolecular base pairingcan occur, resulting in elaborate secondary structure

This gives rise to diverse functionality (e.g., ribozymes,riboswitches, tRNA, rRNA, )


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RNA serves many functions

final product = RNA molecules

Gene expression refers to the biosynthesis of either

DNA-encoded protein, or non-coding RNA

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From an evolutionary

perspective, RNA may have been

the original, self-replicatingbiopolymer

Ribozymes may have developedthe ability to direct protein

synthesis

DNA is probably a relative

newcomer, usurping RNAs role

in information storage


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Transcription is the DNA-directed biosynthesis of a

single, complementary RNA molecule

RNA is much shorter than DNA. RNA polymerase carries out transcription. RNA polymerase does not need a primer. Many RNA polymerases can transcribe a

single gene at the same time.

Transcription does havesimilarities to replication.

As for DNA, RNA issynthesized in the 5to 3

direction.


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E k i i i diff f b i l


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Eukaryotic transcription differs from bacterial

transcription in a few ways

1. Bacteria have only 1 RNA pol. Eukaryotes have 3.

2. Eukaryotic RNA polymerases require a bunch of accessory proteinscalled the general transcription factors (GTFs) to initiate

transcription.3. Control mechanisms are more complex in eukaryotes in part because

genes are much further apart. This allows more sophisticated gene

regulation.4. Eukaryotic transcription has to deal with more compact chromatin

structure.

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B i l d h ifi


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Bacterialpromotersand terminatorshave specific

sequences recognized by RNA polymerase

The promoter orients RNA pol and tells it where to start and which way to go. All bacterial genes have promoter and terminator sequence similar to those shown

below.

C i f DNA ll RNA l *


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Certain sequences of DNA tell RNA polymerase

where to start (promoters) and stop (terminator/stop site)

In bacteria, the sigma

factor, a subunit of the

RNA pol, recognizes the

promoter.

This is a dynamic process.

In bacteria

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B i l T i i


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Bacterial Transcription*

Ei h d f DNA l


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Either strand of DNA can act as a template,

but the promoter is asymmetrical

Th G l T i i F *


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The General Transcription Factors

Assemble on the promoter. Position RNA polymerase. Open DNA. Launch the RNA polymerase.

typically ~25 bp upstream

of start site; mostpromoters have this

transcription initiation

complex

Both opens DNA and

phosphorylates and

releases RNA pol from

initiation complex

TBP is a

subunit ofTFIID that

distorts

DNA,

forming

landmark

All components are then laterrecycled to be used again and

again

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Figure 7-12 (part 1 of 2) Essential Cell Biology ( Garland Science 2010)

~ 25 bp upstream

from transcription

start siteTBP distorts DNA

TFIIB provides scaffold

TFIIH separates strands

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TATA B Bi di P t i di t t th d bl h li


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TATA Box Binding Protein distorts the double helix*


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~ 25 bp upstream

from transcription

start siteTBP distorts DNA

TFIIB provides scaffold

TFIIH separates strands


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TFIIF phosphorylates tail

domain of RNA pol II

. launches polymerase

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Control of transcription initiation

is the most common wayorganisms regulate control gene

expression.

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Eukaryotic RNAs must be processed and


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In eukaryotes, transcriptionoccurs in nucleus, translation

occurs in cytoplasm. The

exportof RNA occurs via

nuclear pore complexes in thenuclear envelope.Prior to nuclear export,RNA

processingoccurs as the RNAmolecule is being synthesized.

Eukaryotic RNAs must be processed and

exported to cytoplasm

RNA i


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Occurs as RNA is beingmade.

Processing machinery isrecruited to the

phosphorylated taildomain of the eukaryotic

RNA polymerase. Different types of processing

occurs depending of what

type of RNA is being made.

RNA processing *


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*

E k ti RNA i


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Addition of 5-caps and 3-polyadenylation tails (poly-A tails)

(also intron splicing) . 5-caps and poly-A tails:

1. Increase stability.2. Identifies the molecule as mRNA.3. Marks the mRNA as being

complete.

Eukaryotic mRNA processing

Usually gets trimmed back firstbefore few hundred Aadded.

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Introns in eukaryotic mRNA are


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Eukaryotic genes are often interrupted by noncodingsequences (introns). Need to remove/splice these introns out to

get finished/meaningful message. Exons-expressed sequences Introns-intervening/nonexpressed sequences

Introns in eukaryotic mRNA are

removed by RNA splicing

Splicing can happen in prokaryotes, but rarely does.

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Introns are removed by RNA splicing


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Occurs during transcription after 5-capping. Can occur before during or afteraddition of poly-A tail.

Involves cutting out introns and stitchingexons back together.


Unlike exons, most of intron sequence appearsto be unimportant. There are a few short

sequences at or near each intron end that act as

cues for removal.

carried our primarily by catalytic RNA

molecules (small nuclear RNAs; snRNAs)

coupled to a few proteins to form small nuclear

ribonucleoprotein particles (snRNPs)forming

the core of the Spliceosome.

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This process carried our primarily by catalytic RNA

molecules (small nuclear RNAs; snRNAs) coupled to a fewproteins to form small nuclear ribonucleoprotein particles

(snRNPs)forming the core of the Spliceosome.

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snRNPs bind to specific

sequences at both ends of

the intron.The 2hydroxyl of a

conserved Aattacks 5

splice site forminglariat

3hydroxyl of first exon

attacks 3splice site,

knitting exons together

Lariat is degraded

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Alternative splicing leads to greater protein diversity fromsingle gene. ~60% of human genes undergo alternative splicing. Could have helped speed evolution of eukaryotes

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Rea Lec 9 DNA Replication