Rea Lec 8 DNA Replication

8/13/2019 Rea Lec 8 DNA Replication

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Various types of machinery are needed to

adjust the structure of chromatin rapidly

Chromatin-remodeling complexescan decondensechromatin

A molecular motor Needed for both replication

and gene expression. Hence, chromatin structure

is dynamic.

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Covalent modifications of histones regulate

chromatin structure (histone code) These modifications function, in part, by recruiting other

chromatin-remodeling complexes.*


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Covalent modifications of histones regulate

chromatin structure (histone code) These modifications function in part by recruiting other

chromatin-remodeling complexes.

Can also modify core

histones to help alter

chromatin (e.g., HATs)Still learning what

the codesmean

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Interphase chromosomes contain both

condensed and more extended chromatin Euchromatinmore extended, higher gene expression Heterochromatincondensed, low gene expression ~10% of interphase chromatin

Located around centromere and telomeres There are heterochromatin-specific proteins

Heterochromatin can

spread to coding regions

and silencethe gene.Barrier DNA prevents

heterochromatin spread

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Figure 5-29 Essential Cell Biology ( Garland Science 2010)

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Maternal Gene SilencingOne must be silenced,

so that expression in

females and males is

similar.Selection appears to

be randomSilenced X is passedalong the cell lineageFemales are Mosaics

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Chromatin structure can be inherited

(epigenetic inheritance)

Specific histone modifications are passed on. DNA methylation patterns are passed on. This process occurs during differentiation of cells (committed)

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Chapter 6DNA Replication, Repair, and

Recombination

EssentialCell Biology

Third Edition

Copyright Garland Science 2010


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Template-directed DNA Synthesis *


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Base-paring enables DNA replication

new strand

old strand

Watson-Crick base pairing; A-T and G-C


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How to distinguish between these 3 possibilities ?


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Meselson and Stahl Experiment

~ 1.2 % heavier

*14N15N


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14

N

15N


*


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14N

15N


(~ 1 generation)


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14

N

15N


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14N

15N


showed DNA replication is NOT conservative *


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1

223 3

numberof

generations

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Meselson & Stahl (1958)


showed DNA replication IS semiconservative

3rd2nd1st

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DNA replication happens during S Phase *


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DNA replication begins at replication origins

Need to break H-bonds

Typically have veryspecific sequencesA-T rich

A-T rich

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Origin of Replication : Bacteria vs Human

Bacteria (~6 million bps) have single origin of replication intheir circular genome.

Human (~3 billion bps) have ~10,000 origins. Why so many origins?

bacteria human

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DNA synthesis starts at replication forks

Y-shaped structures (replication forks)

Bidirectional

S-Phase

Chromatin


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DNA polymerase is a core component of the

replication machinery

Synthesizes new DNA using one of parental strandsas template.

Catalyzes the addition of nucleotides to the 3-endof a growing DNA strand by forming

phosphodiester bonds between the 3hydroxyl

group and the 5-phosphate group of the incoming

nucleotide. The nucleotides come initially as high energy

nucleoside triphosphates (also a nucleotide). provides the energy for polymerization.

Thus,DNA is synthesized in the 5-to-3direction.

DNA polymerase remains bound to template strandand slides along the DNA.


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Must have 3hydroxyl

to elongate the strand !!

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ATP hydrolyzed to AMP can be used

if more energy is needed

!G0= 32.2 kJ/mol

!G0

= 33.5 kJ/molinorganic

pyrophosphatase

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DNA polymerase is self-correcting

Makes only ~1 error every 107bps. This is beyond the predicted accuracy.

DNA polymerase has 3-to-5proofreadingactivity. Monitors new base-pairing. Can correct mistake using a nuclease

that cleaves the phosphodiester

backbone.

Occurs during DNA synthesis.

Polymerization and proofreading are

carried out by 2 different domains of

DNA polymerase.


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Proofreading corrections are possible

only if DNA is synthesized 5-to-3


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The DNA replication fork is asymmetrical

Solution: DNA polymerase uses backstitchingto piece together short

strands of DNA called Okazaki fragments.


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Answer: DNA polymerase uses backstitchingto piecetogether short strands of DNA called Okazaki fragments.

Thus, this lagging strandis made discontinuously. These

short fragments are subsequently joined together.*


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Short RNAs are primers for DNA synthesis

Q: Since DNA pol (polymerase) can only join a nucleotide to abase-paired nucleotide in DNA, how does it start?A: It needs a primer nucleotide sequence.This priming is done by the enzymeprimase. This enzyme does

not need a base-paired end.Primase makes short stretches of RNA (~10 bps)!primersfor

DNA synthesis.

Primase is an example of anRNA polymerase-an enzyme

that makes RNA using a DNA template. This RNA/DNA

duplex is known as a heterodimer.

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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 anucleasethat recognizes the RNA/DNA

heterodimer. ADNA repair polymerase with

proofreading then fills in the gap (end ofOkazaki is primer).

The completed fragments are finallyjoined/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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many proteins to form the replication machine *


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Lagging strand cannot be completed

Must have a base-paired residue

with a 3hydroxyl to be

synthesized by the DNA

polymerase

Primase 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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"

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Failure to repair DNA mistakes can have

serious consequences

Can lead to permanent changes in the DNAmutations.

Ex. Sickle Cell Anemia


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

BAD

WORSE

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

GOOD

GOOD

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DNA mismatch repair

Distorts dsDNA; hence

can be recognized as

different

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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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Thymine Dimers can form as consequence of UV radiation

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D d DNA i i lf


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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 the

two strands. Proteins (nucleases) involved in Step 1

vary 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*


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Homologous recombinationduring meiosis

During meiosis, chromosomal crossovers lead tothe exchange of genetic information.

During meiosis recombination preferentially occurs

between maternal and paternal chromosomes rather

than between newly replicated, identical DNA

strands like when HR repairs dsDNA breaks.

(meiosis-specific proteins)


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Gene Conversion Crossover

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Barbara McClintock(1902 1992)

Discovery of Genetic TranspositionJumping GenesTransposons

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M bil G i El (T )


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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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M bil G i El (T )


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Mobile Genetic Elements (Transposons)

inverted repeats5---GACTGCGCAGTC---3

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Mobile Genetic Elements encode the


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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


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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 relativelyrecently.

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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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Vi th lti t bil DNA


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Viruses: the ultimate mobile DNA

Vi th lti t bil 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.

Vi a d th h t ll a hi


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Viruses commandeer the host cell s machinery

This lysis can cause an immune response.

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Retroviruses make DNA from an RNA template


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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*

Rea Lec 8 DNA Replication

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