Chem 40 April29 Replication

8/7/2019 Chem 40 April29 Replication

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Chemistry 40

Replication(DNA Synthesis)

Chemistry 40

How Is DNA Replicated?

DNA replication is semiconservative

DNA replication is bidirectional

Replication requires unwinding of theDNA helix

DNA replication is semidiscontinuous

The lagging strand is formed fromOkazaki fragments


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Chemistry 40

Fig. 10-2, p.242


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Chemistry 40

Features of DNA Replication

DNA replication is bidirectional

Bidirectional replication involves tworeplication forks, which move in oppositedirections

DNA replication is semi-discontinuous

The leading strand copies continuously

The lagging strand copies in segments(Okazaki fragments) which must be joined

Bidirectionalreplication of theE. colichromosome. (a)If replication isbidirectional, auto-radiograms ofradioactivelylabeled replicatingchromosomes

should show tworeplication forksheavily labeledwith radioactivethymidine. (b) An

autoradiogram ofthe chromosomefrom a dividing E.colicell showsbidirectionalreplication. (Photocourtesy of David M.Prescott, Universityof Colorado.)


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Chemistry 40

Fig. 10-4, p.244

Chemistry 40

Features of ReplicationMostly in E. coli, but many features are general

Replication is bidirectional

The double helix must be unwound - byhelicases

Supercoiling must be compensated - by DNAgyrase

Replication is semidiscontinuous

Leading strand is formed continuously

Lagging strand is formed from Okazakifragments - discovered by Tuneko and Reiji "O"


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Chemistry 40

Fig. 10-5a, p.245

Chemistry 40

Fig. 10-5b, p.245


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Chemistry 40

Fig. 10-10, p.249

Chemistry 40

Fig. 10-11, p.250


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Chemistry 40

Chemistry 40

Table 10-2, p.242


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Chemistry 40

DNA Polymerase III

The "real" polymerase in E. coli At least 10 different subunits

"Core" enzyme has three subunits - , , and

Alpha subunit is polymerase

Epsilon subunit is 3'-exonuclease

Theta subunit is involved in holoenzymeassembly

The beta subunit dimer forms a ring around

DNA Enormous processivity - 5 million bases!

Chemistry 40


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Chemistry 40

Chemistry 40

How Is DNA Replicated in Eukaryotic Cells?

The cell cycle controls the timing of DNAreplication

Eukaryotic cells contain a number of differentDNA polymerases

DNA polymerase d is the principal DNA

replicase


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The eukaryotic cell cycle.The stages of mitosis andcell division define the Mphase (Mfor mitosis). G1(Gfor gap, not growth) is

typically the longest part ofthe cell cycle; G1, ischaracterized by rapid

growth and metabolicactivity. Cells that arequiescent, that is, notgrowing and dividing (suchas neurons), are said to be

in G0. The S phase is thetime of DNA synthesis. Sis followed by G2, arelatively short period ofgrowth when the cellprepares for cell division.Cell cycle times vary fromless than 24 hours (rapidly

dividing cell such as theepithelial cells lining the

mouth and gut) tohundreds of days.

Chemistry 40

How Are the Ends of ChromosomesReplicated?

Telomeres, the structures at the ends ofeukaryotic chromosomes, consist of 5-8 bptandemly repeated G-rich nucleotidesequences

Telomeres are 1-12 kbp long

Telomeres are replicated by an RNA-

dependent DNA polymerase calledtelomerase


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Telomere replication. (a) In replication of the lagging strand, short RNA primers are added(pink) and extended by DNA polymerase. When the RNA primer at the 5-end of each strandis removed, there is no nucleotide sequence to read in the next round of DNA replication.The result is a gap (primer gap) at the 5-end of each strand (only one end of the chromosome

is shown in this figure). (b) Asterisks indicate sequences at the 3-end that cannot be copiedby conventional DNA replication. Synthesis of telomeric DNA by telomerase extends the 5-ends of DNA strands, allowing the strands to be copied by normal DNA replication.

Chemistry 40

Eukaryotic DNA Replication

Like E. coli, but more complex

Human cell: 6 billion base pairs of DNA tocopy

Multiple origins of replication: 1 per 3- 300kbp

DNA polymerase alpha - four subunits,polymerase (processivity = 200) but no 3'-exonuclease

DNA polymerase beta - similar to alpha


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Chemistry 40

More Eukaryotic polymerases

DNA polymerase gamma - DNA-replicating enzyme of mitochondria

DNA polymerase delta has a 3'-exonuclease as well as proliferating cellnuclear antigen (PCNA)

PCNA give delta unlimited processivityand is homologous with prokaryotic pol III

DNA polymerase epsilon - highlyprocessive, but does not have a subunitlike PCNA

Chemistry 40

How Are RNA Genomes Replicated?

Many viruses have genomes composed ofRNA

DNA is an intermediate in the replication ofRNA viruses

The viral RNA serves as a template for DNAsynthesis

The RNA-directed DNA polymerase is calledReverse Transcriptase


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Figure 28.16

The structures of AZT (3-azido-2,3-dideoxythymidine). This nucleosidewas the first approved drug fortreatment of AIDS. AZT is

phosphorylated in vivo to giveAZTTP (AZT 5-triphosphate), asubstrate analog that binds to HIVreverse transcriptase, HIV reversetranscriptase incorporates AZTTPinto growing DNA chains in place of

dTTP. Incorporated AZTMP blocksfurther chain elongation because its3-azido group cannot form aphosphodiester bond with anincoming nucleotide. Host cell DNA

polymerases have little affinity forAZTTP.

Chemistry 40

Reverse Transcriptase

Primer required, but a strange one - a tRNAmolecule that the virus captures from the host

RT transcribes the RNA template into acomplementary DNA (cDNA) to form aDNA:RNA hybrid

All RNA tumor viruses contain a reverse

transcriptase


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Chemistry 40

Reverse Transcriptase Activities

Three enzyme activities RNA-directed DNA polymerase

RNase H activity - degrades RNA in the

DNA:RNA hybrids

DNA-directed DNA polymerase - which makes

a DNA duplex after RNase H activity destroys

the viral genome

HIV therapy: AZT (or 3'-azido-2',3'-

dideoxythymidine) specifically inhibits RT

Chemistry 40

Table 10-3, p.250

Chem 40 April29 Replication

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