Molecular Biology Fifth Edition Chapter 21 DNA Replication II: Detailed Mechanism Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill.

Molecular BiologyFifth Edition

Chapter 21

DNA Replication II:

Detailed Mechanism

Lecture PowerPoint to accompany

Robert F. Weaver

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

21-2

21.1 Initiation

• Initiation of DNA replication means primer synthesis

• Different organisms use different mechanisms to make primers

• Different phages infect E. coli using quite different primer synthesis strategies

• Coliphages were convenient tools to probe DNA replication as they are so simple they must rely primarily on host proteins to replicate their DNAs

21-3

Priming in E. coli

• Primosome refers to collection of proteins needed to make primers for a given replicating DNA

• Primer synthesis in E. coli requires a primosome composed of:– DNA helicase– DnaB– Primase, DnaG

• Primosome assembly at the origin of replication, oriC, uses multi-step sequence

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Priming at oriC

Source: Adapted from DNA Replication, 2/e, (plate 15) by Arthur Kornberg and Tania Baker.

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Origin of Replication in E. coli

Primosome assembly at oriC occurs as follows:– DnaA binds to oriC at sites called dnaA boxes

and cooperates with RNA polymerase and HU protein in melting a DNA region adjacent to leftmost dnaA box

– DnaB binds to the open complex and facilitates binding of primase to complete the primosome

– Primosome remains with replisome, repeatedly primes Okazaki fragment synthesis on lagging strand

– DnaB has a helicase activity that unwinds DNA as the replisome progresses

21-6

Priming in Eukaryotes

• Eukaryotic replication is more complex than bacterial replication

• Complicating factors– Bigger size of eukaryotic genomes– Slower movement of replicating forks– Each chromosome must have multiple origins

• Started study with a simple monkey virus, SV40

• Later consider yeast

21-7

Origin of Replication in SV40

• The SV40 origin of replication is adjacent to the viral transcription control region

• Initiation of replication depends on the viral large T antigen binding to: – Region within the 64-bp ori core– Two adjacent sites

• Exercises a helicase activity that opens up a replication bubble within the ori core

• Priming is carried out by a primase associated with host DNA polymerase

21-8

Origin of Replication in Yeast

• Yeast origins of replication are contained within autonomously replicating sequences (ARSs)

• These are composed of 4 important regions:– Region A is 15 bp long and contains an 11-bp

consensus sequence highly conserved in ARSs

– B1 and B2– B3 may allow for an important DNA bend

within ARS1

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21.2 Elongation

• Once a primer is in place, real DNA synthesis can begin

• An elegant method of coordinating the synthesis of lagging and leading strands keep the Pol III holoenzyme engaged with the template

• Replication can be highly processive and rapid

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Speed of Replication

• The Pol III holoenzyme synthesizes DNA at the rate of about 730 nt/sec in vitro

• The rate in vivo is almost 1000 nt/sec

• This enzyme is highly processive both in vitro and in vivo

21-11

The Pol III Holoenzyme and Processivity of Replication

• Pol III core alone is a very poor polymerase, after assembling 10 nt it falls off the template

• Takes about 1 minute to reassociate with the template and nascent DNA strand

• Something is missing from the core enzyme– The agent that confers processivity on

holoenzyme allows it to remain engaged with the template

– Processivity agent is a “sliding clamp”, the -subunit of the holoenzyme

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The Role of the -Subunit

• Core plus the -subunit can replicate DNA processively at about 1,000 nt/sec– Dimer formed by -subunit is ring-shaped– Ring fits around DNA template– Interacts with -subunit of the core to tether the whole

polymerase and template together

• Holoenzyme stays on its template with the -clamp

• Eukaryotic processivity factor, PCNA forms a trimer, also forms a ring that encircles DNA and holds DNA polymerase on the template

21-13

Model of the dimer/DNA complex

21-14

The Clamp Loader

• The -subunit needs help from the complex to load onto the DNA template– This complex acts catalytically in forming this

processive complex– Does not remain associated with the complex during

processive replication

• Clamp loading is an ATP-dependent process– Energy from ATP changes conformation of the loader

so that-subunit binds to one of the -subunits of the clamp

– This binding opens the clamp and allows it to encircle DNA

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The Clamp and Loader

21-16

Lagging Strand Synthesis

• The pol III holoenzyme is double-headed• There are 2 core polymerases attached through

2 -subunits to a complex– One core is responsible for continuous synthesis of

the leading strand– Other core performs discontinuous synthesis of the

lagging strand– The complex serves as a clamp loader to load the

clamp onto a primed DNA template– After loading, clamp loses affinity for complex

instead associating with core polymerase

21-17

Model for simultaneous strand synthesis• The complex and

clamp help core polymerase with processive synthesis of an Okazaki fragment

• When fragment completed, clamp loses affinity for core

• Associate clamp with complex which acts to unload clamp

• Now clamp recycles

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Lagging Strand Replication

Source: Adapted from Henderson, D.R. and T.J. Kelly, DNA polymerase III: Running rings around the fork. Cell 84:7, 1996.

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21.3 Termination

• Termination of replication is straightforward for phage that produce long, linear concatemers

• Concatemer grows until genome-sized piece is snipped off and packaged into phage head

• Bacterial replication – 2 replication forks approach each other at the terminus region– Contains 22-bp terminator sites that bind specific

proteins (terminus utilization substance, TUS)– Replicating forks enter terminus region and pause– Leaves 2 daughter duplexes entangled– Must separate or no cell division

21-20

Decatenation: Disentangling Daughter DNAs

• At the end of replication, circular bacterial chromosomes form catenanes that are decatenated in a two-step process– First, remaining unreplicated double-helical

turns linking the two strands are melted– Repair synthesis fills in the gaps– Left with a catenane that is decatenated by

topoisomerase IV

• Linear eukaryotic chromosomes also require decatenation during DNA replication

21-21

Termination in Eukarytoes

• Unlike bacteria, eukaryotes have a problem filling the gaps left when RNA primers are removed at the end of DNA replication

• If primer on each strand is removed, there is no way to fill in the gaps– DNA cannot be extended 3’5’ direction– No 3’-end is upstream– If no resolution, DNA strands would get

shorter with each replication

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Telomere Maintenance• At the ends of eukaryotic chromosomes are special

structures called telomeres• One strand of telomeres is composed of tandem

repeats of short, G-rich regions whose sequence varies from one species to another– G-rich telomere strand is made by enzyme telomerase– Telomerase contains a short RNA serving as template for

telomere synthesis• C-rich telomere strand is synthesized by ordinary

RNA-primed DNA synthesis– This process is like lagging strand DNA replication

• This mechanism ensures that chromosome ends can be rebuilt and do not suffer shortening with each round of replication

21-23

Telomere Formation

21-24

Telomere Structure

• All eukaryotes protect their telomeres from nucleases and ds break repair enzymes

• Eukaryotes from yeast to mammals have a suite of telomere-binding proteins that protect the telomeres from degradation, and also hide the telomere ends from DNA damage factors that would otherwise recognize them as chromosome breaks

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Mammalian Telomere Binding Proteins

• In mammals, the group of telomere-binding proteins is known as shelterin, because it ‘shelters’ the telomere

• Six known mammalian proteins: TRF1, TRF2, TIN2, POT1, TPP1 and RAP1

• Other proteins besides shelterin binds to telomeres but they can be distinguished from the others in three ways: they are found only at telomeres, they associate with telomeres throughout the cell cycle and they function nowhere else in the cell

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Mammalian Telomere Binding Proteins

• TRF1 and 2: bind to the double-stranded telomeric repeats

• POT1: binds to the single-stranded 3’ tail of the telomere

• TIN2: organizes shelterin by facilitating interaction between TRF1 and TRF2 and tethering POT1, via its partner, TPP1, to TRF2

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Mammalian Telomere Binding Proteins

• Shelterin affects telomere structure in three ways:

• 1 - it remodels telomeres into t-loops, wherein the single-stranded 3’-tail invades the double-stranded telomeric DNA, creating a D-loop - in this way, the 3’-tail is protected

• 2 - it determines the structure of the telomeric end by promoting 3’-end elongation and protecting both 3’ and 5’-telomeric ends from degradation

• 3 - it maintains the telomere length with close tolerances

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The role of shelterin in suppressing inappropriate repair and cell cycle arrest

• Unmodified chromosome ends would look like

broken chromosomes and cause two potentially

dangerous DNA repair activities, HDR and NHEJ

• They would also stimulate two dangerous

pathways (the ATM kinase and the ATR kinase)

leading to cell cycle arrest

• Two subunits of shelterin, TRF2 and POT1, block

HDR and NHEJ, as well as repress the two cell

cycle arrest pathways

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Telomere Structure and Telomere-Binding Protein in Lower Eukayotes

• Yeasts and ciliated protozoa do not form t-loops, but their telomeres are still associated with proteins that protect them

• Fission yeasts have shelterin-like telomere-binding proteins

• Budding yeasts have only one shelterin relative, Rap1, which binds to the double-stranded part of the telomere plus two Rap1-binding proteins and three proteins that protect the ss 3’-end of the telomere

21-30

The role of Pot1

• In 2001 proteins that bound to the single-stranded tails of telomeres were reported in S.pombe and the gene was named pot1, for the protection of telomeres

• In S.pombe, Pot1, instead of limiting the growth of telomeres, as mammalian POT1 does, plays a critical role in maintaining their integrity

• The loss of Pot1 can cause the loss of telomeres from this organism

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The role of Pot1

• S.pombe Pot1 binds to telomeres and protects them from degradation

• Without Pot1, telomeres in this organism are eliminated

• With time, the few cells that survive without Pot1 circularize their chromosomes so telomeres are no longer needed