1. Molecular Biology Fifth Edition
Lecture PowerPoint to accompany
Molecular Biology Fifth Edition
Robert F. Weaver
Chapter 21
DNA Replication II:
Detailed Mechanism
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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

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

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

5. 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

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

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 a

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

9. 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

10. 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

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 b-subunit of the holoenzyme

12. The Role of the b-Subunit
Core plus the b-subunit can replicate DNA processively at about 1,000 nt/sec
Dimer formed by b-subunit is ring-shaped
Ring fits around DNA template
Interacts with a-subunit of the core to tether the whole polymerase and template together
Holoenzyme stays on its template with the b-clamp
Eukaryotic processivity factor, PCNA forms a trimer, also forms a ring that encircles DNA and holds DNA polymerase on the template

13. Model of the b dimer/DNA complex

14. The Clamp Loader
The b-subunit needs help from the g complex to load onto the DNA template
This g complex acts catalytically in forming this processive adb 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 d-subunit binds to one of the b-subunits of the clamp
This binding opens the clamp and allows it to encircle DNA

15. The b Clamp and Loader

16. Lagging Strand Synthesis
The pol III holoenzyme is double-headed
There are 2 core polymerases attached through 2 t-subunits to a g complex
One core is responsible for continuous synthesis of the leading strand
Other core performs discontinuous synthesis of the lagging strand
The g complex serves as a clamp loader to load the b clamp onto a primed DNA template
After loading, b clamp loses affinity for g complex instead associating with core polymerase

17. Model for simultaneous strand synthesis
The g complex and b clamp help core polymerase with processive synthesis of an Okazaki fragment
When fragment completed, b clamp loses affinity for core
Associate b clamp with g complex which acts to unload clamp
Now clamp recycles

18. 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.

19. 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

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. 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

22. 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

23. Telomere Formation

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

25. 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

26. 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

27. 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

28. 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

29. 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

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

31. 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