L5: Eukaryotic chromosome replication III

Cell cycle control of DNA replication:

• DNA replication is tightly controlled during the cell division cycle

  • entire genome is replicated precisely once in S phase

  • separation of the replicated chromatids occurs in mitosis

BOTH EVENTS ARE STRICTLY SEPARATED

The licensing factor model: explaining how DNA is only replicated once

1. activator made

2. binds to DNA

3. bound activator stable and the free is destroyed

4. replication initiates at activator

5. activator destroyed by initiation or by fork

How re-initiation prevented?

• helicase loading and activation under cell cycle control

What controls the start of DNA synthesis in eukaryotic cells?

• cyclin-dependent protein kinase CDK complexes

→ kinases→ will phosphorylate the cyclins→ acts as an activation switch

These are related to

• the complex of cyclin B and CDK1

• that controls mitosis

Prime candidates iin vertebrates

1. cyclin A-CDK2

2. cyclin E-CDK2 complexes

What is also important:

Dbf4-Cdc7 protein kinase DDK

• crucial for origin activation

and

• initiation of DNA replication

Cell cycle control machinery: cyclin expression

1. mid-G1 cyclin CDKs

   • cyclin D-CDK4

   • cyclin D-CDK6

2. late G1 cylin-CDK

   • cyclin E-CDK2

3. S-phase cyclin CDK

   • cyclin A-CDK2

4. mitottic cyclin-CDKs

   • cyclin A-CDK1

   • cycli B-CDK1→ very high especially

In order to ensure replication if only once per cell cycle, you need to be able to…

1. Initate replication only once per cell cycle

AND ALSO

2. Ensure that it is fully once

How to ensure that replication is FULLY once 

• many origins that are spread out

• some are loaded

• some are not loaded

  • stochastic nature

• can be used as fall back if needed

• Have many more than you actually need

1. Not only have many origins replicating at once→ so it is fully done and not too slow

2. Also→ have things to fall back on→ ensure that it will be all fully done

Once replication is initiated at an origin…

• re-initiation is prevented

• stops it happening twice in a replication fork

how…?

How is re-initiation of DNA replication prevented

• at each origin is the essential pre-replication complex (pre-RC)

• replication licence

→ assembled following exit from mitosis

What is the licensing factor? (MODEL)

1. present at un-replicated chromatin at origins

2. required for origin activation/initiation

3. becomes inactivated during origin activation

4. absent on replicated chromatin until mitosis

i.e can only be licsensed BEFORE start→ coz environment changes other wise→ temporal and spatial separation between licensing and initiation

What fits this model:

1. MCM2-7/Cdt1 proteins fit the model best (seen above)

2. Oxidised double strand DNA also fits

1. MCM2-7/Cdt1 What does it consist of

1. ORC

2. cdc6

3. Cdt1

4. MCM (minichromosome maintenance) proteins

required for initiation

What happens after DNA replication is initiated

1. pre-RC is dismantled

   • Cdc6 and Cdt1 are degraded by proteolysis

   • MCM complexes are displaced from replication DNA

→ reformation of new pre-RCs and re-initiation of DNA rep are therefore prevented

• until exit from mitosis

First level of control is exerted by CDKs:

• High CDK activity is essential for origin firiing in S phase

• and for preventing pre-RC re-assembly in S and G2

  • CDK activities are low in G1

This basic mechanism is conserved from yeast to humans…

Second indpenendent level of control (found in mutlicellular organism) involves…

• Cdt1

• protein Geminin

1. Geminin

• binds to and inactivates remaining Cdt1 in S and G2 phase

• prevents re-assembly of new pre-RCs after initiation of DNA replication

In mitosis, for the subsequent G1 phase…

1. Geminin is degraded (perhaps due to CDKs)

2. allows Cdt1 to assemble new pre-RCs

3. for the subsequent G1 phase

the degredation and remaking of Geminin must be more efficient than the making and degredation of cdt1

2. Oxidised double strand DNA also fits

Modification of cytosine:

• the oxidiased 5f-cC is only found at unreplicated origins

• The oxidation reactions are inhibited by bobcat399

  • bobcat STOPS replication

2. Evidence for this…

1. Stain with MSO→ the replicating cells will be green

2. Add bobcat399 enzyme→ no replicating cells (often released in S phase)

3. Release and add bobcat399 again→ some cells are now replicating

   • allows oxidation

THEREFORE: this is a cause and effeect experiement

2. Times when the DNA is modified

1. quiesnce→ after mitosis

2. late G1 phase→ % modified DNA still high

3. very low in S phase→ replicating at this point

2. Step by step of this modification

1. G1→ modified so dense

2. initiation S phase→ the daughter strands are not dense/modified

3. S phase→ eongation→ some modification

4. S/G2→ methylation and DNMT→ becoming more dense

5. coming back into G1→ oxidation TET→ dense again

i.e the DNA modifications itself are helping to regulate when replication happens, to ensure that it is happening only once

Sister chromatid cohesion: newly replicated sister chromatin fibres are …

• physically held together until the metaphase to anaphase transition in mitosis

COHESION

How is this sister chromatid cohesion mediated

• by cohesins

  • → proteins are belonging to the class of ;structural maintenance of chromosomes’ proteins (SMCs)

sister chromatid cohesion by SMC proteins

• before they have become the kelsisin complexes

• similar to cohesins seen previously but from different genes

Chromosome assembly: during chromosome replication in S phase, what must happen

1. entire genomic DNA must be replicated

2. Chromatin strucutrre also replication

Key observations on replicating chromatin fibres:

1. nucleosomes are present on both unreplicated parentala and replicated daughter DNA strands

2. New nucleosomes are present on replicated DNA already within a few hundred base pairs past the fork

THEREFORE:

• Nucleosome assembly needs to be fast and efficient

How does this happen?

1. in front of advancing replication form, chromatin partially diassembles

2. parental nucleosoms are transfered past the replication fork machinery

3. their histones are recycled→ on one of the strands

4. new histones are synthesised during S phase of the cell cycle

5. assembled into nucleosomes on replicated DNA by assembly factors

In order to ensure that the histone modifications are preserved for the next DNA strand

• this partial degrading helps ensure that each ‘new’ histone STILL has components of old ones

• Means that the histone modifications are preserved

• the cell maintains its identiity

  • → still some after modification after to ensure back to normal 

Issue after DNA repair?

• DNA repair might mean old histone are lost or changed

• so new DNA stand may not take up the whole old identity of the old histone

• may lose some knowledge of expression etc of the cell type

Histones and DNA can, in principle…

• self assemble to form nucleosome cores

but…

THis process is mediated by…

• other proteins in the cell→ chromatin assmebly factor

Example: Xenopus embryos

• proteins called N1  and nucleoplasmin

• associated with histones

• will assemble nucleosome cores at physiological ionic strength in vitro

Example: in human and other cells (SV40)

• Chromatin assembly factor CAF-1 (chromatin assembly factor)

• facilitiates replication-dependent nucleosome assembly in the SV40  replication system

How does it do this

1. interacts with replication fork protein PCNA

2. targets newly synthesised histones H3 and H4 to the replication fork

3. Other assembly proteins

   • Asf1 and NAP-1/2

   • act synergistically with CAF-1 to assemble entire new nucleosomes

OVERALL: 2 steps for histone subunits:

1. H3-H4 on DNA via PCNA (which was used previously in replication)

2. H2A-H2B dimers bind to NAP-1onto the DNA

CAF-1 structure

Made up on:

• p60

• p48

• p150

CAF-1 and H3/H4 cryo-EM strucuture

• H3/H4 are in handshake arrangement

• p150 loop kinda resembles the structure of DNA

  • in extendedn configuration

3D model of nucleosome→ histone DNA contacts

• similar to the contacts being made between p150 and the histone

This suggests that…

• CAF-1 mimics DNA

• so easily hands over the histone back onto the DNA

co-localises with DNA replication foci

Evidence for this

1. replication loci marked

2. CAF-1 (p60)→ shown in green

3. Merge→ shows orange so must be both

   • CAF-1 p60 colocalises with DNA replication foci

so yes this does happen in the cell

What happens to old tetramers of histones H3 and H4

1. stay together during replication

2. after transfer to a replicated DNA daughter strand

3. can associated with either new or old dimers of histones H2A and H2B

4. Linker hitones H1 associate later and higher order strucutres are forme

Chromatin remodelling

Once assembled, chromatin fibres are not static:

• Factors remodel chromatin in an ATP-dependent manner

  • slide nucleosomes along the DNA fibre

• Nucleosomes compete with DNA binding proteins

  • can therefore inhibit DNA trnasaction

    • transciption, replication initiation, repair

Types of remodelling

1. histone exchange

2. nucleosome sliding 

These remodelling factors are usually…

• large multi-subunit complexes

1. Histone exchange

• exchange (and evict) histones within assembled nucleosomes

• utilising histone chaperones as co-factors

1. What does this allow for?

• an exchange of histone types or reprogramming of epigenetic marks

  • e.g histone modifications

UPPER: H2A/H2B have been switched out with a modified other

LOWER: whole histone has benn reoved and replaced with histone with new core

2. Nucleosome sliding

• extends and makes a loop around the histone

• moves around like an inchworm

• gradually moving the histone along

→ takes alot of energy

• each cycle moves it only by one base pair

takes energy so needs chromatin assembly factors to help

DNA translocation model (SWI/SNF)

• how the nucelosome sliding works

Overall: chromatin remodelling allows…

• chromatin fibre to be dynamic

• therefore→ can react to metabolic requirements arising from:

  • DNA replication, repair and transcription

A mechanistic feature of this is that…

• DNA binding factors will thus be able to gain access to sites on DNA

• which might be otherwise occluded by nucleosomes