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

What controls the start of DNA synthesis in eukaryotic cells?

• cyclin-dependent protein kinase CDK complexes

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

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 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

2. allows Cdt1 to assemble new pre-RCs

3. for the subsequent G1 phase

Sister chromatid cohesion: newly replicated sister chromatin fibres are …

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

How is this sister chromatid cohesion mediated

• by cohesins

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

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

1. entire genomic DNA must be replicated

2. Chromatin strucutrre also replication

How does this happen?

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

2. parental ucleosoms are transfered past the replication fork machinery

3. their histones are recycled

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

5. assembled into nucleosomes on replicated DNA by assembly factors

Histones and DNA can, in principle…

• self assemble to form nucleosome cores

but…

THis process is mediated by…

• other proteins in the cell

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

• 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

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

v

Chromatin remodelling

Once assembled, chromatin fibres are not static:

• Factors remodel chromatin in an ATP-dependent manner

  • slide nucleosomes along the DNA fibre

These remodelling factors are usually…

• large multi-subunit complexes

Several remodelling factors can…

• exchange (and evict) histones within assembled nucleosomes

• utilising histone chaperones as co-factors

What does this allow for?

• an exchange of histone types or reprogramming of epigenetic marks

  • e.g histone modifications

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