L3: Eukaryotic chromosome replication I

Replication origins, centromeres, telomeres

How does chromosome replication differ in eukaryotes to prokaryotes

1. replication initiates at many sites per chromosome

2. one round of rpelication and cell division is completed before the next starts

3. parental & progeny DNA is assembled into nulceosomes and higher order chromatin strucutres

The three important elements on chromosomes for DNA cycle

1. Relication origin

2. Centromere

3. Telomere

What are the DNA replication initiation sites called

Replication origins

Why is it useful for origin to be specific sites

• so that replication cannot just keep happen all the time

• need to be specific r=time at specific sites

• helps to regulate it

what we know about replication origins in eukaryotes?

• We know more about prokaryotic, animal viruses and lower eukaryotes (e.g yeast0 replication origins

The replicon model: start with a conceptual idea of how replication of circular bacterial chromosomes might be regulated

Processes at a replicator

1. unwound

2. primed

3. elongated

where is knowledge of higher eukaryotic origins from?

• biochemical

• genetic analyses of few example cases

• high thorughput DNA sequencing analyses at genome-wide resolution

Is the model supported?: Replication origins of animal viruses: they do nothave all the elements

e.g SV40 and polyoma virus

• organised as minichromosomes

what do they have→ only the control elements for their own replication:

1. origin sequences

2. initiator protein

Imagese of replicating circular SV40 DNA

How do they get the other factors required for replication?

• recruited from the infected host cell (monkey or human)

therefore:

• they becaome the first simple and useful model systems for eukaryotic chromosome replication

What is the initiator protein

‘Large T antigen’

What does it do to initiate replication

1. bind to unique site on viral genome

   • 65bp control region containing:

     1. a 27 bp inverted repeat (a potential hairpin)

     2. conserved A/T rich element is needed for origin function

Origin unwinding by T antigen

• see the single stranded DNA is tagged with proteins in the EM

→ THEREFORE: this matches the replicon model

Overall genomic map of SV40

1. Large T antigen (early genes)

2. Late genes

3. Control region + replication origin with origin sequence

Why are virus origins not valid models for cell chromosomal orgins?

• virus must replicate many times in one cell cycle for propagation

• → therefore: defies cellular controls

Replicating chromatin image (drosphila)

How are chromosomal origins different

1. Linear chromosomes

2. Many origins per chromosome

3. activated only once per cell cycle

Yeast: plasmids

2 mu plasmid

• organised as spisomal minichromosomes

Yeast plasmids: what does they replication require

1. specific DNA seqeunce element→ autonomously replicating sequencse ARS element

What does the ARS element act as

• replication origin

• allows initiiation

ARS elements were isolated from random genomic DNA fragments by…

• their ability to confer the ability to replicate

• as an autonomous plasmids in yeast cells→ after ligation to an origin-less circular plasmid

How experiment worked

1. Get a plasmid vector containing a selectible marker

   1. i.e HIS gene required to make histidine

2. Add randomly selected yeast DNA segment

3. If does not contain ARS→ rare transformants obtained: these contain plasmid DNA that has integrated into a yeast chromosome

   • some will be able to survive but the offspring of this will not have the plasmid so will not→ that is why you get SOME colonies but not all of them

4. If it does contain ARS→ high frequency transformants obtained

   1. contain plasmid DNA circles replicating free of the host chromosome

Mutant analysis: Deletion and point mutants in typical ARS element define…

• an essential consensus

  • A box

• for ARS activity

Mutant analysis: What are B elements

• Flanking sequencing

• affect the efficieny of ARS function

What does this mutant analysis overall show

• Get an idea of wchih parts are more important than others

• which parts can/cannot stand mutation

OVERALL: Gives ideas of the spacing of components along the ARS

What is the DNA sequence conservation at the A element: 

11bp consensus sequence (ACS)

How many ARS elements are found in chromosomal DNA of budding yeast

• over 200 ARS elements

In this context, they also function as

• origins

However…

• replication does not always initiate at all possible yeast origins

How are the A and B elements not degraded by nnuclease?

Experiment: with nuclease digestion in vivo

• show DNA sequences are protected by associated proteins

How are yeast origins recognised

• sequence-dependent manner

• by initiator protein complex 

What is the initator complex called

• origin recognition complex ORC

How does the ORC work

This diagram shows the spacing that the mutant analysis hinted at

1. recognises origin

2. binds with A box in presenece of ATP

3. cdc6, cdt1, MCM complex associate with ORC in a stepwise manner

   • occupying additional positions e.g B elements

overall: forms the pre-replicative complex 

Why is yeast a good model

1. Small

2. acts like bacteria→ colonies, plasmids, etc

3. Eukaryotic

4. 5-6 thousand genes

   • analagous to human genes

   • humans: 20000 genes→ will be duplicates and divergents of the yeast ones

Replication origins in higher eukaryotes: human chromosomes are big, meaning with two divergent forks

• take a month to replicate from a single initiation site

• moving at 3kb/min

Features in mammalian replication:

1. okasaki fragments

2. Transition point: initiation site, origin of replication

3. Forward nascent strands (leading strands)

How come replication only lasts less than 10 hours?

• have several thousand replication origins present per chromosome

• tens or hundres of thousand origins per genome

EM analysis of replicating chromosomes showed that

• there are replication bubbles emanating from activated origins

  • at intervals of 30-300kb

Site specific replication origin is defined and mapped at high precision by

• determining the transition point 

• between leading and lagging  strand synthesis

What technologies have made mapping DNA replication origins in vertebrate cells easier?

• high-throughput DNA sequencing

  • e.g SNS-seq

  • e.g ini-seq

• computational analyses

identified some tens or thousand potential origin sites in the human genome

Genome-wide origin mapping techniques

1. Sequencing of short nascent strands (SNS-seq)

2. Sequencing of initiation sites (Ini-seq)

3. Sequencing of Okazaki fragments (OK-seq)

What is SNS-seq

1. small nascent DNA leading strands tagged so know they are new strands that have been replicated

2. isolated from replicating cells

3. sequenced to localise replication origins

Also a bulk experiment

SNS-seq sequence mapping on the human genome

What is ini-seq

1. DNA replication initatied in nuclei of cells synchronised

   • in late G1 phase of cell cycle (just before DNA rep)

2. DNA allowed to replicate only for a short time following initiation

3. labelled by modified nucleotide

4. initiation-site associated replicated DNA is isolated and sequenced

SNS-seq and ini-seq have both

• yielded consistent result

• detailing tens of thousands of defined replication origins in the human genome

OK-seq: Okazaki fragments

Sequence mapping on the human genome OK-seq

Sequence mapping on the human genome OK-seq compared to the other techniques: what does it show?

Initiation zone:

• many inefficient origins

• often flanked by efficient ones

→ conglomerate of many weak origins

What characterises active DNA replication origins?

• not defined by consensus DNA sequences

  • unlike seen in yeast or SV40

What are these replication origin correlate to?

1. Proximity to gene promoter sites

   • but outside the genes

2. genomic sites with high GC content

   • including CpG islands

   • G quadruplexes

   • oxidised methyl-cytosines

3. Distinct patterns of histone modification

4. Open chromatin (hypersensitive to DNAse I)

Many of them!

2. Features of the genomic sites with high GC content

1. May form G quadruplex strucutres

2. May contain modified bases

   • methylated

   • hydroxymethylated cytosines

Additional experimental approaches

1. genome-wide profiling of Osaki fragments

2. isolation of small replication bubbles

These techniques have suggested that…

• several individual origins tend to aggregate into

• larger initiation zones

Prominent origin sites (and initiation zones) are often present in…

• actively transcibed euchromatic regions of the genome

Where are they frequenctly located

• at or upstream of Transciption Start Sites

What does this mean?

• when DNA replication initiates at or near active gene promoters

• replication and transciption elognation complexes move in the same direction

• and any detrimental head-on collisions between are avoided

  • over that transcribed gene body!

Genetic and epigeneic factors that determine or influence specification of an origin site on a higher eukaryotic are…

• still unclear and currently under investigation

Unlike in yeast…

• No strict DNA consensus motifs are known (to date)

however

• likely candidates of origin specification elements include:

  • short GC-rich DNA sequence motifs

    • that may lead to unusal DNA strucutures (i.e G quadruplexes)

  • include specific DNA and histone modifications

Centromeres: what do they do

• attach chromosomes to mitotic or meiotic spindles

In yeast: why are they needed

• Plasmids containing ARS elements replicate

BUT

• are gradually lost without strong selective pressure

Centromere sequence (CEN)

• stabilise them

• We only x2 the DNA

• so in order to enusre that each new cells has a copy of each chromosome→ need to make sure the DNA is exactly split in two

• so need centromeres

unlike to bacteria which could just replicate loads of times and then hope that each cell will have a copy o each gene it needs.

A CEN conist of

1. Three conserved DNA sequence elements:

   • I, II and III

   • Needed for SPECIFIC attatchment points for microtubule

2. AT-rich element n the middle

   • just needs to be the right size

   • doesn’t matter about sequence

   • so the microtubule can fit between the points

Centromes serve as what?

• the attachment sites for 

  • centromeric proteins

  • spindle microtubules

to form part of…

• the kinetochore complex

• where in mitosis, the chromatids of condensed chromosomes are attached to mitotic spindle

i.e microtubule does not attach directly→ instead does it through kinetochores

Molecular interactions at kinetochores

Centromeres in higher eukaryotes 

• Larger and more complex

• you can see that they are similarin strucutre to the yeast ones

  • when interacting with other proteins

Centromeric DNA contains…

• alpha-satellite DNA elements

• further assembled into specific centromeric heterochomatin

  • spreads even further into chromosomes

Centric heterochromatin contains…

1. sections with centromere-specific histone H3 varianet

2. sections with normal histone H3 that is di-methylated  at lysine 4

Where centromeres are in the chromatin

What do telomeres provide

• stable chromosome ends

Why need Telomeres

1. Protect from exonuclease degredation of ends

2. Stops the termination problem issue

   • when okaski fragments cannot be extended until the end

   • so causes degredation

   • telomeres→ before useful DNA so that it is only rubbish repeated regions that get degraded

telomerase can extend these in cells that do not really have a lifespan: Germ cells, cancer cells and induced in tissue culture cells

Linear plasmids in yeast need

1. CEN

2. ARS

3. Telomere (TEL)→ functional chromosome end

in order to replicate and segregate to daughter cells

Telomere DNA sequence repeats

telomeres contain…

• simple repeating sequences

  • forming 3’-single stranded DNA overhang ends

→ FORMS into Terminal loop T-loop

model of T-loop

What are these 3’-single stranded DNA overhanging ends in different things

1. Yeast repeat TG(1-3)

2. human repeat TTAGG

3. Arabidopsis repeat TTTAGGG

stabilised by proteins

→ becomes a loop so no end anymore!

Models of the unsual strucutre of the DNA ends

1. hairpin

2. loop model

To explain the DNA ends as no free ends are detectable

EM has shown human telomere terminal loop ‘T-loop’

How was telomeric DNA assembled into

• specilaised telomeric heterochromatin 

• attracts a protective protein complex called→ shelterin

Telomeric heterochromatin spreads from…

• telomeric DNA

• further into the chromosomal DNA

It is possible to generate…

YACs→ yeast artificial chromosomes by using

• all three elements

What are YACs used for

• cloning huge DNA fragments

  • e.g

  • during the original human genome sequencing project

Hman sticky ends that can stick plasmid→ can transcribe useful human genes and get alot of DNA into it (unlike in bacteria genes)