L4: Eukaryotic chromosome replication II

What is the first enzymatic step of DNA replication

1. localised separation 

   • or unwinding of the two DNA strands at the replication origin

   • catalsed by DNA helicases

2. Unwound DNA is stabilised by single-stranded binding proteins

3. DNA polymerases and additional proteins are recruited thhat built up active DNA replication forks

This is seen in SV40 origin unwinding

• with large T antigen

The replicative DNA helicase: the eukaryotic helicase is…

• a core complex of six MCM proteins

• with many other associated proteins:

  • Cdc45

  • GINS complex

1. Loading the MCM complex (the helicase)

1. MCM double hexamer complex is loaded at replication origins in an ATP-dependent manner

   • by origin recognition complex ORC

2. Involving Cdc6 and cdt1 proteins

   • (ORC binds to Cdc6→ 6 subunits hexamer complex)

3. Loads Mcm on DNA

4. The complex can now move along DNA→ similar to what homohexmaer in T antigen does

5. Removes cdt1 (accessory protein)

6. recruits another copy of the hexamer (again using a cdt1 to help it load)

7. The double hexamers can move away from eachother (but have to be activated first)

2. Activation of the MCM helicase in S phase

1. Loaded but inactive MCM double hexamer complex is converted into  an active form

2. With protein kinases CDK and DDK

   • with association with several other proteins:

   • cdc45 and GINS DDK pol epsion (pol subunit)

3. Complex: CMG complex 

   • (Cdc45, MCM, GINS)

4. Mcm10 added

5. origin melting,

6. MCM ring opening,

7. Double hexamer separation

8. Helicase activation

3. What does DNA helicase activation lead to

1. local unwinding 

2. separation of the double hexamer complexes

3. ATP used to migrate and push

4. Where do each of the two CMG  helicase complexes travel

• travel with one of the two emerging replication forks away  from the initiation site

5. After unwinding, the active CMG DNA helicase…

1. translocates on the DNA leading strand

   • in 3’ to 5’ direction 

2. Dependent on ATP hydrolysis

THEREFORE→ displacing the complementary DNA strand

6. What does this cause?

1. Around activated helicases, functional DNA replication fork complexes are assempled

2. Involving the recruitment of the DNA polymerases and replication factors

Conservation

• Strucutre of eukaroytic DNA helicase:

  • E1 protein of papillomavirus and ssDNA

• similar strucutres have been obtained for the cellular MCM2-7 hexamer complexes

• NOTE: no origin consensus sequences in vertebreates (unlike in yeast) BUT ORC cdc6, cdt1 and MCMs are strucutrually and functionally concerved from yeasts to vertebrates

• How we know about protein funtions

  • mutant genetics

  • biochem→ reductionist→ until pure protins and so test function

Comparing SV40 viral genome to eukaroytic cellular genomes

1. SV40 viral genome

   • one protein for both binding and helicase activity

2. Eukaryotic cellular genomes

   • two protein complexes:

     • one for origin binding and helicase loading (ORC etc)

     • one for helicase acitivity (MCM/CMG etc)

Aims of DNA replication

Types of DNA polymerases→ 6 major DNA polymerases (pol) in eukaryotes

• polyermisation in 5’ to 3’ direction by DNA polymerases

→ because the chemistry of DNA is uni-directional

Three main types of DNA polymerases

1. alpha

   • contains purine

   • needed for priming (RNA synthesis)

   • limited primer extension (DNA synthesis)

   • → starts off the replication

2. Delta

   • Highly processive replication

   • lagging strand synthesis (Okazaki fragments)

   • → helps complete the replication coz it can do longer stretches

3. Epsilon

   • highly processive replication

   • leading strand synthesis

   • → helps complete the replication in the LEADING

DNA polyermases delta and epsilon contain:

• proof-reading exonuclease activity

How does DNA polymerase acitivty work: hand fist anaology

1. Open lose grip hand

2. with incoming deoxynucleoside triphosphate nearby

3. closed fist

4. positions the triphosphate

5. nucleotide incorporated

6. open again

7. phosphates leave

perhaps need to look at how detailed the text book is of this process for more info!

But, DNA polymerases make mistakes, they are corrected by…

1. Proof-reading exonuclease activity

2. by mismatch repair systems

can tell that there is a mismatch becase the DNA doesn’t bind as well and is bulky

diagram shows→ the polymerase shuffles back along the DNA to re-do the mistake it just made

Error rates of the pol

• pol alpha is the worst

• pol gamma or epsilon

• pol gamma or epsilon 

Why need low error rate in eukaroytes?

• multicellular organism

• other cells rely on these cells

• → can get benefits from duplicated genes that are mutated and do not have such a detrimental effect

• in bacteria etc→ mutations are actually fab

Replication factors: other crucial proteins of the eukaryotic DNA replication fork include:

1. DNA helicases→ unwind the two DNA strands, generate ssDNA templates

2. RPA→ single strand binding protein, stabilities the unwound strands, recruits pol alpha/primase

3. PCNA→ sliding clamp, binds  to pol gamma and epsilon, Fen-1 and others

4. RFC→ loads and unloads PCNA

5. Fen-1→ flap endonnuclease, removes sort primers

6. Dna2→ endonuclease, removes long primer flaps

7. DNA ligase I→ joins Okazaki fragments

8. DNA topoisomerases→ release superhelical stress

there is co-opertation of replication fork proteins with DNA polymerases

Please note:

• my notes from BoC are so much better than this

On top of this…recent proteomic analysis of isolated DNA replication fork complexes have identified…

• large amounts of additional proteins which play a role in

  1. maintaining replication fork stability

  2. facilitating replication of damaged DNA 

  3. replication of chromatin templates

DNA synthesis at DNA replication forks: concerted action of these core replication proteins during DNA strand synthesis in eukaryotes

1. INitiation and elongation of DNA strand synthesis

   1. → applies to both leading and lagging strand

2. Maturation of Okasaki fragments (lagging strand)

1. INitiation and elongation of DNA strand synthesis

1. Replication Protein A (RPA)→ cover the single-stranded DNA

   • used to protect the DNA from endonucleases

   • AND signals for polymerase alpha

   • in open hand atm

2. Pol alpha PRIMES the DNA

3. Pol alpha primes and extends the leading strand

but then BIG pol delta/epison needs to get onto and stay on DNA, how?

4. Replication factor-C RF-C→ detect end

5. displaces alpha polymerase

6. Recruits PCNA→ acts as sliding clamp (forms a circle around DNA to pull it through

7. binds to epsilon/alpha

8. pol d and e now on the DNA

9. Thousands of base pairs can now easily be polymerased

Crystal structure of sliding clamp

How does RF-C work to recuit PCNA

• opens the PCNA ring like a spring washer

→ kinda slides open

2. Maturation of Okasaki fragments (lagging strand)

1. pol delta ploughs through

2. gets to the double stranded ends from the other lagging strand/fragment

3. RPA is recuited→ stabilises the singlge stranded strands that is on this flap

4. This reuicts Dna2→ endonucleauses

5. cuts the DNA NON PRECISE

6. RPA leaves and Fen-1 is recruited→ flap endonuclease

7. this removes primers PRECISE

8. Fen-1 leaves

9. PCNA remains and recruits DNA ligase 1 to close the gap→ binds phosphate back bone together

To establish a DNA replication fork…

• both leading and lagging strand synthesis are coupled

• the lagging strand is looped back to obtain co-linearity

  • → trombone model

DNA replication is localised in areas

• the DNA itself must be moved by this enzymatic activity

• the DNA itself doen’t move by itself obvs

Historic 2D model of a replication fork (SV40)

• however

→ this causes circular movement!

The proteins of the replication fork thus form…

• a complex ‘molecular machine’

→ co-linear leading and lagging strand synthesis is now enabled with this formation

DNA topoisomerases: The immense length of DNA in the nucleus generates topological problems…

1. Winding

   •  Average human chromosome (150Mbp)

   • DNA strands wind around each other (1.4 ×107 times)

   • These turns must be removed during replication

2. Supercoils

   • separation of DNA strands during transciption and DNA replication generates positive supercoils

   • ahead of the moving polymerase complexes

   • would eventually pprevent further elongation

How do DNA topoisomerases resolve this issue

• altering the number of times DNA strands wind around each other

How does DNA topoisomerase I work?

1. nicks one strand of a DNA duplex

2. attaches a DNA phosphate group to a tyrosine residue in its active centre

3. covalently forming a new ester bond

4. Allows roatation of the free end of the cut strand around the uncut single-strand

5. seals the nick

   • breaks the ester bond of the DNA

   • with tyrosine

   • re-ligating the DNA without requiring ATP

   • these are trans-esterifications

This process can therefore…

1. Remove strain imposed on a molecule by local helix unwinding

   • as found in front of active DNA or RNA polymerases

Topoisomerase biochemistry

Problem when two forks meet

• Catenation

Solution to this

• Topoisomerase II

→ two subunits, two DNA strands cut

HOw does DNA topoisomerase II work?

1. cuts both strand 

2. bridges the gap

3. allowing other regions of DNA duplex to pass through before

4. resealing, removing supercoils from the DNA

This enzyme can also…

• Separate interlocked DNA rings (concatemers or catenanes)

This property is essential in

• the final stages of DNA replication

• and during mitosis

Replication of telomeres: termination problem

• mechanism of co-ordinated leading and lagging strand synthesis

• leads to loss  of DNA at the linear ends of the chromosomes→ telomers

  • shorted after each cycle

How is this loss counteracted?

1. Enzyme telomerase can elongate the ends by

2. synthesising and adding new telomere repeats onto the ends

3. using own RNA template

from diagram:

1. Binding→ RNA template pairs with DNA primer

2. Polymerisation→ RNA template directs addition of nucleotides to 3’ end of DNA primer

3. Translocation→ enzyme moves to new 3’ end of template

What cells have telomerase

1. germ cells

2. cancer cells

3. immortalised cells→ HeLa