Part 4: Replication and evolution of genomes

10.Genome replicationThe replication process and regulation of

eukaryotic genome replication

•Genome replicationidentical copies of cells

•Genome undergoes changes:•Nucleotide sequence alterations by mutation•Occasional errors in replication•Sequence rearrangements due to recombination•Chromosome rearrangements

molecular evolution

Genome replication

Watson and Crick 1953: the specific pairing suggests a possible copying mechanism, each strand acts as template

Big Question: human chrom.1 = 250Mb = 25 million rotationshow does it unwind? – ‘topological problem’

Semiconservative replication model

Genome replication

Proposal by Max Delbrück 1954: DNA double helix does not unwind, but breaks to separate the strands. The new daughter molecule is partly made up of parental DNA

Genome replication

Proof of semiconservative replication: experiments of Meselson and Stahl (1958) – labeling of DNA with 15N (heavy isotope of N) followed by density gradient centrifugation

Genome replication

The DNA topoisomerases – a solution to the topoligical problem

• Carry out breakage-and-reunion reaction

Genome replication

http://www.youtube.com/watch?v=EYGrElVyHnU

Topoisomerase I solves the problem caused by tension generated by winding/unwinding of DNA. It wraps around DNA and makes a cut permitting the helix to spin. Once DNA is relaxed, topoisomerase reconnects broken strand

Genome replication

Rolling circle replication in circular genomes

Genome replication

Genome replication

The replication process

• Initiation – recognition of the position where replication begins – origin of replication (ORI)

• Elongation – parent nucleotide is copied• Termination – after completed replication

Genome replication

Initiation of genome replication• Begins at origin of

replication• Two replication forks

emerge• Bidirectional replication• Several ‘origin of

replication’ in eukaryotic genomes (yeast: every 40 kp, human: every 150 kb)

Genome replication

Initiation at the E. coli origin of replication (oriC)

13-nucleotide motifs9-nucleotide motifs – DnaAbinding sites

Melted region

Barrel of DnaA proteins through torsional stress

DnaC and DnaB (helicase) bind at oriCcopying begins

Genome replication

Genome replication

•In yeast: -ORS = origin recognition sequence – 40bp in total-ORC= origin recognition complex (6 proteins) bind-react to regulatory signals within the cell cycle-subdomain B2 and B3, similar to oriC-Protein ABF1 attaches to B3 and melts B2

ORSB3 B2

ABF1 ORC

•Mammalian genomes contain replication origins equivalent to those in yeast

Genome replication

•Helicases break of base pairs and unzipp the helix, separating two annealed nucleic acid strands. Energy is supplied by hydrolisis of ATP•Topoisomerases relieves torsional stress•‘Re-closing’ of the helix is prevented by single stranded binding proteins (SSBs) in bacterias and replication protein A (RPA) in eukaryotes,

Genome replication

The elongation phase of replication

• Enzyme = DNA polymerase• Catalyse 5’3’ synthesis of DNA

polynucleotide• Often combined with exonuclease activity

(usually 3’5’) proofreading activity• 3 bacterial DNA polymerases, 5 eukaryotic

DNA polymerases (repair, replication, mitochondrial replication)

Genome replication

The lagging strand synthesis

•Okazaki fragments in bacteria: 1000-2000 bp; in eukaryotes: 200 bp•Primer necessary, DNA polymerase III can not synthesize from single stranded DNA•Primers are made from RNA by primase, a special RNA polymerase•At leading strand: priming only once at replication origin

Genome replication

Joining of adjacant Okazaki fragments

• DNA polymerase III does not have 5’-3’ exonuclease activity

• • Primer removal and

completion of DNA synthesis by DNA polymerase I

• Ligation of fragments by DNA ligase

Genome replication

Termination of replication

• Eventually the replication fork reaches the end of the molecule or meets a second replication fork running in opposite direction

Terminator sequencesRecognition site for DNA binding protein TUS

Genome replication

Genome replication

• Termination in eukaryotes is not well understood

• There exist no termination sequences or Tus proteins

• Termination might involve simply ligation of the ends of the new polynucleotides

Genome replication

Maintaining the ends of a linear molecule

• Lagging strand – priming sites are always 200 bp apart last priming at 200 bp form 3’ end

3’ 5’

5’ 3’Leading strand

3’5’5’3’ Lagging strand

3’ 5’

5’ 3’Granddaughter molecule

Molecule has become shorter

3’

3’ 5’5’

Genome replication

•Telomeres = short repeat motifs at end of eukaryotic chromosomes

Genome replication

•Most part of the telomere is replicated in a normal fashion•Telomeres can be extended by independent mechanism catalyzed by the enzyme ‘telomerase’, composed of protein and RNA•Extension by copying mechanism

Genome replication

•Embryonic stem cells express telomerase, which allows them to divide repeatedly and form the individual.

•In adults, telomerase is highly expressed in cells that need to divide regularly (e.g., in the immune system), whereas most somatic cells express it only at very low levels in a cell-cycle dependent manner.

A variety of premature aging syndromes are associated with short telomeres (Werner syndrome).

Genome replication

3’5’5’3’

3’5’5’3’

Telomerase extends the 3’-overhang

New DNA

3’5’

Okazaki fragment

When enough DNA has been synthesized, a new Okazaki fragment can be primed

Genome replication

Regulation of eukaryotic genome replication

• Coordination of replication by cell cyletwo copies are available when cells divide

Mitosis – nucleus and cell divideGap1 (G1) phase – interval, when transcription, translation etc. occursSynthesis (S) phase – genome is replicatedGap2 (G2) phase – a second interval period

Genome replication

•Cell cycle checkpoints before phase S and M cycle becomes arrested if critical genes involved in cell cycle control are mutated•Cell cycle is controled by kinases, which phosphorylate and activate enzymes•Kinases are controlled by cyclinscyclin dependent kinases

http://nobelprize.org/educational_games/medicine/2001/index.html

Questions• Which processes lead to changes in the genome?• Explain the three models of replication• Which enzymes are needed for replication

initiation and where does replication start?• What is the ‘proofreading activity’ of a DNA

polymerase?• What is the ‘Okazaki fragment’?• What is the function of the telomerase?• Which are the four phases of the cell cycle and

which enzymes control it?