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DNA Ends: Just the Beginning Nobel Lecture
- Dec. 7, 2009
DNA Ends: Just the Beginning Nobel Lecture Dec. 7, 2009 Jack W. - - PowerPoint PPT Presentation
DNA Ends: Just the Beginning Nobel Lecture Dec. 7, 2009 Jack W. - - PowerPoint PPT Presentation
DNA Ends: Just the Beginning Nobel Lecture Dec. 7, 2009 Jack W. Szostak HHMI, MGH, HMS Two Telomere Problems: 1. DNA ends are reactive 2. Incomplete Replication Telomeres have been known to be special since the 1930s McClintock, Genetics
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McClintock, Genetics 26: 234-282 (1941) “No case was found of the attachment of a piece of one chromosome to the end of another [intact chromosome]” McClintock, Missouri Agr. Exp. Sta. Res. Bull. 163, 1-48 (1931) McClintock, Genetics 23: 315-376 (1938)
Telomeres have been known to be special since the 1930s
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Incomplete Replication of DNA Ends
5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ 5’ lagging strand is incomplete
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Setting the stage: Molecular analysis of the reactions of DNA ends.
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Non-homologous end-joining in yeast
Orr-Weaver and Szostak, PNAS, 1983
cut ligation, +/- deletion
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Double-strand breaks in DNA stimulate recombination
Orr-Weaver et al., PNAS, 1981 very few recombinants cut abundant recombinants
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Double-strand break repair model for recombination
resection strand invasion, repair synthesis repair synthesis, branch migration Holliday Junction resolution double-strand break
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GGGGTT
Blackburn and Gall, J. Mol. Biol. 120: 33-53 (1978)
Telomeres from Tetrahymena: stable DNA ends that are fully replicated
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A very special piece of DNA:
rDNA: high copy, symmetrical dimer 35S rRNA primary transcript 5’ 3’ splicing
(GGGGTT)50-70
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Tetrahymena telomeres in yeast:
A collaborative study to ask whether the biochemistry of telomeres is widely conserved.
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gene
- ri
- ri
gene
t t
plasmid cut Szostak and Blackburn, Cell 29: 245-255 (1982)
Moving Tetrahymena Telomeres into Yeast
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- ri
gene
t t
cut Szostak and Blackburn, Cell 29: 245-255 (1982)
Cloning Yeast Telomeres
y
- ri
gene
t y
ligate
- ri
gene
t y
chromosomal DNA
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A digression: Yeast Artificial Chromosomes
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First attempt to make an artificial chromosome
Murray et al., Nature, 1983
cut
CEN gene ORI gene ORI CEN Tr Tr Tr Tr stable in yeast unstable in yeast!
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Successful attempt to make an artificial chromosome
Murray et al., Nature, 1983
gene ORI CEN Tr Tr
unstable in yeast
gene ORI CEN Tr Tr λ λ
add extra DNA stable in yeast
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Recombination based models for telomere replication
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Telomere Lengthening by Recombination
incomplete replication generates 3’ overhang
Telomere Lengthening by Repair Synthesis
3’ strand invasion allows extension of 3’ overhang
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Telomere Replication by Holliday Junction Resolution
5’ 3’ 5’ 3’ 5’ 3’
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telomere maintenance in yeast points to the correct solution
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- ri
gene
t t
- ri
gene
t t y y
GGGGTT GGGTGTGGTGTGG
Shampay et al., Nature 310: 154-157 (1984)
Yeast adds new DNA to Tetrahymena Telomeres
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Correct Structure of Telomeric DNA Ends
3’ 5’ G-rich 3’-overhang
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New Model for Telomere Shortening, and the Role of Telomerase in Telomere Maintenance
5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ 5’ leading strand is incomplete, but overhang is regenerated by telomerase G-rich overhang 3’ 5’ 3’ 5’ 3’ 5’ 3’
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Cells without telomerase have limited division potential, Cells with telomerase can divide without limit.
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Lundblad and Szostak, Cell 57: 633-643 (1989)
Senescence of Yeast EST-1 Cells
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After telomeres: Directed Evolution of RNA and Protein
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Laboratory Evolution of Aptamers
An ATP binding RNA molecule
Sassanfar and Szostak, Nature 364: 550-553 (1993)
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Class I Ribozyme Ligase
Bartel and Szostak, Science 261: 1411-1418 (1993)
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An HDV ribozyme in the human genome
HDV hCPEB3
Salehi-Ashtiani et al., Science, 2006 hu rh m d
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ATP Binding Protein
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Current focus: Origin of Life
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A simple cell might be based on a replicating vesicle for compartmentalization, and a replicating genome to encode heritable information. A complex environment provides nucleotides, lipids and various sources of energy. Mechanical energy (for division), chemical energy (for nucleotide activation), phase transfer and osmotic gradient energy (for growth) may be used by the system.
Schematic Model of a Protocell
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
Mansy et al., Nature, 2008
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Montmorillonite can bring RNA into Vesicles
Hanczyc et al., Science, 2003
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Cycles of growth and division
Zhu and Szostak, JACS, 2009
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Self-Replicating Genetic Polymers
It seems likely that informational replication will be achieved in the next decade, and that it will throw new light on the origins of life. Leslie Orgel, 1992
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Typical monomer for spontaneous synthesis
O O NH N N N O NH2 N N P O O- NH2
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Typical monomer for spontaneous synthesis and corresponding polymer
O O NH N N N O NH2 N N P O O- NH2
N O NH P O O O O- N O NH P O O O- N O NH P O O O-
2'-NP-DNA
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Origin of Telomerase in Spontaneous Copying Chemistry?
5’ 3’ 5’ 3’ 5’ activated nucleotides 3’ 5’ 3’ activated nucleotides 5’ 3’ 3’ 5’
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