Final Journal ClubMonday April 27 & Wed April 291.New techniques for genome editing & other purposes• CRISPR• TALEN• Zn Finger• Cre-Lox

2.New techniques for DNA sequencing• Illumina• Ion Torrent• Nano-Pore• Pac-Bio• Mass-Spec• Other?

3.Molecular biology for fun and profit

Telomeres• sequences at chromosome ends • humans have 250-7,000 repeats of TTAGGG• special proteins bind them

Telomeres• sequences at chromosome ends • humans have 250-7,000 repeats of CCCTAA• special proteins bind them• can't replicate 5' end of lagging strand since remove primer

Telomeres• can't replicate 5' end of lagging strand since remove primer• telomeres lose ~ 200 bp each S

Telomeres• can't replicate 5' end of lagging strand since remove primer• telomeres lose ~ 200 bp each S• telomerase replaces missing bases

Telomerase• telomeres lose ~ 200 bp each S• telomerase replaces missing basesreverse transcriptase with attached RNA template

Telomerase• telomeres lose ~ 200 bp each S• telomerase replaces missing basesreverse transcriptase with attached RNA template1) RNA bonds leading strand

Telomerasereverse transcriptase with attached RNA template1) RNA bonds leading strand2) Forms template to extend leading strand

Telomerasereverse transcriptase with attached RNA template1) RNA bonds leading strand2) Forms template to extend leading strand3) Translocates6 bases & repeats

Telomerase1) RNA bonds leading strand2) Forms template to extend leading strand3) Translocates6 bases & repeats4) Extend lagging strandwith primer & DNA pol

Telomere structureBound by complex called shelterin or the telosome

Telomere structureBound by complex called shelterin or the telosomeProtects DNA, prevents recombination

Telomere structureBound by Shelterin/telosome complexProtects DNA, prevents recombinationDNA proposed to form T-loop structure IF long enough

Telomere structureBound by Shelterin/telosome complexProtects DNA, prevents recombinationDNA proposed to form T-loop structure IF long enoughG-rich SS-DNA invades and displacesC-rich strand

Telomere structureBound by Shelterin/telosome complexProtects DNA, prevents recombinationDNA proposed to form T-loop structure IF long enoughG-rich SS-DNA invades and displacesC-rich strand POT1/TPP1 binds & protects D-loop

Telomere structureBound by Shelterin/telosome complexProtects DNA, prevents recombinationDNA proposed to form T-loop structure IF long enoughG-rich SS-DNA invades and displacesC-rich strand POT1/TPP1 binds & protects D-loop If can’t form T-loop trigger apoptosis

Telomere Length MaintenanceBound by shelterin/telosome complex Proposed to form T-loop structure IF long enoughIf can’t form T-loop trigger apoptosisTelomere length proposed to be regulated by TRF1 & PINX1

Telomere Length MaintenanceTelomere length proposed to be regulated by TRF1 & PINX1 If too short, not enough TRF1bind so telomerase is active

Telomere Length MaintenanceTelomere length proposed to be regulated by TRF1 & PINX1 If too short, not enough TRF1bind so telomerase is activeOnce long enough TRF1 bindstelomere

Telomere Length MaintenanceTelomere length proposed to be regulated by TRF1 & PINX1 If too short, not enough TRF1bind so telomerase is activeOnce long enough TRF1 bindstelomere PINX1 then binds TRF1 & stops telomerase

Telomeres

Aging theory:” mature” cells lose telomerase

lose DNA each S

Telomeres

Aging theory:” mature” cells lose telomerase

lose DNA each S

Die when lose too much: Hayflick limit ~50 divisions for 1˚ cultures

Telomeres

Aging theory:” mature” cells lose telomerase

lose DNA each S

Die when lose too much: Hayflick limit ~50 divisions for 1˚ cultures

Ramunas et al (2015) DOI: 10.1096/fj.14-259531 found that transient expression of TERT in cultured cells added 1000 bp to telomeres and allowed 28 x more divisions in fibroblasts

Telomeres

Aging theory:” mature” cells lose telomerase

lose DNA each S

Die when lose too much: Hayflick limit ~50 divisions for 1˚ cultures

Ramunas et al (2015) DOI: 10.1096/fj.14-259531 found that transient expression of TERT in cultured cells added 1000 bp to telomeres and allowed 28 x more divisions in fibroblasts

Cancer cells must maintain telomere length

•90% reactivate telomerase

Telomeres

Cancer cells must maintain telomere length

•90% reactivate telomerase

• [serum telomerase] can diagnose cancer

Telomeres

Cancer cells must maintain telomere length

•90% reactivate telomerase

• [serum telomerase] can diagnose cancer

• Inhibiting telomerase kills most cancer cells

Telomeres

Cancer cells must maintain telomere length

•90% reactivate telomerase

• [serum telomerase] can diagnose cancer

• Inhibiting telomerase kills most cancer cells

• Telomerase is symptom cf cause of cancer

• not viewed as good target for cancer therapy

Telomeres90% of cancer cells reactivate telomerase

[serum telomerase] can diagnose cancerInhibiting telomerase kills most cancer cellsTelomerase is symptom cf cause of cancer

10 % of cancer cells lengthen telomeres by alternative mechanism involving homologous recombination

Telomeres10 % of cancer cells lengthen telomeres by alternative mechanism involving homologous recombination•normally inactive: therefore good target for cancer therapy

Telomeres10 % of cancer cells lengthen telomeres by alternative mechanism involving homologous recombination•normally inactive: therefore good target for cancer therapy•See high frequencyof SCE at telomeres in ALT cells

Telomeres10 % of cancer cells lengthen telomeres by alternative mechanism involving homologous recombination•normally inactive: therefore good target for cancer therapy•See high frequency of SCE at telomeres in ALT cells•Also see high frequency of telomere fragments• Substrates for HR and telomere elongation

Telomeres10 % of cancer cells lengthen telomeres by alternative mechanism involving homologous recombination•normally inactive: therefore good target for cancer therapy•See high frequency of SCE at telomeres in ALT cells•Also see high frequency of telomere fragments• Substrates for HR and telomere elongation

Key common feature = chromatin remodeling allowing HR•Perhaps due to depletion of shelterin, especially TRF2

DNA damageDNA gets damaged a lot!

DNA damageDNA gets damaged a lot!

DNA damageDNA gets damaged a lot! >200,000 events/human cell/day

DNA damageOccurs 2 ways

1) spontaneously

DNA damageOccurs 2 ways

1) spontaneously2) mutagens : damage DNA

Spontaneous mutations1) In Sa) mispairsC tautomersbind A T tautomersbind G

Spontaneous mutations1) In Sa) mispairsC tautomersbind A T tautomersbind G1/106

Spontaneous mutations1) In Sa) mispairsC tautomersbind A T tautomersbind G1/106

6000/S

Spontaneous mutationsWobbling: no numbers!

Spontaneous mutations1) In Sa) mispairsb) misaligned bases

Spontaneous mutations1) In Sa) mispairsb) misaligned bases

-> indels

Spontaneous mutations1) In S

a) mispairsb) misaligned bases -> indels

2) Hydrolysisa) C deaminates to U

Spontaneous mutations2) Hydrolysis

a) C deaminates to U100-500/cell/day

Spontaneous mutations2) Hydrolysis

a) C deaminates to Ub) 5-meC deaminates to T!

Spontaneous mutations2) Hydrolysis

a) C deaminates to Ub) 5-meC deaminates to T!c) Adenine deaminates to hypoxanthine: bonds C!

Spontaneous mutations2) Hydrolysis

a) C deaminates to U100-500/cell/dayb) Depurination (lose A or G) (also depyrimidation, but 20 x less)

Spontaneous mutations2) decomposition

a) C deaminates to U100-500/cell/dayb) Depurination (lose A or G) (also depyrimidation, but 20 x less)Replace missing base randomly

decompositiona) C deaminates to U 100-500/cell/dayb) Depurination (lose A or G) (also depyrimidation, but 20 x less)Replace missing base randomly 10,000 – 50,000 depurinations /cell / day1,000 – 3,000 depyrimidations /cell / day

Spontaneous mutations2) decomposition3) Oxidation: > 20 base lesions due to oxidation have been identified

Spontaneous mutations2) decomposition3) Oxidation: > 20 base lesions due to oxidation have been identified•G is most reactive: most frequent lesion is 8-hydroxy G

Spontaneous mutations2) decomposition3) Oxidation: > 20 base lesions due to oxidation have been identified•G is most reactive: most frequent lesion is 8-hydroxy G•8oxoG (syn) bonds A well enough to evade proofreading!

Spontaneous mutations3) Oxidation: > 20 base lesions due to oxidation have been identified•G is most reactive: most frequent lesion is 8-hydroxy G•8oxoG (syn) bonds A well enough to evade proofreading! •(8oxoG (anti) bonded to C gets removed!)