SOLEXA aka: Sequencing by Synthesis 2) Attach each adaptor to a flow cell to create ssDNA templates....

SOLEXA aka: Sequencing by Synthesis

2) Attach each adaptor to a flow cellto create ssDNA templates. Aftereach round of amplification, the strands are denatured to createmore templates.

1) Randomly cut DNA fragments and ligateadaptors to both ends

3) Add four labeled reversible terminators, primers, and DNA polymerase.

4) Wash off all of the unbound components, excite the laser, and read the emitted fluorescence of the first base for each cluster.

5) The blocked 3’ terminus and fluorophore has to be removed before repeating to determine the next base

434 Sequencing

• Capable of sequencing 400-600 megabases of DNA in a 10 hour run

• Applications:1.Whole genome sequencing2.Amplicon sequencing3.Transcriptome sequencing4.Metagenomics

• Advantages: low cost, longer reads and higher accuracy than the Sanger chain-termination method

434 SequencingConcept

• Large-scale parallel pyrosequencing, “sequencing by synthesis”:

1.Each nucleotide is added in turn, producing PPi

2.Sulfurylase uses PPi to produce ATP, that is used by luciferase to produce light

3.Apyrase denatures the remaining dNTP at each step

4.The light produced with the addition of each nucleotide can be plotted to form a sequence

434 SequencingTechnology

• DNA is fractionated, 5’ biotin labeled and attached to streptavidin coated beads. The non-labeled strand is released.

• The DNA on each bead is amplified onto the bead

• One bead is deposited in each well

• Pyrosequencing takes place in the Genome Sequencer FLX instrument

Applied Biosystems SOLiD System

© Copyright 2008 Applied Biosystems. All Rights Reserved.

* The position on the DNA at which replication start points are found. (4 methods discussed)

* A DNA sequence that when added to a non-replicating DNA causes it to replicate.

* A DNA sequence whose mutation abolishes replication.

* A DNA sequence that in vitro is the binding target for enzyme complexes known to function in initiation of DNA replication.

Characteristics used to define Origins of Replication

1) In synchronously replicating cells, density transfer experiments can reveal which sequences replicate earliest. What is the direction of fork

movement here?

Time

% o

f p

rob

e in

HL

DN

A0

100

A B C

C B A

Genes on achromosome

TR50

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8

Hr of S phase

% c

um

ula

tiv

e r

ep

lic

ati

on

Gene A

Gene B

TR50 - Time at which 50% of the locus is replicated

In the example below,‘probe A’ has a TR50 of 1.25hr (80% at 2hr, 0% at 0hr)‘probe B’ has a TR50 of 6.33hr (100% at 8hr, 40% at 6hr)

TR50 Calculation for human chromosomal probes on genome tiling arrays (ENCODE project)

ProbeProbe

Time Point Probe A Probe B

0-2 .80 .10

2-4 .10 .10

4-6 .10 .20

6-8 0 .60

Example:

Predicted origins, UCSC genome browser, ENCODE: UVa DNA replication tracks

Neerja KarnaniChris TaylorAnkit Malhotra

EcoRIBamHI

BamHI EcoRI

2) Electron microscopy of bubbles after restrictionenzyme digestion

3) Hybridizing lagging strand nascent DNA to strand-specific fragmentsshows site of switch from leading to lagging strand synthesis

A B C DUpper strand fragments

Lower strand fragments

E F G H

Nascent DNA used to probe DNA fragments:

A B C D

E F G H

A B C D

E F G H

DNA spotted on membrane

Autorad after hybridizing nascent strands

3b) Hybridizing of leading strand nascent DNA shows sites near origins that are enriched in nascent strand

A B C DProbes E FG H

Nascent DNA 500-2000 base long used to probe fragments along chromosome

DNA spotted on arrayNascent strand peak at origin

Okazaki fragments: <500 bases longLeading strand nascent strands: > 500 bases longLeading strand nascent strands near origins < 2000 bases long

A B C DE F G H

A B C DE F G H

Nas

cent

str

and

abun

danc

e

4) 2D Gel electrophoresis of replication intermediates followed byhybridization with a DNA fragment reveals whether a replication

bubble originates in the fragment

Dimension 1: separates by size; dimension 2: separates by shape

Sequence that when added to a non-replicating DNA causes it toreplicate; this approach was used to identify

Autonomously Replicating Sequences (ARS) in yeast.

Sequence that when mutated causes a replicating DNA tofail to replicate; this approach was used to identify

essential elements in an (ARS) in yeast

Yeast containing Leu2 on a plasmid

Plate yeast on Leucine minus plates to estimate rate of plasmid loss

Grow under non-selective conditions

Linker scanning mutations in yeast ARS1

% U

RA

+ c

olo

nie

sExamples of mutants

AB1B2B3WT-ARS1

OriC in E. coli chromosomal DNA

Initiation of DNA replication in E. coli

Nature Structural & Molecular Biology – 13, 676 - 683 (2006) Berger JM

Two primosomes for E. coli chromosomal replication

Site of primosome assembly oriC, A-site n'-pas, D-loop, R-loop

Recognition DnaA protein PriA protein

Auxilliary proteins for (HU for oriC) PriBloading helicase PriC

DnaT

Helicase DnaB/DnaC

Priming Primase

DnaA and oriC independent replication initiation in E. colifrom hairpins, D-loops and R loops

SV40: a polyomavirus that is very useful forstudying eukaryotic replication enzymes

Early genes : proteins essential for viral DNA replication

T antigen binding sites cluster around the ori

* 98% nuclear: NLS * origin-specific DNA binding * DNA independent ATPase * ATP dependent DNA helicase * Binds to DNA polymerases, AP2, p53, Rb * Undergoes phosphorylation etc.

Properties of SV40 T antigen

COS cells (infected with SV40)(expresses T Ag)

+ plasmid + dNTP + rNTP+ 32PdATP

37o C, 60 min

Analyze products by electrophoresis and autoradiography

SV40 ori

SV40 - + ori - +

Li & KellySV40 DNA replicationIn vitro.PNAS (1984) 81:6973

What else could they doto validate their system?

Fractionation to homogeneity

COS cells (infected with SV40)(expresses T Ag)

T Antigen frombaculovirus expression system

Human HeLa or 293 cells

cell lysate

PC

0.2 M KCl 0.66 M KCl

QS

0.2 M KCl 0.4 M KCl (PCNA)

ssDNA

0.6 M KCl 1 M KClRPA

+

(RF-C, pol + pol )

Prokaryotic Function Eukaryotic

SSB ss DNA coating RPA

PRIMASE RNA primer synth. Primase subunitof DNA POL ,

5'-3' polymerase DNA POL ,DNA POL

POL III CORE 3'-5' exonuclease DNA POL 5'-3' exonuclease Fen1

COMPLEX ATP dependentclamploader

RF-C

CLAMP processivity factor PCNA

LIGASE seal nicks Lig1

DnaB Helicase ?

ELONGATION FACTORS ARE CONSERVED

RF-C is a five-subunit complexAll subunits are related in sequence and have ATP binding motifsATP hydrolysis by RF-C is associated with the loading of PCNARF-C is the functional homolog of the clamp-loader complex

RF-C

RF-C

Polymerase switching occurs even on lagging strands; pol does most of DNA synthesis

Dna2 endonuclease is also necessary for this step

How do you think the PCNA is removed after ligation?

PCNA interacts with RF-C, pol , Fen1, DNA ligase,CAF1 and MCMT

Several of these have a common motifused in the interaction: Q-X-X-L/I/M-X-X-F-F/Y

p21/CIP1/WAF1, a protein induced by the tumor suppressor p53 uses the same motifto interact with PCNA

What effect is p21 expected to have on DNA replication/repair?

FEN1 interacts with PCNA

DNA Helicase

* Helicase binds ss DNA

* Hydrolyzes ATP to move alongssDNA and peel of substrate DNA

* Can move 5‘ to 3’ or 3‘to 5’ or in both directions on ssDNAand is classified as such

* Dimers or hexamers

P P

5‘ 3‘

3‘ 5‘ 5‘3‘

Substra

te

Boiled su

bstra

te

+he

licas

e

+helic

ase+

ATP

15mer 30mer

15mer30mer

Conserved helicase motifs:

Includes the following for ATPbinding and hydrolysisI = Walker A motif GxGxGKTII = Walker B motif DEad

Binds ATP

Tight conformation

Hydrolyzes ATP

Relaxed conformation

Helicases are ATP driven molecular motors

Mutation in a yeast gene that causes a failure in MiniChromosome Maintenance (MCM genes)

Yeast containing Leu2 on a plasmid

Plate yeast on Leucine minus plates to estimate rate of plasmid loss

Grow under non-selective conditions

MCM8

MCM9

Form hexamer

Associates with MCM2-7

Newbies of unknown function

MCM homolog fromM. thermoautotrophicum(an archaebacterium)has helicase activity.

What direction?

Mt MCM formsdouble hexamers

T antigen helicase domain: XS Chen, Nature, 2003;423:512-8

MtMCM-N terminal (non-helicase) domain: XS Chen, Nature Str. Biol. 2003;10:160-7

Model of replicative double-hexameric helicase action : XS Chen, Nature, 2003;423:512-8

SOLEXA aka: Sequencing by Synthesis 2) Attach each adaptor to a flow cell to create ssDNA templates....

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