SOLEXA aka: Sequencing by Synthesis 2) Attach each adaptor to a flow cell to create ssDNA templates. After each round of amplification, the strands are denatured to create more templates. 1) Randomly cut DNA fragments and ligate adaptors 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 megabases of DNA in a 10 hour run Applications: 1. Whole genome sequencing 2. Amplicon sequencing 3. Transcriptome sequencing 4. Metagenomics Advantages: low cost, longer reads and higher accuracy than the Sanger chain- termination method

434 Sequencing Concept 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 Sequencing Technology 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?

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) Probe Time PointProbe AProbe B Example:

Predicted origins, UCSC genome browser, ENCODE: UVa DNA replication tracks Neerja Karnani Chris Taylor Ankit Malhotra

2) Electron microscopy of bubbles after restriction enzyme digestion

3) Hybridizing lagging strand nascent DNA to strand-specific fragments shows site of switch from leading to lagging strand synthesis 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 ABCDProbes EF GH Nascent DNA base long used to probe fragments along chromosome DNA spotted on arrayNascent strand peak at origin Okazaki fragments: <500 bases long Leading strand nascent strands: > 500 bases long Leading strand nascent strands near origins < 2000 bases long A B C DE FG H A B C DE FG H Nascent strand abundance

4) 2D Gel electrophoresis of replication intermediates followed by hybridization 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 to replicate; this approach was used to identify Autonomously Replicating Sequences (ARS) in yeast.

Sequence that when mutated causes a replicating DNA to fail to replicate; this approach was used to identify essential elements in an (ARS) in yeast Yeast containing Leu2 on aplasmid Plate yeast on Leucine minus plates to estimate rate ofplasmid loss Grow under non-selective conditions

Linker scanning mutations in yeast ARS1 % URA+ colonies Examples of mutants A B1B2B3 WT-ARS1

OriC in E. coli chromosomal DNA

Initiation of DNA replication in E. coli

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

Two primosomes for E. coli chromosomal replication Site of primosome assemblyoriC, A-siten'-pas, D-loop, R-loop RecognitionDnaA proteinPriA protein Auxilliary proteins for(HU for oriC)PriB loading helicase PriC DnaT HelicaseDnaB/DnaC PrimingPrimase DnaA and oriC independent replication initiation in E. coli from hairpins, D-loops and R loops

SV40: a polyomavirus that is very useful for studying 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

Li & Kelly SV40 DNA replication In vitro. PNAS (1984) 81:6973 What else could they do to validate their system?

Fractionation to homogeneity

ELONGATION FACTORS ARE CONSERVED

RF-C is a five-subunit complex All subunits are related in sequence and have ATP binding motifs ATP hydrolysis by RF-C is associated with the loading of PCNA RF-C is the functional homolog of the clamp-loader  complex 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 motif used 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 motif to 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 along ssDNA and peel of substrate DNA * Can move 5‘ to 3’ or 3‘to 5’ or in both directions on ssDNA and is classified as such * Dimers or hexamers

Conserved helicase motifs: Includes the following for ATP binding and hydrolysis I = Walker A motif GxGxGKT II = Walker B motif  DEad Helicases are ATP driven molecular motors

Mutation in a yeast gene that causes a failure in MiniChromosome Maintenance (MCM genes) Yeast containing Leu2 on aplasmid Plate yeast on Leucine minus plates to estimate rate ofplasmid loss Grow under non-selective conditions

MCM8 MCM9 Form hexamer Associates with MCM2-7 Newbies of unknown function

MCM homolog from M. thermoautotrophicum (an archaebacterium) has helicase activity. What direction?

Mt MCM forms double 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