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III.

DNA: Concepts and Applications Transcription / Translation DNA Library Mutation / Cancer Sequencing / Genome projects / Crime / Paternity Microinjection / Antisense / Gene silencing Cyclic nucleotides and cell signaling PCR Transposons 1. Cell cycle / DNA replication / Mismatch repair 2. Cloning 3. DNA Micro-array 4. Promoter CpG methylation / Gene silencing 5. Reporter genes / Transgenics / Gene therapy 6. DNA/protein interaction
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RNA: Concepts and Applications

RNA in translation 7. RNA interference 8. RNA modification

1a. The cell cycle

Central theme of molecular biology

1b. Prokaryotic DNA replication

Things to happen 1. 2. 3. Parent DNA unwinds Long leading strand Short pieces of lagging strand

Involved enzymes DNA gyrases / helicases DNA polymerases DNA ligases DNA repair enzymes5

1b. Eukaryotic DNA replication A dimerized coiled-coil domain and an adjoining part of Geminin interact with two sites on Cdt1 for replication inhibition
July 23, 2004 Molecular Cell

Yuan et al. (2004).

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S
MCM

G1

G2

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ORC

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S
MCM

G1

G2

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CDC6 ORC

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S
MCM

G1

G2

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CDC6 ORC Cdt1

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S
MCM

G1
MCM

G2

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CDC6 ORC Cdt1

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Geminin role in cell cycle


Geminin Degradation Cdt1 CDC6 ORC Replication Machinery MCM CDC6 ORC Cdt1 MCM MCM

S G1 M
Degradation
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G2

ORC

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Crystal structure of geminin

N93

C152

N93

C152

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Geminin + Cdt1 complex structure

Mouse Geminin (79157), mouse Cdt1 (172368) Human Geminin (82-160), human Cdt1 (159-338) Lee, C. et al. (2004). Nature, 430, 913.

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1c. Signaling from DNA mispairs to mismatch-repair excision sites despite intervening blockades

Huixian Wang and John B Hays* Department of Environmental and Molecular Toxicology Oregon State University, Corvallis, OR, USA The EMBO Journal, 23, 21262133 (2004).

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Types of DNA Repairs Direct repair 1. Thymine dimer formation by UV No transcription; no replication; can kill E.coli 2. Nucleotide alkylation (O6-Methyl guanine) Highly mutagenic; forms more G:T Excision repair 1. Nucleotide excision repair (NER)- excess bases 2. Base excision repair (BER)- modified nucleotide Mismatch repair- MutS, MutL, MutH SOS response- cell division stops Double strand break repair
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Mismatch repair - prokaryotes

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MutS

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Mismatch repair - eukaryotes


G

MutS

MutL

Exonuclease Cofactors

or
Excision

Pol

DNA synthesis 18

2. Cloning Dolly was the first mammal cloned from a cell from an adult animal. She was derived from cells from the udder of a 6-year old Finn Dorset ewe. Individual cells were then fused with unfertilised eggs from which the genetic material had been removed. Implanted into surrogate Scottish Blackface ewes. One gave rise to the live lamb, Dolly, 148 days later. Dolly was born on 5 July 1996. Put to sleep on Feb. 14, 2003.
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Natural

Cloning

Take an egg

Remove the nucleus

Take the nucleus from any cell and place in the egg

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3. DNA micro-array

Normal cell

Cancerous cell

Active

Active Inactive Active Inactive

Inactive

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3. DNA micro-array

http://www.sumanasinc.com/webcontent/animations/content/dnachips.htm

Courtesy: Affymetrix

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DNA micro-array data GAS1 growth arrest-specific 1 plasminogen activator, urokinase receptor /// plasminogen activator, urokinase receptor G0/G1switch 2 hairy and enhancer of split 1, (Drosophila) -26.6224

PLAUR G0S2 HES1

-20.5745 -16.6496 -10.8398 3.33123 3.6362

SQSTM 1 Sequestosome 1 MT2A metallothionein 2A Potassium channel tetramerisation domain containing KCTD7 7

4.66503
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4. CpG methylation and gene silencing Promoter CG CG CG Transcription start

CG methylation Methylated DNA binding proteins McCP2 MBD 1-4 Epigenetic gene silencing Pyro sequencing

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Change of CpG methylation status of a gene in tumor

Courtesy: Biotage

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Pyro sequencing

Courtesy: Biotage

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5a. Transgenics / Reporter genes

GFP/X-Gal

Gene to be checked for expression

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5b

red orange yellow

red orange yellow green

green wild type light wild type dark - Prof. Z. Gong


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5c. Gene Therapy Conventional treatment Diabetes Insulin injection Hemophilia Factor VIII injection

Gene therapy Take the gene responsible for the protein Introduce into a vector (say, a non-infectious virus) Target to the cell where the protein can be synthesized

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6. Protein-DNA complex

Protein-DNA interaction is very important for various biochemical processes. Swaminathan et al. Nature Structural Biology, 1997

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7. Short interfering RNA Most of the free forms of RNA, mRNAessenger RNA molecules in particular, are single strands. tRNAs and selected RNA regions are double-stranded. Many viruses, however, form long stretches of double-stranded RNA when they replicate. When our cells find double-stranded RNA, it is often a sign of an infection. However, plant and animal cells have a more targeted defense that attacks the viral double stranded RNA directly, termed RNA interference.
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Courtesy: Protein Data Bank

Dicer

Viral double-stranded RNA are cut into pieces (about 21 base-pairs), called small interfering RNA (SiRNA) by the protein Dicer (top panel). The argonaute protein, shown here at the bottom, strips away one strand from the siRNA, and then looks for any messenger RNA that matches it. If it finds some, it cleaves the RNA, destroying it. In this way, the cell removes any messenger RNA that is the same as the original double-stranded piece found and processed by dicer. Based on this principle, we can synthesize a nonnatural interfering RNA, then insert it into a cell to destroy any messenger RNA that we desire. Researchers use these small RNA molecules to fight disease, for instance, using them to knock out cancer genes. 32 Courtesy: Protein Data Bank

Argonaute

8a. RNA modifications Pseudo-uridine synthase


Fig 1

rRNA from 30S

RsuA RsuA S4
- ve charge + ve charge
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- Prof. J. Sivaraman

8b. RNA modification PARN in complex with a poly(A) oligo

- Prof. H. Song PARN is a processive, poly(A)-specific 3 exoribonuclease in 34 mammalian cells responsible for poly(A) shortening (deadenylation)