protein purification and making dna (pre midterm)

lectures 6-9

chromatography

• components are separated based on their intraction with a solid material

• speed depends on size and strength of interactions with medium

gel filtration chromatography

• based on size and shape of proteins

• solid gel beads have tiny pores for proteins to pass through

  • beads are in a liquid

• larger proteins will flow to the bottom faster

• spherical prtoeins move slower

ion exchange chromatography

• separates proteins based on electric charge

• charged gel beads bind to oppositely charged proteins

• weakly bound proteins are releaed by running an aqueous alt through the column

• ion exhange

  • protein gets displaced by salt ion because they compete to bind to the gel beads

antibody affinity chromatography

• uses specific binding between antibody and antigen

• all non-target proteins get wasshed away

• at the end, separate antigen-antibody complex by lowering pH

PAGE

• polyacrylamide gel electrophoresis

• uses free solution with an electric field to separate proteins basedon net charge/mass ratio

SDS

• sodium docecyl sulfate is an anionic detergent

• denatures proteins when its hydrophobic tail interacts with proteins

• bind with other SDS, causing protein to become covered and unravel

does sds-page involve dyeing protein?

yes, to make them visible

immuo/westernblotting

• often combined with SDS-PAGE

• proteins from SDS-PAGE gel are tranferred to a membrae

  • membrane has a coating that prevents non-specific antibodies from binding

• membrane is incubated and primary antibody binds to target protein

• secondary antibody is labelled somehow and will bind to to primary

co-immunoprecipitation

• method of detecting protein-protein interaction

• specific antibody immunprecipitates a protein of interest

  • if target has a friend protein, the friend will come too

immunofluorescence

• introduce specific antibody

• introduce secondary antibody that is fluorescent

CMG helicase

• hexamer of MCM

• has accessory subunits

  • Cdc45and GNS complex

• bind to the leading strand

RPA

• replication protein A

• binds ssDNA to prevent secndary structures

DNA polymerase epsilon

• synthesizes leading strand

PCNA

• homotrimeric protein that prevent polymerase epsilon or delta complex  from dissassociating from the template strand

primase/polymerase alpha complex

• forms RNA part of primer

• extends primer with DNA

Rfc/PCNA complex

• Rfc loads the PCNA

  • opens PCNA ring and loads it at a primer on the dna

ribonuclease H and FEN-1

• displace the RNA part thats at the end of Okazaki fragments

• polymerase delta replaces RNA w DNA

ORC

• origin recognition complex

• 6 subunit protein that loads helicase onto dna

• mcm gets loaded during G1

  • activated through phosphorylation and protein onteraction during S

hydrolytic depurination

• dna change that occurs 2000-10 000 times daily in each cell

cytosine deaminatinon

• dna change that occurs every 5 days per cell

guanidine oxidation

• dna change that occurs every 5 days per cell

adenine methylation

• dna change that happens about 600 time daily in each cell

proofreading exonuclease

• increase activity efficiency by 100 fold

• causes there to be a mistake in dna every 1/1million

how often does dna pol make a mistake in adding nucleotides?

every 1 in 10 000 nucleotide

how much does mismatch repair increase efficiency?

• by 1000 fold

• makes mistakes 1/1billion

how does mismatch repair work?

• error recognized, MSH2 and MSH6 bind to the daughter strand

• binding of MLH1 endonuclease is triggered, will cut near the mistake

• dna helicase unwinds, dna exonuclease digests mistake in daughter strand

• dna pol delta and ligase repair gap

how often do parents give mutations to offspring?

• fathers give more mutations as they age

• because of the continuous germ cell production

which polymerases have proofreading exonuclease activity in eukaryotes?

• dna pol epsilon and delta

steps of base excision repair

1. DNA glycosylase hydrolyzes bond btwn incorrect base and phosphate backbone

2. APE1 cuts DNA backbone

3. APlyase removes the deoxyribose phosphate

4. dna pol beta fills the gap, dna ligase seals the sugar phosphate backbone

steps of nucleotide excision repair

1. 23B and XP-C complex recognize a lesion

2. TFIIH is recruited to catalyze dna unwinding

3. XP-F recruited and works with XP-G to make cuts 24-32 bp apart

4. ssDNA gap is filled by DNA pol and sealed by DNA ligase

translesion synthesis

• last resort

• translesion polymerases bypass lesions and use damaged dna as a template

• no proofreading exonucleases, error prone

methods for fixing double strand dna breaks

• non homologous end joining (NHEJ)

• homologous recombination (HR)

method for fixing single strand breaks

dna ligase

chromosomal translocation

• region with rearranged dna 

• usuaLLY FROM 2 NON-HOMOLOGOUS CHROMOSONE

• can be caused by NHEJ bringing the wrong pieces together

what type of repair is used during each part of the cell cycle

G1: NHEJ

S: HR, MMR and translesion synthesis

G2: HR

BER and NER always active