Bio SI Before Exam 3Herceptin:- Another example of a targeted cancer therapy- Used to treat HER2 (which is involved in breast cancer – body overexpressing HER2 in breast cancer cells)o Negative effect is not targeted to specific receptor, will bind to whatever receptor it can bind too Get a mutation in promoter gene that gets receptor to be overly expressed- Monoclonal antibody (not a small molecule – antibodies are huge)o Gleevec is a small molecule inhibitor- Targets all HER2 receptorsAntibodies:- Proteins that got produced by white blood cells- Function is to bind to antigens (which are foreign bodies (non-self) ex: viruses)o Antibodies mark them for later destruction- Variable domain – where we get different variants of the antibodies- Constant domain – always the same- Made up of heavy chain & light chain (one variable of each, one constant of each = 4 subunits)- Disulfide bond makes antibodies very stable- Antigen bonds to variable because antigens can be anything, can be different types, so need antibody to be susceptible to bind to these- How does variable region become variant?o VDJ recombinationAntibodies – VDJ Recombination:- Variable region made up of V segments, D segments, and J segmentso Regions in the DNA- Recombination of the DNA makes up the variable region- Can make up to ~4,000 arrangements- How can cell make sure it adds more variability to variant region?o TDT – polymerase that adds random nucleotides in between the segmentso More than 4,000 antigens so need to add different nucleotides between segments to increase number of variability so can bind to all those antigens- How does this cutting happen?o RAG1 & RAG2 cut DNA to make different segments and put them togethero Same way CML cutOverview of Cell Cycle:- G1 phase – growth (“gap” phase)o Could duplicate organelleso Cell checks environment: Growth factor signals present? Enough nutrients? Enough energy? DNA  check for damageo Restriction point / START / G1-checkpoint (“prepare ingredients”) Make nucleotides Make proteins required for replicationo Once replication starts cells now in S phase- S phase – “synthesis phase”o DNA replication- G2 phase – 2nd growth phaseo Check for DNA damageo Finish replicationo Prepare for M phase- M phase – chromosome segregationo Cytokinesis: splitting of the cytosol- G0 phase – non-dividing stateCell Cycle Control System:- CDK: enzyme called Cyclin Dependent-Kinaseo Cyclin is binding partnero “Master cell cycle regulator”o Any point in cell cycle that cells decide they need to stop  inhibit CDK- CDK is regulated in several ways:o Cyclin regulation – different cyclins for different phases of the cell cycle G1 cyclins telling CDK to target G1 proteins S phase cyclins M phase cyclinso Phosphorylation & dephosphorylation by kinases and phosphataseso CDK inhibitors- Another way of regulation is the regulation of cyclin levelso Play with levels of cyclins themselves- Cyclin levels rise & fall throughout cell cycle via:o Transcription & translationo Degradation through ubiquitin-mediated proteolysiso Eventually cause regulation of CDK – not regulating CDK levels, regulating the cyclin levels here(things that bind to CDK) Open response question on exam probably **Cell Cycle Checkpoints:- G1o Check for DNA damage (before replication begins)o Check for favorable environmento Must have enough material to create a duplication of the cell- So Check for errors, incompletely duplicated DNA- G2o Damaged or incorrectly replicated DNAo Enough resources to continue- Mo Proper chromosome and microtubule attachment and arrangement*G0 is rest phase – when DNA damage too much to be fixed / didn’t get fixed / etc.P53 (DNA Damage Response):- P53 is a tumor suppressor – transcription factor that when activated promotes the transcription of the P21 gene & other genes, P21 gene is a CDK inhibitoro G1/S-CDK & S-CDK inhibitor specifically inactivated by P21Restriction Point (Rb/E2F):- Restriction point (Start, decision point) point of no returno Happens in G1o Protein levels rise (Cyclin D)o E2F is family of transcription factorso E2F up-regulates proteins needed for S phaseo E2F is kept inhibited by Rb until there is enough cyclin levels in the cell (the cell makes the decision to continue with the cell cycle)o Once Cyclin D/CDK complex reaches a certain concentration, Rb releases E2F and the cell can enter the cell cycle and express proteins for S phaseControl of the Replicative Cycle:- Origin of replication complex (Pre-RC) binds to origin replicationo Pre-RC made of ORC, CDC6, CDT1- ORC (origin of replication) recruits a protein called Cdc6, which recruits Mcm complex (helicase)- CDT1 recruits Mcm (which is a helicase) to the Pre-RC complexo Mcm only protein involved in DNA replication- This occurs from G1  S phase- Once you start replicating, in S phase- How make sure replication doesn’t happen back to back or twice at a time?o S-CDK regulation S-CDK regulates ORC complex, CDC6, Mcm- Phosphorylation of ORC prevents association of ORC with CDT1 & Mcm- Phosphorylation of Cdc6 leads to degradation- Phosphorylation of Mcm leads to export from the nucleusG2 Phase: Prepping for Chromosome Segregation:- During G2 cyclin levels rising & M-CDK complexes forming but not active- M-phase cyclin CDK activity not occurring yet because of positive feedback loop & M-CDK pathway- Mitotic CDK binds to M-cyclin, makes an inactive M-CDK, Wee1 (inhibitory kinase) & Cak (activating kinase) adds a phosphate each to M-CDK complex, CDC25 (activating phosphatase) takes off inhibiting phosphate from Wee1 from M-CDK = active M-CDK complex o CDC25 not active all the timeo To activate more CDC25, need the positive feedback loop- * Longer you have M-CDK inactive, the longer you have to grow the cell, therefore bigger the cell will beo Faster you are in activating M-CDK, less time to have right size causing you to get a smaller cello Inactive Wee1 = smaller cell (less time to activate M-CDK)o Inactive CDC25 = bigger cell (longer time to activate M-CDK)Positive Feedback Loop:- Inactive CDC25 phosphatase gets phosphate from M-CDK complex, makes it an active CDC25, then goes and finds another inactive M-CDK complex and takes the phosphate, etc. forever and ever- Q: How does CDK trigger onset of M phase?o CDK phosphorylates laminin, which leads to breakdown of nuclear envelopeo Would lead into M phase (segregation, chromosome segregation)o