Describe cell cycle checkpoints

• compentency (G0-G1A)- DNA intact and proteins are functional
• viability (G1A-G1B)
• replication competency (G1C-S)- after passing this point, its irreversible

• G1A- cyclin dep. kinases
• G1B- protein synthesis dependent
• G1C- protein synthesis indepedent (make most cyclins, kinases)
• S- replication enzymes, nucleotides, histones
• G2- mitotic apparatus

• the lagging strand synthesis is discontinous by laying down segments of DNA called Okazaki fragments

DNA Polymerase mechanism of action in template directed DNA synthesis

• 3'-OH dependent nucleophilic attack using dNTP's that elimnates pyrophosphate molecule
• pyrophosphate hydrolyzed by water into two phosphate ions
• must have RNA primer

• mechanism of action- bind to:
  • ssDNA
  • nicked DNA
  • gapped DNA
• function- repair enzyme
  • gap filling
  • mismatch
  • short patch repair
  • Okazaki RNA primer repair
• components
  • 5-3 exonuclease
  • 3-5 exonuclease

• 5'-3'- clears damaged DNA or RNA primers (ex: if hypermethylated GC rich regions and it decides it doesn't want to see these particular regions)
• 3'-5'- proofreader (if you put in wrong nucleotide, it will step back and cut it back out)

• function- repair enzyme
  • ssDNA gap filling
  • replace RNA primer regions in Okazaki fragments
• components- 3-5 exonuclease activity

• Enzyme activity (essential for replication)
  • 5-3 polymerase
  • 5-3 exonuclease
  • 3-5 exonuclease
• Structure (oligomeric complex)
  • core enzyme (8 subunits)
  • holoenzyme (14 subunits)
  • beta subunit

Describe the fidelity of DNA polymerases

• prokaryotes- one
• eukaryotes- many

ALL THESE ORIGINS ARE SEQUENCE SPECIFIC!!!!!

Describe sequence that you will see at origins of replications

How does initiation of replication occur? (Process)

1. Phosphorylation of origin recognition proteins in a cell cycle dependent signaling fashion
2. this induces supercoiling
3. this will cause denaturing of AT rich regions due to stress
4. leads to recruitment of replication dependent proteins
   1. ex: SSB, helicase, topoisomerase
5. establish primosome complex (primase)

Function of SSBP's

grab onto ssDNA strand and stabilize it to prevent it from being broken down

Mechanism of action of helicase

1. binds to s.s region of DNA and binds to ATP 
2. helicase hydrolyze ATP
3. leads to changed confirmation
4. cause unwinding of DNA (continually pulls one strand of DNA through its active site)

• SSBP's
• DNA helicase
• primase

Fate of RNA primers in DNA replication

DNA Polymerase I (exonuclease activity) in prokaryotes and it is replaced using the same enzyme using its own polymerase activity. RNase in humans gets rid of RNA primer and our equivalent of "DNA pol. II) would fill in gap.

Mechanism of action of ligase

1. enzyme will bind to ATP and cleave off pyrophosphate to yield E-AMP
2. the enzyme will attach the AMP onto the DNA to create AMP-P-5' DNA
3. AMP-P-5' DNA combine with DNA 3' OH to yield AMP and DNA 3'-O-P-5' DNA

1. cut single strand
2. DNA bind to the Tyr in enzyme active site
   1. allow for CB btw enzyme and phosphate of nucleotide
3. single strand (driven by DNA's physical properties), go around to relax the supercoil
4. seal phosphodiester bond

Mechanism of action of topoisomerase II

1. cut both strands
2. one DNA segment will bind
3. ATP binds and a second segment of DNA comes in at a 90 degree angle
4. confirmation change involves cutting both strands in one segment
5. allows second dsDNA to pass through without cutting
6. hydrolysis reaction to seal DNA

• ciprofloxacin
• novobiocin
• nalidixic acid
• camptothecin (chemotherapy)

Mechanism of termination of DNA replication in prokaryotes

• if replisome sees many methyl sequences at AT regions, termination
• due to bidirectional replication forks, the replisomes could run into each other, "fall off" the DNA, short patch repair by DNA Pol. I, topoisomerase resolves product

SLIDING TEMPLATE MECH. 

1. elongation of DNA sequence laying down telomerase RNA and binds to replication sequence in active site
2. translocation of telomerase complex as telemerase reverse transcriptase lays down complementary sequence
3. continued elongation and translocation until finish
4. eventually the complex will loop the around to form a four way hydrogen binding network consisting of guanine residues from reduntant telomeric sequencs

Mechanism of action of ciprofloxacin

• binds to topoisomerase
• prevents reversal of the covalent bond between the tyrosine and the phosphate, so the tyrosine stays bound to the DNA strand