mutation rate per replication and

replication time

• 1 nucleotide chng per 10⁹ nucleotides for each DNA replication
• 3 nucleotide chnges per cell division in humans
• 50 nucleotides per second are generated

Directionality of DNA synthesis

DRAW IT!

• DNA pol ONLY reads in 3'-5' direction
• New strand grows in 5'-3' direction
• 3'-5' template is copied continuously-new strand grows 5'-3'
• 5'-3' fragment is copied discontinuously in 3'-5' direction
• okazaki fragments: nucleotides added in 5'-3' direction 

• enzyme DNA primase adds RNA primer with 3'OH at one end
• primer added once for leading strand at beginning of replication and at beginning of each okazaki fragment on lagging strand 

• one side binds to back of polymerase
• assembly of clamp around DNA requires ATP hydrolysis by protein complex (clamp loader: C-RPC) 

• DNA repair system removed primer and replaces with DNA
• DNA ligase joins nick

• SSBP composed of 3 subunits not 1 like mammals
• DNA primase is inc. into a multisubunit enzyme, DNA pol alpha
• 1/10 slower in mammals due to nucleosomes
• okazaki fragments synth in intervals of 100-200 in humans vs 1000-2000 in bacteria 

• Carried out by DNA pol just prior to adding a new nucleotide 
• Correct nucleotide has higher affinity for the moving polymerase than an incorrect one
• After binding but before it is covalently added to the growing chain the enzyme must undergo a conformational change
• incorrectly bound nucleotide likely to disassociate
• Allows polymerase to "double check" geometry before catalyzing addition of next nucleotide

• Exonucleolytic 3'-5' proof reading by DNA pol clips off unpaired residues at primer terminus
• Self-correcting enzyme that removes its polymerization errors
• Takes place immediately after an incorrect nucleotide is added to the growing chain (rare)

• if synth 3'-5' mistakes could not be corrected
• bare 5' chain end cannot be hydrolyzed away 

• Removes errors that escape the replication machine
• detects potential for distortion in helix due to mismatch
• req'd to detect newly synth strand (methylated in bacteria; eukaryotes new strand distinguished by nicks/ ss breaks)
• Defective copy of mismatch repair gene - high predisposition to hereditary nonpolyposis colon cancer (HNPCC) 
• MutS, MutL

• Depurination - 5000 A or G bases lost daily from each cell by spont. hydrolysis of the sugar-base link
• Deamination: ~100 C's are spontaneously deaminated daily to produce uracil
• ROS: attack purine and pyrimidine rings
• Nonenzymatic methylation of A or G distorts DNA helix + interferes with vital DNA - protein interaction
• UV radiation causes pyrimidine dimers bet. two T's or C and T 

• glycosylases remove altered base
• AP endonuclease and phosphodiesterase remove sugar phosphate
• DNA pol adds new nucleotides
• DNA ligase seals nick

• Repairs damage caused by large structural alterations in helix (ie: pyrimidine dimers)
• nuclease cleaves strand on both sides of the lesion
• lesion is removed and repaired by DNA helicase, polymerase, and ligase 

• non-homologous and homologous end joining

• broken ends are juxtaposed and rejoined by DNA ligation
• loss of 1 or more nucleotides
• emergency solution
• results in mutation but is acceptable in most cases 

• general recombination mechanisms used
• Transfer nucleotide sequence info from intact DNA double helix
• requires special recombination proteins to recognize areas of DNA that match and bring them together
• uses undamaged DNA as a template for transferring info
• difficult but precise