Unit 3 Synthesis of DNA
Origin of Replication
Single site in prokaryotes. Multiple sites along genomic DNA in eukaryotes- usually at AT rich sites.
Replication is bidirectional.
DNA-A: binds and melts dsDNA; DNA helicase: unwinds dbl helix; ssDNA-binding proteins: keep strands separate & protect from nucleases. Moves along template 3'-5'
unwinding DNA forms positive supercoils ahead of replication fork; DNA topoisomerase removes supercoils & facilitates transcription (nicks one of two strands)
Direction of DNA Replication
Leading strand: copied in direction of replication fork, synth. continuously; Lagging strand copied away from replication fork, Okazaki Fragments (small fragments of DNA) discontinuously
Initiation and Elongation (Prokaryotic)
DNA polymerase require RNA primer to initiate synth.- accomplished with Primase. DNA pol III initiates replication using RNA primer, and elongation (highly processive), and proofreading DURING replication
Replacement of RNA primers (Prokaryotic)
DNA pol I: locates space between DNA & RNA, replaces RNA w/ DNA; DNA fragments linked by DNA ligase
Pol I; Pol II; Pol III Table (Prokaryotic)
Pol I: 5'-3' & 3'-5' exonuclease actvty, remove primer in conjunction w/ RNAse H, DNA repair, fill gap after removal of RNA primer; Pol II: 3'-5' exonuclease actvty, DNA repair; Pol III 3'-5' exonuclease actvty, Replication-synth of DNA
forms phosphodiester bond between 3' OH & 5' phosphate of nucleotides w/ ATP, DNA repair actvty, used in recombinant DNA tech
Eukaryotic DNA Replication
Multiple Ori sites, RNA primers removed by RNAse H, DNA replication and cell division coordinated with cell cycle
Cell Cycle (Eukaryotic)
G1- precedes replication, S- replication/ synthesis phase, G2- mitosis, G0- cells that stop dividing
Eukaryotic DNA Plymerases
No Proof Reading- *Pol alpha: contains DNA primase and initiates DNA synthesis, *Pol beta: Repair;
Proof Reading- *Pol gamma: replicates mito DNA, *Pol delta: elongates leading and lagging strands, *Pol epsilon: repair
Shortening of DNA with Replication
No way to fill gap after removal of primer from extreme 5' end- may cause aging
Region of highly repetitive DNA at end of chromosome (TTAGGG)- many Kbp in humans, *buffer consumed during cell division, *protect ends from nucleases, *fixed number of divisions, loss of DNA leads to *senescence & cell death
active in germ, stem, and cancer cells- elongates telomeres. Reactivated by physical exercise (aorta & mononuclear cells), inc. vit. D (in leukocytes), TA-65 telomerase activator; *Ribonucleoprotein complex: proteins & RNA, RNA-dependent DNA pol; extends 3' end of DNA
DNA Repair Rate
up to 500K modification events each day per cell
Action of Mutagens
Indirect: *X-rays form free radicals from H2O; Direct: *cigarette smoke contains carcinogen benzo(a)pyrene- when oxidized forms adducts with guanine residues, *UV leads to dimer formation between thymines on DNA
autosomal recessive, defect in nucleotide excision repair (NER) system, cells cannot repair damaged DNA...skin cancer
E.coli- photoactivating enzymes for Thymine dimers; Nucleotide excision repair: *Endonuclease removes distorted region; Base excision repair: *DNA glycosylases cleave N-glycoside bond, *AP endonuclease cleaves sugar-phosphate strand
Which Strand to Repair?
Prokaryotic: Parental DNA is methylated. Not clearly understood in humans.
Reverse Transcriptase Inhibitors
*didanosine (ddl): dideoxynucleoside converted to ddlTP in cells and *terminates elongation when incorporated by reverse transcriptase.