Unit 3 Synthesis of DNA

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Origin of Replication

Single site in prokaryotes. Multiple sites along genomic DNA in eukaryotes- usually at AT rich sites.

Replication is bidirectional.


Pre-priming Complex

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'


Solving Supercoiling

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


DNA Ligase

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


Xeroderma pigmentosum

autosomal recessive, defect in nucleotide excision repair (NER) system, cells cannot repair damaged DNA...skin cancer


Repair Mechanisms

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.