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semiconservative replication
- each of the parental strands are used as a template for syn of new strands
- the daughter strands hav one new and one old strands
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origin of replication in prokary vs eukary
- single ori in prokary
- multiple in eukary
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final product of replication in pro vs eukary
- in prokary - 2 seperate circular daughter DNAs
- in eukary - sister chromatids that are connected at the centromere, to be seperated during mitosis
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replication forks
- the origin of replication, opens two replication forks that move away from each other
- thus replication is a bidirectional process
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polymerases and nucleases
- polymerases: enzymes that syn nucleic acids by formin phophodiesterbonds
- nucleases: break these bonds
- exonucleases remove nucleotides from either 5` or 3` ends
- endonucleases cut within the strand and release nucleic acid fragments
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features of DNA polymerase
- syn DNA in 5` to 3` direction
- req template is DNA that is copied from 3` to 5` direction
- req deoxy nucleotide substrates
- req primer is a RNA
- has proof readin activity - 3`to 5` exonuclease activity
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features of RNA polymerase
- syn rna in 5` to 3` direction
- reads the DNA template in 3` to 5` direction
- needs no primers
- has no proof reading activity
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initiation of strand syn
DNA polymerases cannot initiate strand syn where as RNA polymerses can do so
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steps of DNA replication
- the base sequence at the origin is recognized
- helicase: unwinds by breaking the hyd bonds - thus two replication forks are formed
- single stranded binding proteins: bind to the single strands preventing them from reassociating and protecting them from degradation
- primase: syn a short RNA primer in 5` to 3` direction startin at the ORI using parental strand as template
- DNA polymerase III: starts makin DNA in 5` to 3` direction starting at 3` end of the primer
- can make continously - leading strand
- or discontinously - lagging strand (each discontinously made fragment called okazaki fragment - initiated with primer and made in 5` to3`)
- RNA primers removed by RNAase H in eukaryotes and an uncharecterized DNA poly fills in the gap with DNA
- in prokary DNA poly I removes both the primer and fills the gap
- both eu and prokary DNA poly hav proof readin activity
- DNA ligase: seals the nicks between okazaki fragments making them continous
- DNA gyrase (topoisomerase II): provides a swivel in front of each replication fork
- replication finished when the 2 replication forks meet on the other side of the circle opp the origin
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DNA gyrase and replication
- provides a swivel in fornt of each of the replication forks
- as helicase unwinds the DNA, the DNA ahead becomes overwound and positive supercoils appear
- gyrase inserts negative supercoils in the strands
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drugs and DNA gyrase
- quinolones block the action of topoisomerases
- nalidixic acid kills bacteria by inhibiting DNA gyrase
- inhibitors of eukaryotic topoisomerse II like etoposide and tenoposide are used as anticancer drugs
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eukaryotic DNA polymerases and their func
- DNA α and δ work together to syn both leadin and lagging strands
- DNA poly γ replicates mitochondrial DNA
- DNA poly β and ε participate primarily in DNA repair
- ε may substitute δ in some cases
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telomers and aging of cells
- telomers - repetitive seq at the ends of linear DNA molecule
- with each replication telomers are shortened as DNA poly cannot syn the 5` end of each strand
- this causes aging of cells...ultimately telomeres become so short that chrmosomes cannot func and cells die
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telomerase
- completes replication of the telomeric sequences at both ends of a eukaryotic chromosome
- present in embryonic cells, fetal cells and certain adult stem cells; but not in adult somatic cells
- inappropriately present in many cancer cells, contributing to their unlimited replication
- it has a short RNA template complementary to the DNA telomers and telomerase reverse transcriptase activity
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syn of the DNA (both leading and lagging strands) in prokary is by
DNA poly III
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removal of RNA primers
- in prokary by dna poly I (5`to 3` exonuclease activity)
- in eukary by RNAase H (5` to 3` exonuclease activity)
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quinolones and fluroquinolones
- prevent DNA gyrase and thus prevent DNA replication
- most active against aerobic gram negative bacteria
- eg levofloxacin, ciprofloxacin, moxifloxacincurrently used for rx of gonorrhea and upper and lower urinary tract infections in both sexes
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Antivirals and DNA replication
- one chemotherapy for HIV is use of AZT (3`azido 2`,3`dideoxythymidine) or struc related comp
- in cells, AZT converted to triphosphate derivative and used as a substrate for viral reverse transecriptase that makes DNA form its RNA genome
- in AZT, an azide gr is at 3` postition instead of hydroxyl gr
- this prevents further replication by effectively causing chain termination
- this is possible as the reverse transcriptase though a DNA polymerse has no proofreading activity
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