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43 Cards in this Set
- Front
- Back
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rules of DNA replication
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semiconservative
bidirectional unwinds helix semidiscontinuous lagging formed from okazaki |
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Untwisting of DNA
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bp-ing ensures nucleot are inserted correct positions as new complement strands are synthesized.
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semiconservative model
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meselson-stahl:
1 from parent, 1 new |
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meselson and stahl experiment
density and generation |
DNA heavy, hybrid, light
density increases L-R |
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DNA replication= bidirectional
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2 replication forks in opp directions
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DNA replication=semidiscontinuous
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leading strand is continuous
lagging copies is segments that need to be joined |
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enzymology of DNA replication
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DNA polymerase I (Pol I)
needs all four deoxynucleotides a template and a primer, a SS-DNA that pairs with template |
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DNA polymerases with ecoli
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ecoli have several
its Pol I has 3 active sites, is its own editor. Pol III replicates its chrom, sits at each replication fork |
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DNA Pol I
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nucleot added 5'-3'
catalyzes 20 cycles before new strand dissociates from template |
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exonuclease activity with Pol I
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3'-5' is proofreading, removes incorrect bases of growing DNA chain
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features of replication
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bidirectional
helix unwound by helicase supercoil compensated by gyrase |
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replication is semidiscontinuous
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as helix is unwound during replication the 3-5 strand can be copied continuously by DNA pol proceeding in the 5-3 direction behind the rep fork
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replication AGAIN
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Pol III uses an RNA primer
primase forms primer Pol I excises the primer ligase seals nicks bw fragment |
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replication fork
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duplex unwould by gyrase and helicase, SS's coated with protein, primase primes synthesis on lagging. Pol I and ligase act downstream on lagging to remove RNA primers, replace with DNA and ligate the fragments
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Pol III
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10 diff subunits
core enzyme has 3 subs(a,e,t) alpha=polymerase epsilon=3'exonuclease theta=holoenzyme assembly beta=ring around DNA |
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mechanism of replication
replisome |
consists of unwinding proteins, priming complex, 2 Pol III
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mechanism of replication
initiation |
DnaA binds to oriC, intitiate separation. DnaB, helicase delivered by DnaC further unwinds. primase binds and contructs RNA primer
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mechanism of replication
elongation |
DnaB helicase unwinds, SSB bind to keep strands separate. Pol replicates each strand
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mechanism of replication
termination |
the ter locus, rich in G and T, signals end of replication. ter protein is a contrahelicase and prevents unwinding
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replication ending of elongation and termination
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topoisomerase II (gyrase) releives supercoiling that remains
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mitosis intermediates
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G1:growth and metabolism
S: DNA replication G2: growth/prep for division M:mitosis |
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model for initiation
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ORC(after anaphase of G1)
ORC w/ CDc6p (opportunity) also add MCM (late G1) activation G1/S S phase G2 and up to metaphase |
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DNA polymerases (greek)
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gamma:rep enzyme of mito
delta:3'exo w/ PCNA PCNA: give delta unlimited processivity and is homo epsilon:highly processive, no subunit like PCNA |
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end of chromosomes replicated
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telomeres:consist of 5-8bp repeated G-rich nucleot seq
telomere:1-12kbp long, replicated by RNA dependent DNA pol called telomerase |
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telomere replication
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rep of lagging, short RNA primers are added and extended by DNA pol.
synthesis of telomeric DNA by telomerase extends the 5' end |
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RNA directed DNA Pol
reverse transcriptase |
primer required: tRNA that virus captures from host
RT transcribes the RNA into cDNA to form hybrid |
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RNA genomes replicated
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DNA is intermediate in the rep of RNA viruses
viral RNA is template for DNA synthesis |
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RT activities (3)
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RNA directed DNA pol
RNase H activity:degrade RNA in the hybrid DNA-directed DNA pol:makes duplex after RNase H destroys viral genome |
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AZT(HIV therapy)inhibits RT
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substrate binds to HIV RT. AZTMP blocks chain elongation because 3' cant form phosphodiester with incoming nucleotide
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genetic recombination
(proteins:RecA,RecBCD,RuvA,RuvB, and RuvC) |
rearranges info, new assoc.
similar DNA=homo homo acheived by general recom recomb :need break and reunion |
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meselson and weigle
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ABC is heavy, abc is light
created recombination |
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progeny bacteriophage
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heteroduplex (+and-)--->
(semiconservative DNA rep) progeny phage of 2 diff geno's |
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holiday model for homo recomb
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+ is 5-3 and - is 3-5
only strands of like polarity exchange DNA during recombination |
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RecBCD-dependent initiation of recombination
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1.bind RecBCd to DNA end,unwind
2.nonspecific cleave, more 3' 3.encounter and cleave 4.displace ssb, bind of RecA enhanced 5' terminal cleavage 5.pair with homo duplex and strand invasion |
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structure of RecA
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352-residue and 38-kD protein
ADP is bound near helices C and D. The filament has 4 turns w/ 6 RecA per turn. |
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homologous recombination promoted by RecA enzyme
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protein aids in invasion of 3'ssDNA into DNA duplex.
forms a d-loop, that is displaced by invasion to form a holiday junction as invastion continues. |
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site-specific recombination
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transposon has inverted nucleotide sequence repeats at the terminal. Creates a staggered cut, ss ends are ligated to transposon. Gaps are filled in and strands are ligated. Transposon insertion generated direct repeats of the target site in host DNA.
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can DNA be repaired...?
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DNA must be preserved
cells require means for repair 2 principal mechanisms:mismatch repair and reversing chemical damage |
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mismatch repair
(ie:methyl-directed pathway of e.coli) |
scan DNA for mismatched bases, excise the mispaired region and replace it.
Methylation occurs post replication, repair proteins identify methylated strand as parent, remove mismatched bases on the other strand and replace them |
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UVA irradiation
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causes dimerization of adjacent thymine bases. Cyclobutyl ring forms bw C's 5&6 of the pyrimidine rings. Normal base pairing is disrupted by the dimers.
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Reversing chemical damage
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pyrimidine dimers repaired by photolyase.
excision repair:DNA glycosylase removes dmaged base, get AP site AP endonuclease cleave backbone, removes serveral residues and gap is repaired by DNA poly and ligase |
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molecular basis of mutation
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point mutations arise by wrong base pairing
can be caused by base anaologs chemical mutagens react with bases in DNA insertion and deletion |
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chemical mutagens
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HNO2 converts C to U
nitrosoamines with nitric acid Hydroxylamine reacts w/ C alkylation of G any alkylation agens |
