- Nov 2022
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www.wikipathways.org www.wikipathways.org
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Bacterial chromosomes have a single origin, termed ori, whereas eukaryotic organisms can have many not sequence specific.
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Can broadly split the process into three parts. Initiation, elongation and termination
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DNAPolymerase A
Primase and DNA polymerase A work in close association.
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CDK2
This is the S-phase CDK.
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Assembly of the pre-replicative complex
- Replication origin licensing.
- All of this occurs in G1 phase
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Orc complex
Orc1-5 complex.
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Cdc6
Cdc6 is only present in G1 phase.
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DNA replication pathway in humans. Orc complex binds the origin of replication.
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en.wikipedia.org en.wikipedia.org
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ChIP
Workflow 1. Cross-linking 2. Chromatin fragmentation 3. Immunoprecipitation of chromatin 4. DNA recovery and purification 5. Sequencing of DNA
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analyze protein interactions
ChIP-seq is concerned with testing for protein DNA interactions.
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www.wikipathways.org www.wikipathways.org
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NER * Helix distortion * ERCC6 and 8 mutation -- Cockayne's syndrome * XP proteins (XPE or DDB2, XPC, XPA) mutated in xeroderma pigmentosum. * TFIIH, the same helicases as seen in DNA replication.
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www.wikipathways.org www.wikipathways.org
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BER * For non-helix distorting base lesions. * Base is not present, a gap is present in DNA. * Specific DNA glycosylases used for identification. * APEX1 and APEX2 (AP endonucleases): responsible for end processing * An AP site (apurinic/apyrimidinic site). * The exposed 3' OH is available to a replicative polymerase. * Ligation performed by ligase * Short or long patch BER is possible.
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www.researchgate.net www.researchgate.net
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In HR, * MRN -- begginings of dsDNA resection. * The PARP1 protein is active on ssDNA. * Free 3' ends made available, crucial for later DNA pol binding. * RPA binds, coats, the ssDNA.<br /> * Rad51 searches for strand for invasion. * Strand invasion carried out by sister chromatid, etc. * D-loop formation.
May have. * DSBR * SDSA * BIR
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NHEJ relies on microhomologies. Doesn't require homologous sequence from another source. * Ku protein instrumental in identification of DSB and recruits DNA-PKcs. * DNa-PKcs autophosphorylates. * DNA ends processed by Artemis. * LIG4 and XCRR4 are needed for strand ligation.
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NHEJ and HR can be compared.
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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TEs are transposable elements.
Transposons are mobile DNA elements.Can move throughout the genome. Can be catagorised as class 1 (retrotransposons) or class 2 (DNA transposons).
Class 1 comprises TEs with LTRs, retroposons (LINE), SINEs.
Class 2 comprises TEs that operate under replicative transposition or non-replicative transposition. Replicative transposition (nick and paste) -- a total of two TEs as an end result, one as part of the donor and one as part of the target sequence. cointegrate.
Non-replicative transposition (cut and paste) - only one TE generated, in the target.
Examples of DNA-only transposon:
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bio.libretexts.org bio.libretexts.org
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The initial sample consisted of 1 band. F = 0. 1st generation = The sample overall less dense, still one band. Intermediate density. dsDNA made of one strand heavy and one light. After 2 generations, there was a band for intermediate density and for strands of just light, N-14, dsDNA.
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Three methods of replication were initially hypothesized: * Conservative * Semi-conservative * Dispersive
Semi-conservative method was eventually determined to be correct based on empirical evidence from Messelson & Stahl, in 1958.
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Parental strands consist of N-15 isotopes. Replicated daughter strands consist of N-14 isotopes. The CsCl ultracentrifugation process creates density gradient. This allows DNA fragments of different densities to migrate and form a band at the point at which their buoyant density equals that of the salt.
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- E.coli grown in an heavy nitrogen, N-15, enriched medium
- Then transferred to N-14 based media to reproduce
- As several points in the experiment, cells were lysed and underwent ultracentrifugation through a CsCl concentration gradient
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bio.libretexts.org bio.libretexts.org
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The several panels show what happens in each cycle. Each cycle consists of a denaturation step at a temperature higher than the melting temperature of the duplex DNA (e.g. 95 oC ), then an annealing step at a temperature below the melting temperature for the primer-template (e.g. 55 oC), followed by extension of the primer by DNA polymerase using dNTPs provided in the reaction. This is done at the temperature optimum for the DNA polymerase (e.g. 70 oC for a thermostable polymerase). Thermocylers are commercially available for carrying out many cycles quickly and reliably
- Denature
- Annealing primer
- Synthesize new DNA with polymerase
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- High speed
- Extreme sensitivity
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