DNA replication

I have to do 3 things in here: 

1. ADD PICTURES

2. EDIT TEXT and improve overall legibility/readability and connect small sentences into whole paragraphs to improve sense of the text. I can choose to do this by any way I want but the information must be retained. Its messy, clear it up

3. EXPAND IT

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-            Bidirectional not un-directional

-          Semi-conservative not conservative nor dispersive as shown in the
      Meselson and Stahl experiment
      and the script

-          One origin in bacteria, multiple (thousands to ten of thousands of) origins in eukaryotes
     It takes about 8hours for DNA to replicate in eukaryotes. 50bp/s in eukaryotes and 1000-2000bp/s in prokaryotes its speed. My lecture notes though differ with this statement, that 1.5hrs in yeast and 24hrs in eukaryotes is needed to replicate the DNA.

-          DNA pol: always polymerize/synthesize in a 5’->3’ direction, adds nucleotides in order. Forms phosphodiester bond, catalyses the addition of nucleotide to the 3’ OH of the last nucleotide of the growing strand
    Ori C (the origin of replication in E. Coli) is 13 base pair sequence rich in AT pairs repeated 5 times.

In E. coli:

I …  in repair and replication

II     DNA repair

III    principal DNA replication enzyme

IV    DNA repair

V     DNA repair

-          Proofreading

-          Sliding clamp

-          Unwinding the helix: topoisomerases, helicase, single strand binding proteins ssbp

-          First initiator proteins in e coli bind to AT rich origin, forms a spiral weakening the double helix and allows helicase and other replication enzymes to attach

-          Priming; dna pol can only extend dna strand at the 3’ end; requires OH to add to. Each new strand starts with a short primer of 10-30 RNA bases complementary to DNA. Primer is formed by an enzyme call primase. Primase does not require an existing 3′ OH for synthesis, unlike DNA polymerase. RNA Primer is later removed by DNA polymerase I and replaced with correct DNA sequence

-          DNA Polymerase can add to RNA primer: Polymerase switching occurs. Primase finishes synthesising the primer. The clamp attaches to the end of the primer. DNA polymerase attaches to the clamp

-          Semi-discontinuous; leading and lagging strand (leading -> continuous: new strand is made without breaks although there are fragments produced on the leading strand because of repair;  lagging -> discontinuous: new strand is made in short fragments/okazaki fragments)

-          Lagging strand in bacteria: Primase synthesizes RNA primer, DNA pol III continuous to synthesize full okazaki fragments and stops when it reaches next primer. Pol III is replaced by Pol I that hydrolyses RNA primer and replaces RNA primer with DNA and leaves a ‘nick’, an unmade phosphodiester bond between discontinuous okazaki fragments. DNA ligase seals the nick by making bond

-          Lagging strand in eukaryotes:  Primase synthesises RNA primer and DNA pol δ continuous to synthesise full okazaki fragment and continuous until it reaches primer then displaces the primer leaving a flap. Flap endonuclease Fen1 cleaves the flap DNA, leaves a nick between discontinuous okazaki fragments that DNA ligase seals the nick by making a bond.

-          DNA polymerase moves in different directions on the leading and lagging strands but overall, polymerase on both strands moves with the replication fork because the lagging strand makes a loop around. Joint regulation of lead and lagging strand synthesis

-          Bacterial replisome: A molecular machine for DNA replication. Contains 2 copies of DNA Polymerase III (Lead & lagging strand). E. coli can replicate its whole genome of 5 million base pairs in 40 min. With 2 replication forks, each replisome works at 1,000 nucleotides/sec. Combines speed with accuracy

-          Eukaryotic Replication Fork:  Eukaryotic replication fork is similar to replisome. Leading and lagging strand synthesis is coupled but not one unit. DNA polymerase δ replicates the leading strand. DNA polymerase ε the lagging strand

-          Like a trombone? Proposed by Alberts et al.In the trombone model, DNA on the lagging strand is looped around, so that the polymerases at the fork are working in the same direction. Every so often, the lagging strand DNA would be released and re-oriented at the fork

-          Chromatin in Eukaryotic replication  Chromatin is the reason replication in eukaryotes is slower. Eukaryotic linear chromosomes of chromatin. Nucleosomes –are disrupted by the replication fork, then re-bind after the fork has passed – why replication.  Half of the parental H3 and H4 go to one daughter, half to the other. Called parental histone segregation. ASF1 guides parental H3 and H4 histones to their new position. New histones delivered with chromatin assembly factor 1 (CAF-1) to replication fork. The correct recruitment of histones, and maintenance of chemical modifications such as methylation underpin epigenetic inheritance

-          Prokaryotic replication: Termination In bacterial circular chromosomes, termination of replication occurs at the ter site. Ter is on the opposite side of the chromosome from ori.  ter is bound by Tus – a replicator terminator protein.  When replication forks hit Tus they stop, and disassembly of the replication complex occurs

-          Eukaryotic replication: Termination   Replication can continue to the end of a chromosome except that the lagging strand requires enzymes (primase) to bind ahead of the replication fork. Therefore the lagging strand will get shorter with every replication. Telomeres are sacrificial ends of chromosomes to protect coding DNA. Telomerase contains RNA and adds telomeric DNA sections onto the 3’ end

-          Chromosome Ends – Telomeres protect chromosome ends. Chromosomes get shorter. But only telomere sequence is lost…for a while. Telomeres range in length from ~100bp to > 20,000bp, depending on species. Telomerase contains RNA that acts as a template for the extension of the DNA with hexamer (TTAGGG) repeats. These provide a site for primer synthesis in replication. Excess hexamer repeats can then be removed. Telomerase extends chromosomes although most normal cells do not express the telomerase and so lose telomeres with each division. In humans telomerase is active in germ cells, epidermal skin cells, follicular hair cells, most cancer cells, some stem cells probably and in vitro immortalised cells.

S  Some pictures good pictures: