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week 7

by: Emma Notetaker

week 7 CELL 2050

Emma Notetaker
GPA 3.975

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week 7 of notes
Dr. Meadows
Class Notes
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Popular in Genetics

Popular in CELL

This 7 page Class Notes was uploaded by Emma Notetaker on Tuesday October 11, 2016. The Class Notes belongs to CELL 2050 at Tulane University taught by Dr. Meadows in Fall 2016. Since its upload, it has received 3 views. For similar materials see Genetics in CELL at Tulane University.


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Date Created: 10/11/16
Thursday, October 6, 2016 Week 7 DNA Replication and Recombination • genetic information must be accurately copied each time cell divides • one error/million bp —> 6400 mistakes every time a cell divides • replication also takes place at high speeds, very efficiently E. coli replicates 100 nucleotides/second • • proposed DNA replication models • conservative • both strands of original DNA serve as template strands, which gives rise to completely new helix • the parental helix is conserved throughout the whole process • dispersive • different bits of the whole molecule are used as templates • each new strand has choppy mixture • semiconservative: actual model - all DNA goes through this • one strand of the parent used for one new cell, the other strand used for the other • after second replication, 2 completely new helices • Meselson and Stahl’s experiment: 2 isotopes of nitrogen 14N common, 1N rare and heavy form • 15 14 • E. Coli were grown first in N media, then transferred to N • cultured E. Coli cells were subjected to equilibrium density gradient centrifugation • spun in centrifuge for days • density gradient develops within tube ( N moves to bottom, 1N moves to top) • which model of DNA replication applies to E. coli? • DNA from bacteria from 15N appeared as single, heavy band ( N original parent) • DNA transfered to normal nitrogen: 14N • after one round of replication, DNA appeared as single band at intermediate weight • is it were conservative, there would’ve been a heavy band and a light band • after 2nd round, DNA was 2 bands - one light and the other intermediate • intermediate made up of original and newly synthezied • light ones are completely new ones IF this were dispersive, it would all have been intermediate band throughout all • rounds • samples taken after additional rounds continued to appear in 2 bands • lighter band becomes thicker because there are more and more completely new ones Repplcattono DNA template Breakage of number of unidirectional/ products Moodell nucleotide replicons bidirectional strand Theta circular no 1 either 2 circular molecules 1 Thursday, October 6, 2016 Replication DNA template Breakage of number of unidirectional/ products Model nucleotide replicons bidirectional strand Rolling circle circular yes 1 uni one circular molecule and one linear molecule that may circularize linear linear no many bi 2 linear molecules • modes of replication • replicons: units of replication • replication origin (one in bacteria, many in eukaryotes) theta replication: • • usually in bacteria • circular DNA, E. Coli • single origin of replication - makes single stranded templates for new DNA synthesis • double-stranded DNA unwinds at replication origin bubble forms with a replication fork at each end • • usually bidirectional replication • at each fork, synthesis of leading strand proceeds continuously in the same direction as unwinding • synthesis of lagging strand is discontinuous in opposite direction leading and lagging strands in circular formation • • products: 2 circular DNA molecules • rolling circle replication • viruses • F factor of E. coli single origin of replication • • replication initiated by break in one of the nucleotide strands • DNA synthesis begins at 3’ end of broken strand • inner is used as template • 5’ end of broken strand is displaced • cleavage or circle releases single stranded linear DNA and double-stranded circular DNA • linear DNA may circularize and serve as a template for synthesis of complementary strand • —> product: multiple circular DNA models • continuous DNA synthesis begins at 3’ end of broken strand • as the DNA molecule unwinds, the 5’ end is progressively displaced • all continuous • linear eukaryotic replication • eukaryotic cells • 1000s of origins • at each origin, DNA unwinds to produce replication bubble • synthesis takes place on both strands at each end of the bubble as forks proceed outward and fuse 2 Thursday, October 6, 2016 • typical replicon: ~200,000 - 300,000 bp • produces 2 linear DNA models • requirements of linear eukaryotic replication • template strand • raw material: nucleotides • enzymes and other proteins • synthesis of new DNA: • new DNA synthesized from dNTPs (deoxyribonucleoside triphosphates) • 3’-OH group attacks 5’ - phosphate group of the incoming dNTP • 2 phosphates cleaved off • phosphodiester bond forms between 2 nucleotides • direction of replication: • DNA polymerase: enzymes that synthesize DNA • adds nucleotides only to 3’ end of growing strand • replication from 5’ —> 3’ • because the strands are antiparallel, DNA synthesis occurs in different directions on each strand • continuous replication: synthesis in the same direction as unwinding • on lower strand, DNA synthesis proceeds continuously in the 5-3 direction (same direction as unwinding) • when lower runs out of template, this one starts again • leading strand • discontinuous replication: synthesis in the OPPOSITE direction of the unwinding • on upper template strand, DNA synthesis begins at the fork and proceeds in the direction opposite that of unwinding (soon runs out of template) • has to restart replication multiple times - gaps form • short fragments of DNA produces by discontinuous synthesis are called Okazaki fragments • lagging strand • result of antiparallel nucleotide strands • bacterial DNA replication • initiation: • 245 bp in oriC (single origin replicon) • initiator proteins (DnaA) recognize oriC, and bind there • causes a short stretch of DNA to unwind • this allows helices and other single-stranded binding proteins to attach to single stranded DNA • unwinding: • initiator protein • DNA helicase binds to lagging strand template at each fork and moves in 5’-3’ direction • breaks hydrogen bonds and moves replication fork • this separates the DNA • single strand binding proteins (SSBs) • bind to single strand and stabilize exposed DNA • DNA gyrase (topoisomerase) • relieves strain ahead of the fork • elongation: • starts with synthesis of primers • primase synthesizes short stretches of RNA nucleotides 3 Thursday, October 6, 2016 • makes a 3’ OH group so DNA polymerase can add DNA nucleotides on leading strand, primer required only at 5’ end of new strand • • because replication is continuous • on lagging strand, new primer needed at the beginning of each Okazaki fragment • because replication discontinuous • primers: existing group of RNA nucleotides with 3’-OH group new nucleotide can be added here • • usually 10-12 nucleotides long • DNA polymerase is the primase • removal of RNA primer: DNA polymerase I • nicks connected after primers are removed by DNA ligase DNA polymerase I: removes RNA primer • • 5’-3’ polymerase and 3’-5’ exonuclease activities • ALSO has 5’-3’ exonuclease activity • removes RNA primers and replaces them with DNA nucleotides by synthesizing in 5’-3’ direction DNA ligase seals the nick • • DNA polymerase III: bacterial elongation • multi-protein complex, large • main workhorse of replication • 5’-3’ activity adds nucleotides in 5’-3’ direction 3’-5’ exonuclease activity allows it to remove nucleotides in 3’-5’ direction • • this enables it to correct errors • backs up to removes excess nucleotides, then resumes 5’-3’ activity)\ • DNA ligase: connects nicks after RNA primers are removed • seals gap with phosphodiester bond between 5’-P group of the initial nucleotide and 3’-OH group of the final nucleotide • catalyzes the formation of phosphodiester bond without adding another nucleotide to strand • fidelity of replication: error rate < 1 mistake per billion nucleotides • proofreading DNA polymerase I • • 3’-5’ exonuclease activity removes incorrect pairs • mismatch repair: corrects errors after replication • requires ability to distinguish old and new strands of DNA (enzymes need to determine which of the wrong pairs to remove) termination: replication fork meets termination protein • Coompponnentt Function initiator protein binds to origin and separates strands of DNA to start replication DNA helicase unwinds DNA at replication fork single-stranded binding proteins attach to sing stranded DNA and prevents secondary structures from forming DNA gyrase moves ahead of replication fork, making and resealing breaks in the double helical DNA to release torque that builds up 4 Thursday, October 6, 2016 Component Function DNA primase sunthesizes a short RNA primer to provide 3’-OH group provided by primer DNA polymerase III elongates new nucleotide strand from 3’ OH group provided by primer DNA polymerase I removes RNA primers and replaces them with DNA DNA ligase joins Okazaki fragments • eukaryotic DNA replication • autonomously replicating sequences (ARSs) • 100-120 bps (ie origin of replication) • origin-recognition complex (ORC) • bind to ARSs to initiate DNA replication • replication licensing factor: approval of DNA replication • MCM: minichromosome maintenance - binds DNA and initiates replication as a helices on all origins • eukaryotic DNA polymerase • DNA polymerases in eukaryotic cells DNA polymerase 5’—> 3’ nuclease 3’-5’ exonuclease cell function activity activity alpha yes no initiation of nuclear DNA synthesis and DNA repair (primase activity) delta yes yes lagging strand synthesis of nuclear DNA, DNA repair and translesion DNA synthesis epsilon yes yes leading strand synthesis **translesion: templates with abnormal bases, distorted structures and bulky lesions • differs from bacterial replication • eukaryotic DNA is complexed to histone proteins in nucleosomes • have to break off while DNA is replicating • nucleosomes reassembled quickly following replication • creation of nucleosomes requires: • disruption of original nucleosomes on the parental DNA (required to access DNA) • redistribution of pre-existing histones on new DNA • addition of newly synthesized histones to complete formation of new nucleosomes experiment: what happens to histones in eukaryotic DNA replication? • • grow cells in medium with amino acids labelled with heavy isotope • transfer cells to medium with amino acids labelled with light isotope • let them undergo replication • isolate histone octamers before and after replication and subject to density-gradient diffusion • newly-synthesized octamers are less dense and will be higher in the tube 5 Thursday, October 6, 2016 • octamers with mix of old and new (heavy and light) • old octamers are dense and will move towards bottom • —> after DNA replication, new reassembled octamers are random mixture of new and old histones • DNA polymerase is fixed in location and template strand is threaded through it • replication at the ends of chromosomes • circular: • replication around the circle provides a 3’-OH group in from of the primer • nucleotides can be added to 3’OH group when the primer is replaced • linear: • multiple origins of replication • elongation in adjacent replications provides 3’-OH group for replacement of primer • primers at ends can’t be replaced • when primers at the end are removed, there is no 3’-OH group • this forms a gap • —> without special mechanisms, DNA replication would leave gaps due to the removal of primers at the ends of chromosomes (if this were the case, chromosomes would shorten after each replication) • telomerase replicates the ends of chromosomes (fills in the gaps from the removal of RNA primer) • telomerase is part RNA and part DNA • telomere has G-overhang • RNA part of telomerase is complementary to the G-rich strand • pairs with it, providing a template for the synthesis of copies of the repeats • new nucleotides added to 3’ end of G-rich strand (pairing with the RNA of telomerase) • after several nucleotides have been added, RNA template moves along DNA • more nucleotides added • telomerase is removed • synthesis takes place on the complementary strand, filling in the gap • unclear how this strand is made • if organism’s telomerase were mutated and nonfunctional, chromosomes would shorten with each generation • homologous recombination: exchange is between homologous DNA molecules during crossover • holliday junction and single-strand break • homologous chromosomes align and single strand breaks occur in same place on both molecules • free end of each migrates to the other molecule • each invading strand joins to the broken end of the other DNA molecule —> holliday junction • begins to displace original complementary strand • branch migration takes place as the strands exchange positions, creating 2 duplex molecules (heteroduplex DNA) • cleavage in vertical plane makes crossover recombinants • cleavage in horizontal plane makes non crossover recombinants • doyle-strand break model of recombination • 2 double stranded DNA molecules from homologous chromosomes align • double-strand break occurs in one molecule 6 Thursday, October 6, 2016 • nucleotides enzymatically removed, producing some single-stranded DNA on each side • free 3’ end invades and displaces a strand of unbroken DNA molecule • 3’ end elongates, further displacing original strand • displaced strand forms a loop that base pairs with broken DNA • DNA synthesis initiated and 3’ end of the bottom strand (displace loop is used as template) • strand attachment produces 2 Holliday junctions, each can be separated by cleavage and reunion • gene conversion: • process of nonreciprocal genetic exchange • produces abnormal gemete ratio • arises from heteroduplex formation • in recombination, single-stranded breaks occur and strands invade - produces heteroduplex DNA with mismatched bases • in repair, mismatched nucleotides excised and replaced using complementary strand as template • if one strand used, gene conversion results with 3 copies of one allele • if the other used, normal recombination occurs with 2 copies of each allele 7


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