Molecular Biology I
Molecular Biology I MBioS 503
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This 31 page Class Notes was uploaded by Carol McDermott on Thursday September 17, 2015. The Class Notes belongs to MBioS 503 at Washington State University taught by Staff in Fall. Since its upload, it has received 23 views. For similar materials see /class/205938/mbios-503-washington-state-university in Molecular Biosciences at Washington State University.
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Date Created: 09/17/15
Replication eves Lan 09 um m blmreclmnal UNlDIHECTIONAL FlEPLICATlON ORIGIN Replication fork Parental DNA ORIGIN A replication fork is initiated at the origin moves sequentially along DNA Replication is unidirectional when a single replication fork is created at an origin Replication is bidirectional when an origin creates two replication forks that move in opposite directions Does Methylation at the Origin Regulate Initiation oriC contains eleven GATCCTAG repeats that are methylated on adenine on both strands The Dam methylase maintains methylation Methylated Hemi DNA methylated DNA Me G A T C C T A G GNET C Replication Dam methylase C T A G e Replication generates hemimethylated DNA which cannot initiate replication 0 There is a 13minute delay before the GATCCTAG repeats are remethylated Only methylated origins are functional Active origin Replication 1 mmo immmw nac ve origins Dam methylasel o v M gmmmw Aciive origin Origins May Be Sequestered after Replication Squ binds to hemimethylated DNA and is required for delaying rereplication Squ may interact with DnaA As the origins are hemimethylated they bind to the cell membrane they may be unavailable to methylases The nature of the connection between the origin and the membrane is still unclear Membranebound inhibitor binds to Me lG A T C N erhLMJJ 70 TA G hemimelhylated DNA DNAisremelhylaled WW andreleasesinhibimr Me 7 7 V EATCVNNNYNNNN ETAGAN NN KNN w DnaAbindslo miC M The A protein is cisvaming A protein nicks the origin and binds to 5 end y 7 Strand strand Floll ng circle replication displaces minus slra l nd NA synthesis Replication lork passes origin A protein nicks DNA and binds to new 539 and Released plus Strand arms Covalent circle Rolling Circles Are Used to Replicate Phage Genomes The cbX A protein is a cisacting relaxase It generates singlestranded circles from the tail produced by rolling circle replication The F Plasmid s Transferred by Conjugation between Bacteria A free F factor is a replicon that is maintained at the level of one plasmid per bacterial chromosome An F factor can integrate into the bacterial chromosome Its own replication system is suppressed The F factor codes for specific pili that form on the surface of the bacterium 0 An F pilus enables an F positive bacterium to contact an F negative bacterium initiate conjugation Fpili connect mating bacteria Conjugation Transfers SingleStranded FM donors conjugate wlth FH reclplants TraYI nick DNA al ONT V 3 on A 5 TraYl muliimei migrates around circle unwinding DNA 1 RECIPIENT S 539 Single strand anters recipient Complementary strands are n e C Donor gap is closed Recipient circuian zes DNA Transfer of an F factor is initiated when rolling circle replication begins at oriI The free 539 end initiates transfer into the recipient bacterium The transferred DNA is converted into doublestranded form in the recipient bacterium The F factor initiates a DONOR EACTEF llUM FACTOR briT rra region r F factor is nlcked aronr 5 a Coll Chromosome Single strands are convened to nsler ur a single strand HEClPiENT BACTEFUUM 5 end leads single Strand 39 i into reciplen 39 5 double strands in both bacteria recipient genome When an F factor is free conjugation quotinfectsquot the recipient bacterium with a copy of the F factor When an F factor is integrated conjugation causes transfer of the bacterial chromosome Transfer continues until the process is interrupted by random breakage of the contact between donor and recipient bacteria The Bacterial Ti Plasmid Causes Crown Gall Disease in Plants Infection with the bacterium A tumefaciens can transform plant cells into tumors The infectious agent is a plasmid carried bythe bacterium TDNA Carries Genes Required for TDNA integrates into he plan genome I n i O n Agrobaclenum V PlanlWll T s W deg39 Part of the DNA ofthe Tl r r plasmid is transferred to the E TDNAlo pram if quotquotquotquotquotquot quot i plant cell nucleus TVDNA l Plant calls grow into tumor 4 opines on which u bacterium can grow Transfer of TDNA Resembles Bacterial TDNA is ganarated like a rolling circle First nickqa Endonucle lt ase TVDNAI 7 7 i E eleased To plant nucleus Conjugation TDNA is generated when a nick at the right boundary creates a primer for synthesis of a new DNA strand The preexisting single strand that is displaced by the new synthesis is transferred to the plant cell nucleus Transfer is terminated when DNA synthesis reaches a nick at the left boundary The T DNA is transferred as a complex of single stranded DNA with the VirE2 single strand binding protein The single stranded T DNA is converted into double stranded DNA integrated into the plant genome The mechanism of integration is not known T DNA can be used to transfer genes into a plant nucleus Replication ls Connected to the Cell Cycle Bacterial Cell Division Bacteria divide by binary fission the single circular bacterial chromosome is replicated replication begins at the origin of replication and proceeds bidirectionally new chromosomes are partitioned to opposite ends of the cell a septum forms to divide the cell into 2 cells Gmwlh repllcallun and segregation are connected A unit cell has a circular chromosome Replication initiales A n cell passes crilical size Replication generates catenalad daughter 1 Daughterchmmoscmes quot are separa ed Septum divides cell C Daugmer cells separale L The doubling time of E coli can vary over a 10x range depending on growth conditions 0 It requires 40 minutes to replicate the bacterial chromosome at normal temperature 0 Completion of a replication cycle triggers a bacterial division 20 minutes later Replication s Connected to the Cell Cycle The doubling time of E coli can vary over a 10 fold range depending on growth conditions It requires 40 minutes to replicate the bacterial chromosome at normal temperature Completion of a replication cycle triggers a bacterial division 20 minutes later If the doubling time is lt60 minutes a replication cycle is initiated before the division resulting from the previous replication cycle Fast rates of growth therefore produce multiforked chromosomes Bacteria can have multiforked chromosomes 3 inmaiion 0 34 15 s D Termination 0 A replication cycle is initiated at a constant ratio of massnumber of chromosome origins There is one origin per unit cell of 17 pm in length The Septum Divides a Bacterium into Progeny That Each Contain a Chromosome The septum divldes the cell Cell suns with annulus al midcenler New annuli are genera e ew armuu move in polar direction New annuli slop movement Central annulus develops into septum Cell divides Side View shows we annulus extends around circumference OI cell Crossrsecllon Shows that annulus connects membranes Outer membrane lCeli wall inner membrane Septum formation is initiated at the annulus The annulus is a ring around the cell where the structure of the envelope is altered New annuli are initiated at 50 of the distance from the septum to each end of the bacterium 0 When the bacterium divides each daughter has an annulus at the mid center position Septation starts when the cell reaches a fixed length The septum consists of the same peptidoglycans that comprise the bacterial envelope Bacterial DNA may be linked to the membrane Bacterial DNA may be linked to the memurane Origins of repiiaating ch romosomes attached to memhrane Daughter chromosomes anached to envelope Septum grows between chromosomes Septum divides ceil mes distributed to daughter ceus Mutations in Division or Segregation Affect Cell Shape fts mutants form long filaments because the septum fails to form to divide the daughter bacteria quot5 mutants give long manems Minicells form in mutants that produce too many septa they are small and lack DNA Anucleate cells of normal size are generated by partition mutants The duplicate chromosomes fail to separate par mutants give anucleate Celts V Cell Division Targeting of cell division to cell midplane is very precise FtsZ Ring l E e 39 e e T FtsZ Ring 6 a FtsZ s Necessary for Septum Formation The product offtsZis required for septum formation at preexisting sites FtsZ is a GTPase that forms a ring on the inside of the bacterial envelope It is connected to other cytoskeletal components min Genes Regulate the Location of the Septum The location of the septum is controlled by minC D and E 0 The number and location of septa is determined by the ratio of MinEMinCD MinCD ls controlled by MInE Annuii ETD l Septum Poles derived from septum of division before lasl Poles derived irom sepmm cl last division D l or Seplumfom ling capacity k Sepmm forms to Sepiumforming capacity The septum forms where MinE is able to form a ring At normal concentrations MinCD allows a midcenter ring prevents additional rings of MinE from forming at the poles Plasmid genomes may recombine Monomeric circles Recombinalion Dimsric circle Recombination Monumeric circles Plasmid genomes may recombine
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