Introduction to Introduction to Microbial GeneticsMicrobial Genetics

Microbiology 221Microbiology 221

A Historical OverviewA Historical Overview

The scientists who provided the clues to The scientists who provided the clues to the nature of DNAthe nature of DNA

Friederich Meischer – DNA isolatedFriederich Meischer – DNA isolatedLuria and Delbruck – Bacteriophages Luria and Delbruck – Bacteriophages Stanely Giffiths( 1928) The idea of the Stanely Giffiths( 1928) The idea of the transforming “ substance” – Avery, transforming “ substance” – Avery, MacLoed, and McCarty( 1944) – the MacLoed, and McCarty( 1944) – the nature of transformationnature of transformationHershey and Chase – Bacteriophage – Hershey and Chase – Bacteriophage – DNA as the hereditary materialDNA as the hereditary materialChargaff – A= T and C=GChargaff – A= T and C=GMaurice Wilkins and Rosalind Franklin – Maurice Wilkins and Rosalind Franklin – x-ray crystallography of DNAx-ray crystallography of DNAWatson and Crick – Double helixWatson and Crick – Double helix

GriffithsGriffiths

Luria and Delbruck at Luria and Delbruck at Cold Spring Harbor in Cold Spring Harbor in

19531953Luria and Delbruck Luria and Delbruck

studied bacterial studied bacterial mutations and mutations and resistance to resistance to infection with infection with bacteriophagesbacteriophages

The characterized the The characterized the virus and its life virus and its life cyclecycle

Alfred Hershey and Martha Chase Alfred Hershey and Martha Chase and the Blender Experimentand the Blender Experiment

Hershey and Chase Hershey and Chase wanted to verify wanted to verify that DNA was the that DNA was the hereditary materialhereditary materialThey used a They used a bacteriophage for bacteriophage for their studytheir studyThey labeled the They labeled the DNA with DNA with Radioactive P( P32) Radioactive P( P32) and the protein with and the protein with radioactive radioactive sulfur( S35)sulfur( S35)

Results of the Results of the ExperimentExperiment

Proved that the radioactivity from the labeled Proved that the radioactivity from the labeled DNA was present in the progeny phage DNA was present in the progeny phage produced from infection of the bacteria.produced from infection of the bacteria.

The Race for the Double The Race for the Double HelixHelix

Rosalind Franklin Rosalind Franklin and Maurice Wilkins and Maurice Wilkins at Kings Collegeat Kings College

Studied the A and B Studied the A and B forms of DNAforms of DNA

Rosalind’s famous Rosalind’s famous x-ray x-ray crystallography crystallography picture of the B picture of the B form held the form held the secret, but she secret, but she didn’t realize its didn’t realize its significancesignificance

The Race for the Double The Race for the Double HelixHelix

Watson and Crick Watson and Crick formed an unlikely formed an unlikely partnershippartnershipA 22 year old PhD A 22 year old PhD and a 34 year old and a 34 year old “want to be” PhD“want to be” PhDembarked on a embarked on a model making model making venture at venture at CambridgeCambridgeUsed the research of Used the research of other scientists to other scientists to determine the determine the nature of the double nature of the double helixhelix

Nucleic Acid Nucleic Acid CompositionCompositionDNA and RNADNA and RNA

DNA – Basic MoleculesDNA – Basic Molecules

a.a. Purines – adenine and guaninePurines – adenine and guanine

b.b. Pyrmidines – cytosine and thyminePyrmidines – cytosine and thymine

c.c. Sugar – DeoxyriboseSugar – Deoxyribose

d.d. Phosphate phosphate groupPhosphate phosphate group

http://http://www.dnai.org/index.htmwww.dnai.org/index.htm -  DNA -  DNA backgroundbackground

Double HelixDouble HelixTwo polynucleotide strands joined by Two polynucleotide strands joined by phosphodiester bonds( backbone)phosphodiester bonds( backbone)Complementary base pairing in the center of Complementary base pairing in the center of the moleculethe molecule

A= T and C G – base pairing. Two A= T and C G – base pairing. Two hydrogen bonds between A and T and three hydrogen bonds between A and T and three hydrogen bonds between C and G.hydrogen bonds between C and G.

A purine is bonded to a complementary A purine is bonded to a complementary pyrimidinepyrimidineBases are attached to the 1’ C in the sugarBases are attached to the 1’ C in the sugarAt opposite ends of the strand – one strand At opposite ends of the strand – one strand has the 3’hydroxyl, the other the 5’ hydroxyl has the 3’hydroxyl, the other the 5’ hydroxyl of the sugar moleculeof the sugar molecule

DNA StructureDNA Structurehttp://www.johnkyrk.com/DNAanatomy.html - DNA structure

Double helixDouble helix( continued)( continued)

The double helix is right handed – the The double helix is right handed – the chains turn counter-clockwise.chains turn counter-clockwise.As the strand turn around each other As the strand turn around each other they form a major and minor groove.they form a major and minor groove.The is a distance of .34nm between The is a distance of .34nm between each baseeach baseThe distance between two major The distance between two major grooves is 2.4nm or 10 basesgrooves is 2.4nm or 10 basesThe diameter of the strand is 2nmThe diameter of the strand is 2nm

Complementary Base Complementary Base PairingPairing

Adenine pairs Adenine pairs with Thyminewith Thymine

Cytosine pairs Cytosine pairs with Guaninewith Guanine

The end view of DNAThe end view of DNA

This view This view shows the shows the double helix double helix and the outer and the outer backbone with backbone with the bases in the bases in the center.the center.

An AT base An AT base pair is pair is highlighted in highlighted in whitewhite

Double helix and anti-Double helix and anti-parallelparallel

DNA is a directional moleculeDNA is a directional molecule

The complementary strands The complementary strands run in opposite directionsrun in opposite directions

One strand runs 3’-5’One strand runs 3’-5’

The other strand runs 5’ to 3’The other strand runs 5’ to 3’

( the end of the 5’ has the ( the end of the 5’ has the phosphates attached, while phosphates attached, while the 3’ end has a hydroxyl the 3’ end has a hydroxyl exposed)exposed)

RNA structureRNA structure

Polynucleotide – nucleic acid - Polynucleotide – nucleic acid - Single stranded molecule that Single stranded molecule that can coil back on itself and can coil back on itself and produce complementary base-produce complementary base-pairing ( t- RNA)pairing ( t- RNA)Four bases in RNA are Adenine Four bases in RNA are Adenine and Guanine ( purines) and and Guanine ( purines) and Cytosine and Cytosine and Uracil( pyrimidines)Uracil( pyrimidines)Sugar – riboseSugar – ribosePhosphatesPhosphates

RNARNA

Three types of RNAThree types of RNA

a.a. MessengerMessenger

b.b. TransferTransfer

c.c. RibosomalRibosomal

d.d. nc- non coding RNA’snc- non coding RNA’s

Prokaryote DNAProkaryote DNA

Tightly coiledTightly coiledCoiling maintained by molecules Coiling maintained by molecules similar to the coiling in eukaryotessimilar to the coiling in eukaryotesCircular ds moleculeCircular ds moleculeBorrelia burgdoferiBorrelia burgdoferi ( Lyme Disease ( Lyme Disease )has a linear chromosome)has a linear chromosomeOther bacteria have multiple Other bacteria have multiple chromosomeschromosomesAgrobacterium tumefaciens Agrobacterium tumefaciens ( Produces Crown Gall disease in ( Produces Crown Gall disease in plants)plants) has both circular and linear has both circular and linear

Prokaryote Prokaryote chromosomeschromosomes

Circular DNACircular DNA

E. coliE. coli – most often – most often studied in molecular studied in molecular

biology of prokaryotesbiology of prokaryotesThe genes of The genes of E. coliE. coli are located on a are located on a circular chromosome of 4.6 million circular chromosome of 4.6 million basepairs. This 1.6 mm long molecule is basepairs. This 1.6 mm long molecule is compressed into a highly organized compressed into a highly organized structure which fits inside the 1-2 structure which fits inside the 1-2 micrometer cell in a format which can micrometer cell in a format which can still be read by the gene expression still be read by the gene expression machinery.machinery. Bacterial DNA is supercoiled by DNA Bacterial DNA is supercoiled by DNA gyrase. Chemical inhibition of gyrase gyrase. Chemical inhibition of gyrase without allowing the cells to reprogram without allowing the cells to reprogram gene expression relaxes supercoiling and gene expression relaxes supercoiling and expands the nucleoid, suggesting that expands the nucleoid, suggesting that supercoiling is one of the tools used to supercoiling is one of the tools used to compress the genomecompress the genome

CoilingCoiling

Coiling maintained by GyraseCoiling maintained by Gyrase

Relaxation of the coils by Relaxation of the coils by TopoisomeraseTopoisomerase

Nucleosome Nucleosome formationformation

DNA is more highly DNA is more highly organized in organized in eukaryote cellseukaryote cells

The DNA is associated The DNA is associated with proteins called with proteins called histones.( eukaryotes)histones.( eukaryotes)

These are small basic These are small basic proteins rich in the proteins rich in the amino acids lysine amino acids lysine and/or arginineand/or arginine

There are five There are five histones in eukaryote histones in eukaryote cells, H1, H2A, H2B,H3 cells, H1, H2A, H2B,H3 and H4.and H4.

. .

Beads on a StringBeads on a String

The DNA coils around the ellipsoid The DNA coils around the ellipsoid approximately 1 ¾ turns or 166 base approximately 1 ¾ turns or 166 base pairs before proceeding to the next.pairs before proceeding to the next.The DNA + the histone proteins The DNA + the histone proteins arranged in this formation are referred arranged in this formation are referred to as a nucleosome.to as a nucleosome.The stretch of DMA between the beads The stretch of DMA between the beads varies in length from 14 to 100 base varies in length from 14 to 100 base pairs.pairs.H1 appears to associate with the linker H1 appears to associate with the linker regions to enable the nucleosome to regions to enable the nucleosome to supercoilsupercoilWhen folding of the structure reaches a When folding of the structure reaches a maximum, the chromosomes can be maximum, the chromosomes can be visualizedvisualized

Chromosome Chromosome structurestructure

http://www.johnkyrk.com/chrohttp://www.johnkyrk.com/chromosomestructure.htmlmosomestructure.html

Eukaryote replicationEukaryote replication

The nature of The nature of DNA DNA replication replication was was elucidated by elucidated by Meselson and Meselson and StahlStahl

Meselson and Stahl Meselson and Stahl experimentexperiment

1. Grew bacteria in heavy Nitrogen – N-15

2. Transferred bacteria to N-14

3. Before bacteria reproduce in new media, all bacteria contain heavy DNA

4. Samples were taken after one round of replication and two round of replication

Semiconservative Semiconservative replicationreplication

Each original strand Each original strand serves a template or serves a template or pattern for the pattern for the replication of the replication of the new strand.new strand.

The new strand The new strand contains one original contains one original and a newly and a newly synthesized strandsynthesized strand

Eukaryote replicationEukaryote replicationMultiple linear chromosomesMultiple linear chromosomes

Each chromosome has more than one origin of Each chromosome has more than one origin of replication replication

Approximately 1400 x as long as bacterial DNAApproximately 1400 x as long as bacterial DNA

Multiple replicons on a chromosomeMultiple replicons on a chromosome

Oris along the length – every 10 to 100 umOris along the length – every 10 to 100 um

Replication forks and bubbles are formed. Replication forks and bubbles are formed. Replication proceeds bidirectionally until the Replication proceeds bidirectionally until the bubbles meetbubbles meet

This shortens the length of time necessary to This shortens the length of time necessary to replicate eukaryote chromosomesreplicate eukaryote chromosomes

The process of elongation occurs at a speed of 50-The process of elongation occurs at a speed of 50-100 base pairs/minute as compared to 750 to 1000 100 base pairs/minute as compared to 750 to 1000 base pairs/ minutebase pairs/ minute

http://www.johnkyrk.com/DNAreplication.hthttp://www.johnkyrk.com/DNAreplication.htmlml

The origin of replication The origin of replication and replication forksand replication forks

Eukaryote replicationEukaryote replication

During the S phase, there are 100 During the S phase, there are 100 replication complexes and each one replication complexes and each one contains as many as 300 replication forks. contains as many as 300 replication forks. These replication complexes are These replication complexes are stationary. The DNA threads through stationary. The DNA threads through these complexes as single strands and these complexes as single strands and emerges as double strands.emerges as double strands.

DNA PolymerasesDNA Polymerases

Fourteen DNA polymerases Fourteen DNA polymerases have been observed in have been observed in human beings as compared human beings as compared to three in to three in E. coli.E. coli.

Prokaryote Prokaryote ReplicationReplication

Enzyme Type Source Properties

Bacterial topoisomerase I ( protein)

I E. coliRelaxation of negative but not positive supercoils

Vaccinia virus topoisomerase I

I Vaccinia virusRelaxation of positive and negative supercoils

Eukaryotic topoisomerase I

I Calf thymusRelaxation of positive and negative supercoils

Reverse gyrase I Thermophilic bacteriaIntroduces positive supercoils into DNA

Topoisomerase V IHyperthermophilic

bacteriaRelaxation of positive supercoils

DNA gyrase II E. coliIntroduces negative supercoils into DNA

Topoisomerase IV II E. coliDNA relaxation and potent decatenation

T4 topoisomerase II II Bacteriophage T4Relaxation of positive and negative supercoils and decatenation

Eukaryotic topoisomerase II

II S. cerevisiaeRelaxation of positive and negative supercoils and decatenation

Bidirectional Bidirectional replicationreplication

There is an origin There is an origin of replicationof replicationTwo replication Two replication forks are formedforks are formedReplication occurs Replication occurs around the circle around the circle until they have until they have opened and opened and copied the entire copied the entire chromosomechromosomeReplicon- contains Replicon- contains an origin and is an origin and is replicated as a replicated as a unitunit

Ori – Origin of Ori – Origin of replicationreplication

Characteristics used to define Origins:Characteristics used to define Origins:

The position on the DNA at which The position on the DNA at which replication replication startstart points (see right) are found. points (see right) are found.

A DNA sequence that when A DNA sequence that when added to a non-replicating DNAadded to a non-replicating DNA causes it to causes it to replicate. replicate.

A DNA sequence whose A DNA sequence whose mutationmutation abolishes abolishes replication. replication.

A DNA sequence that in vitro is the A DNA sequence that in vitro is the binding target for enzyme binding target for enzyme

TopoisomerasesTopoisomerasesTopoisomeraseTopoisomerase

When the double helix of DNA, which is When the double helix of DNA, which is composed of two strands, separates, composed of two strands, separates, helicase makes these two strands rotate helicase makes these two strands rotate around each other. around each other. The DnaB protein is the helicase most The DnaB protein is the helicase most involved in replication, but the n’ protin involved in replication, but the n’ protin may also participate in unwinding.may also participate in unwinding.The single stranded binding proteins SSBP The single stranded binding proteins SSBP help to keep the strand openhelp to keep the strand openBut there is a problem due to the But there is a problem due to the topological reason that the unreplicated topological reason that the unreplicated part ahead of the replication fork will part ahead of the replication fork will rotate around its helical axis when the two rotate around its helical axis when the two strands separate at the replication fork strands separate at the replication fork

Topoisomerase actionTopoisomerase action

It causes strong strain in the helix It causes strong strain in the helix (1). Thus, it is impossible to unlink (1). Thus, it is impossible to unlink the double helical structure of the double helical structure of DNA without disrupting the DNA without disrupting the continuity of the strands. continuity of the strands. In order to perform unraveling of In order to perform unraveling of a "compensating winding up" a "compensating winding up" DNA, enzymes are required (1). DNA, enzymes are required (1). Topoisomerase changes the Topoisomerase changes the linking number as well as linking number as well as catalyzes the interconversionn of catalyzes the interconversionn of other kinds of topological isomers other kinds of topological isomers of DNA (2). of DNA (2).

InitiationInitiation

InitiationInitiationa. a. oriCoriC - origin of chromosomal replication - origin of chromosomal replicationRecognized by Recognized by DnaADnaA protein - only protein - only recognizes if GATC sites are fully recognizes if GATC sites are fully methylatedmethylatedBinding of DnaA allows DnaB to open Binding of DnaA allows DnaB to open complexcomplexb. b. DnaBDnaB is the replication is the replication helicasehelicasec. Strand separation by helicasec. Strand separation by helicased. d. SSBSSB (single-stranded binding) protein (single-stranded binding) protein keeps strands apartkeeps strands aparte. e. DNA gyraseDNA gyrase - a topoisomerase - puts - a topoisomerase - puts swivel in DNA which allows strands to swivel in DNA which allows strands to rotate and relieve strain of unwindingrotate and relieve strain of unwinding

ExplanationExplanation

Recall that DNA double helix is tightly wound Recall that DNA double helix is tightly wound structure and that bases lie between the two structure and that bases lie between the two backbones. If these bases are the template for backbones. If these bases are the template for new strand, how do the appropriate enzymes new strand, how do the appropriate enzymes reach these bases? By the unwinding of the reach these bases? By the unwinding of the helix. helix.

An enzyme called helicase catalyzes the An enzyme called helicase catalyzes the unwinding of short DNA segments just ahead unwinding of short DNA segments just ahead of the replication fork. The reaction is driven of the replication fork. The reaction is driven by the hydrolysis of ATP. by the hydrolysis of ATP.

Explanation continuedExplanation continued

As soon as duplex is unwound, As soon as duplex is unwound, SSB SSB (single-stranded binding protein)(single-stranded binding protein) binds to binds to each of the separated strands to prevent each of the separated strands to prevent them from base-pairing again. Therefore, them from base-pairing again. Therefore, the bases are exposed to the replication the bases are exposed to the replication system.system.

The unwinding of the duplex would cause The unwinding of the duplex would cause the entire DNA molecule to swivel except the entire DNA molecule to swivel except for the action of a for the action of a topoisomerase (DNA topoisomerase (DNA gyrase)gyrase) which introduce breaks in the which introduce breaks in the DNA just ahead of the unwinding duplex. DNA just ahead of the unwinding duplex. These breaks are then rejoined after a few These breaks are then rejoined after a few revolutions of the duplex.revolutions of the duplex.

The need for a primerThe need for a primer

When DNA template is exposed, DNA When DNA template is exposed, DNA synthesis must begin. But DNA synthesis must begin. But DNA polymerases not only need a template but polymerases not only need a template but also a primer for replication to proceed. also a primer for replication to proceed. Where does the primer come from? Where does the primer come from? After observations that RNA synthesis is After observations that RNA synthesis is required for DNA synthesis, it was required for DNA synthesis, it was discovered that the synthesis of DNA discovered that the synthesis of DNA fragments requires a short length of RNA fragments requires a short length of RNA as a primer.as a primer.Primosome (complex of 20 polypeptides) Primosome (complex of 20 polypeptides) makes RNA primers inmakes RNA primers in E. coli E. coli

Formation of the PrimerFormation of the PrimerPrimosomePrimosome contains contains primaseprimasePrimosome moves along DNA duplex in Primosome moves along DNA duplex in 3'>5' direction (with respect to lagging 3'>5' direction (with respect to lagging strand; follows replication fork) even strand; follows replication fork) even though primer is made in 5'>3' directionthough primer is made in 5'>3' direction(Note: The symbol ">" indicates the (Note: The symbol ">" indicates the direction; that is, the primer is made from direction; that is, the primer is made from 5' to 3'.)5' to 3'.)n' proteinn' protein removes SSB in front of removes SSB in front of primosomeprimosomeDnaB protein organizes some components DnaB protein organizes some components of primosome and prepares DNA for of primosome and prepares DNA for primaseprimasePrimase forms the primerPrimase forms the primer

DNA POLYMERASE IIIDNA POLYMERASE IIIHoloenzymeHoloenzyme

Complex that synthesizes Complex that synthesizes most of the DNA copy most of the DNA copy contains the DNA contains the DNA polymerase enzyme and polymerase enzyme and other proteinsother proteins

The gamma delta The gamma delta complex and the B complex and the B subunits of the subunits of the holoenzyme bind it to the holoenzyme bind it to the template and the primertemplate and the primer

The alpha subunit carries The alpha subunit carries out the actual out the actual polymerization reactionpolymerization reaction

All of the proteins form a All of the proteins form a huge complexhuge complex called the called the replisomereplisome

DNA polymerase IIIDNA polymerase III

This is a This is a stationary stationary complex that complex that probably probably attached to the attached to the plasma plasma membrane. membrane.

The DNA moves The DNA moves through the through the replisome and replisome and is copiedis copied

Elongation of the Elongation of the chainchain

dCTP dCTP dCMP +dCMP +

PPiPPi

Energy is Energy is supplied for supplied for biosynthesis biosynthesis by the by the cleaving of the cleaving of the phosphate phosphate bondbond

Elongation( continuedElongation( continued))

Elongation proceeds in 5' > 3' Elongation proceeds in 5' > 3' direction and requires direction and requires 1) all 4 deoxyribonucleoside 5'-1) all 4 deoxyribonucleoside 5'-triphosphates (dATP, dGTP, dCTP, triphosphates (dATP, dGTP, dCTP, dTTP), dTTP), 2) Mg+ ions, 2) Mg+ ions, 3) a primer made of nucleic acid, 3) a primer made of nucleic acid, and and 4) a DNA template.4) a DNA template.

Rate of elongation = 750 - 1000 Rate of elongation = 750 - 1000 nucleotides per secondnucleotides per secondRate of formation of initiation Rate of formation of initiation complex = 1-2 minutescomplex = 1-2 minutes

ElongationElongationElongationElongationDNA polymerase I, II and III in DNA polymerase I, II and III in E .colE .coliiDNA polymerase III holoenzyme - complex of 7 DNA polymerase III holoenzyme - complex of 7 polypeptidespolypeptides

Replisome - primosome and 2 DNA polymerase III - Replisome - primosome and 2 DNA polymerase III - synthesizes DNA on both strands simultaneously synthesizes DNA on both strands simultaneously without dissociating from DNAwithout dissociating from DNA

DNA polymerase IIIDNA polymerase III catalyzes the addition of catalyzes the addition of deoxyribonucleotide units to end of the DNA strand deoxyribonucleotide units to end of the DNA strand with release of inorganic pyrophosphate (PPi)with release of inorganic pyrophosphate (PPi)(DNA)n residues + dNTP < > (DNA)n + 1 residues + (DNA)n residues + dNTP < > (DNA)n + 1 residues + PPiPPiAttachment of new units is by their a-phosphate Attachment of new units is by their a-phosphate groups to a free 3'-hydroxyl end of preexisting DNA groups to a free 3'-hydroxyl end of preexisting DNA chain. chain.

The lagging strand and The lagging strand and discontinuous discontinuous

replicationreplicationThe replication on the 5’ to 3’ strand The replication on the 5’ to 3’ strand differsdiffers

The template strand still must be read The template strand still must be read from 3’ to 5’from 3’ to 5’

The reading begins at the replication The reading begins at the replication forkfork

Occurs at the same time as the Occurs at the same time as the synthesis of the lagging strandsynthesis of the lagging strand

Same steps in synthesis of DNASame steps in synthesis of DNA

But DNA is synthesized in pieces about But DNA is synthesized in pieces about 1000 to 2000 bases in length. These 1000 to 2000 bases in length. These are known as Okazaki fragmentsare known as Okazaki fragments

Okazaki fragmentsOkazaki fragments

After the lagging strand has been After the lagging strand has been duplicated by the formation of Okazaki duplicated by the formation of Okazaki fragments, DNA Polymerase I or fragments, DNA Polymerase I or RNase H removes the RNA primer. RNase H removes the RNA primer. Polymerase I synthesizes the Polymerase I synthesizes the complementary DNA to fill the gap complementary DNA to fill the gap resulting from the RNA delection. resulting from the RNA delection.

The polymerase removes one The polymerase removes one nucleotide at a time and then replaces nucleotide at a time and then replaces itit

AMP( RNA nucleotide) replaced by dAMP( AMP( RNA nucleotide) replaced by dAMP( DNA nucleotide)DNA nucleotide)

DNA ligaseDNA ligaseLigaseLigase can catalyze can catalyze the formation of a the formation of a phosphodiester bond phosphodiester bond given an unattached given an unattached but adjacent 3'OH and but adjacent 3'OH and 5'phosphate. 5'phosphate. This can fill in the This can fill in the unattached gap left unattached gap left when the RNA primer when the RNA primer is removed and filled is removed and filled in. in. The DNA polymerase The DNA polymerase can organize the bond can organize the bond on the 5' end of the on the 5' end of the primer, but ligase is primer, but ligase is needed to make the needed to make the bond on the 3' end. bond on the 3' end.

The End of ReplicationThe End of Replication

DNA replication stops when the DNA replication stops when the polymerase complex reaches a polymerase complex reaches a termination site on the DNA in termination site on the DNA in E. coliE. coli

The Tus protein binds to the ter site The Tus protein binds to the ter site and halts replication.and halts replication.

In many prokaryotes the replication In many prokaryotes the replication process stops when the replication process stops when the replication forks meetforks meet

Plasmid replicationPlasmid replicationColE1 is a naturally occurring ColE1 is a naturally occurring plasmidplasmid of of E. coliE. coli. Its . Its replication is controlled independently of the replication is controlled independently of the replicationreplication of the host chromosome. of the host chromosome. Two plasmids with the same origin of replication can Two plasmids with the same origin of replication can not coexist in the same cell. not coexist in the same cell. The ColE1 origin, defined by molecular genetic The ColE1 origin, defined by molecular genetic methods, is in a region from which two RNAs are methods, is in a region from which two RNAs are transcribed. transcribed. An active RNase H gene is required for ColE1 An active RNase H gene is required for ColE1 replication. RNase H cleaves the RNA II transcript. replication. RNase H cleaves the RNA II transcript. The remaining RNA serves as The remaining RNA serves as primerprimer for initiation of for initiation of replication. replication. RNA I binds to 5' sequences of RNA II via RNA I binds to 5' sequences of RNA II via pseudoknots and regular complementary pairing. pseudoknots and regular complementary pairing. This binding is stabilized by the ROP or ROM protein. This binding is stabilized by the ROP or ROM protein. The binding prevents changes in the conformation The binding prevents changes in the conformation of RNA II that would otherwise result in RNAse H of RNA II that would otherwise result in RNAse H cleavage.cleavage.

Rolling Circle Replication – Occurs in Conjugation in E. coli.

How can one account for the high How can one account for the high fidelityfidelity of replication?of replication?

The answer is based on the fact that DNA The answer is based on the fact that DNA Polymerase absolutely requires 3'-OH end of base-Polymerase absolutely requires 3'-OH end of base-paired primer strand on which to add new paired primer strand on which to add new nucleotides. nucleotides.

DNA polymerase III has 3' > 5' exonuclease activity. It DNA polymerase III has 3' > 5' exonuclease activity. It was discovered that DNA polymerase III actually was discovered that DNA polymerase III actually proofreads the newly synthesized strand before proofreads the newly synthesized strand before continuing with replication. When incorrect nucleotide continuing with replication. When incorrect nucleotide is incorporated, DNA polymerase III, by means of the is incorporated, DNA polymerase III, by means of the 3' > 5' exonuclease activity, "backs up" and 3' > 5' exonuclease activity, "backs up" and hydrolyzes off the incorrect nucleotide. The correct hydrolyzes off the incorrect nucleotide. The correct nucleotide is then added to the chain and elongation nucleotide is then added to the chain and elongation is resumed.is resumed.

All 3 DNA polymerases have 3'>5' exonuclease All 3 DNA polymerases have 3'>5' exonuclease activityactivity

Proofreading ability - 1 error in 10 millionProofreading ability - 1 error in 10 million

Exonucleases and Exonucleases and repairrepair

DNA polymerase IDNA polymerase I also has 5'>3' also has 5'>3' exonuclease activity which removes exonuclease activity which removes RNA primer and 5'>3' polymerase RNA primer and 5'>3' polymerase activity which fills in the gapactivity which fills in the gap

This causes a single-stranded break in This causes a single-stranded break in the DNA - called a nickthe DNA - called a nickDNA ligaseDNA ligase repairs nick by creating a repairs nick by creating a phosphodiester bond phosphodiester bond

Genes and Gene Genes and Gene ExpressionExpression

Genes are written in a code consisting of groups of three Genes are written in a code consisting of groups of three letters called triplets.letters called triplets.There are four letters in the DNA alphabet. There are 64 There are four letters in the DNA alphabet. There are 64 possible arrangements of the four letters in groups of possible arrangements of the four letters in groups of threethreeThe triplets specify amino acids for the synthesis of The triplets specify amino acids for the synthesis of proteins from the information contained in the geneproteins from the information contained in the geneGenes can also specify t- RNA or r- RNAsGenes can also specify t- RNA or r- RNAsThe gene begins with a start triplet and ends with a stop. The gene begins with a start triplet and ends with a stop. The bases between the start and the stop are called an The bases between the start and the stop are called an open reading frame, ORF.open reading frame, ORF.The information in the gene is transcribed by RNA The information in the gene is transcribed by RNA polymerase.polymerase.It reads the gene from 3’ to 5’It reads the gene from 3’ to 5’The template strand is now referred to as the CRICK The template strand is now referred to as the CRICK strand and the nontemplate strand is now known as the strand and the nontemplate strand is now known as the WATSON strandWATSON strandDNA sequences are stored in data bases as the WATSON DNA sequences are stored in data bases as the WATSON strandstrand

Reference - COLD SPRING HARBOR - 2003Reference - COLD SPRING HARBOR - 2003

Promoters are at the beginning of Promoters are at the beginning of the Genethe Gene

RNA polymerase recognizes a binding site in RNA polymerase recognizes a binding site in front of the gene. This is referred to as front of the gene. This is referred to as upstream of the gene. upstream of the gene.

The direction of transcription is referred to as The direction of transcription is referred to as downstreamdownstream

Different genes have different promoters. IN Different genes have different promoters. IN E. coli the promoters have two functionsE. coli the promoters have two functions

The RNA recognition site for transcription The RNA recognition site for transcription which is the consensus sequence for which is the consensus sequence for prokaryotes is prokaryotes is

5’ TTGACA3’ ( Watson strand) which means on 5’ TTGACA3’ ( Watson strand) which means on the reading strand 3’ AACTGT5’ ( Crick strand)the reading strand 3’ AACTGT5’ ( Crick strand)

The Pribnow Box and Shane -The Pribnow Box and Shane -DalgarnoDalgarno

The RNA binding site has a consensus sequence ofThe RNA binding site has a consensus sequence of

5’ TATAAT 3’ ( -) and 3’ ATATTA 5’ (+)5’ TATAAT 3’ ( -) and 3’ ATATTA 5’ (+)

This is where the DNA begins to become unwound This is where the DNA begins to become unwound for transcriptionfor transcription

The initially transcribed sequence of the gene may The initially transcribed sequence of the gene may not reflect doing but may be a leader sequence.not reflect doing but may be a leader sequence.

The prokaryotes usually contain a consensus The prokaryotes usually contain a consensus sequence known as the Shane Delgarno which is sequence known as the Shane Delgarno which is complememtary to the 16s rRNA on the ribosomecomplememtary to the 16s rRNA on the ribosome

( small subunit )( small subunit )

The leader sequence also may regulate The leader sequence also may regulate transcriptiontranscription

The structure of a The structure of a prokaryote geneprokaryote gene

Prokaryote Genes are Prokaryote Genes are

ContinuousContinuousThey do not contain introns like They do not contain introns like eukaryote geneseukaryote genesThe gene consists of codons that will The gene consists of codons that will determine the sequence of amino determine the sequence of amino acids in the proteinacids in the proteinAt the end of the gene there is a At the end of the gene there is a terminator sequence rather than an terminator sequence rather than an actual stop actual stop The terminator may be at the end of a The terminator may be at the end of a trailer sequence located downstream trailer sequence located downstream from the actual coding region of the from the actual coding region of the genegene

The Gene begins withThe Gene begins with

DNA is read 3’ to 5’ and m RNA is DNA is read 3’ to 5’ and m RNA is synthesized 5’ to 3’synthesized 5’ to 3’

3’ TAC is the start triplet3’ TAC is the start triplet

This produces a complementary This produces a complementary mRNA message 5’ AUG 3’ – mRNA message 5’ AUG 3’ –

Groups of three bases in the Groups of three bases in the messenger RNA formed are messenger RNA formed are referred to as CODONSreferred to as CODONS

RNA POLYMERASE

Wobble

•There is wobble in the DNA code – This is a protection from mutations

•More than one codon can specify the same amino acid

• Note arginine - CGU, CGC,CGA, CGG all code for arginine – only the third base in the codon changes

•There are two additional codons for arginine as well AGA and AGG these reflect the degenerate nature of the code

Codon chartCodon chart

Genes for t RNAs and r Genes for t RNAs and r RNAsRNAs

The genes for t RNAs have a The genes for t RNAs have a promoter and transcribed leader promoter and transcribed leader and trailer sequence that are and trailer sequence that are removed prior to their utilization removed prior to their utilization in translation. Genes coding for in translation. Genes coding for tRNA may code for more than a tRNA may code for more than a single tRNA moleculesingle tRNA moleculeThe segments coding for r RNAs The segments coding for r RNAs are separated by spacer are separated by spacer sequencs that are removed after sequencs that are removed after transcription.transcription.

t-RNAt-RNAThe The acceptor stemacceptor stem includes the 5' and 3' includes the 5' and 3' ends of the tRNA. ends of the tRNA. The 5' end is The 5' end is generated by RNase P generated by RNase P The 3' end is the site The 3' end is the site which is charged with which is charged with amino acids for amino acids for translation. translation. Aminoacyl tRNA Aminoacyl tRNA synthetases interact synthetases interact with both the with both the acceptoracceptor 3' end and 3' end and the the anticodonanticodon when when charging tRNAs. charging tRNAs. The anticodon matches The anticodon matches the codon on mRNA the codon on mRNA and is readand is read

3’ to 5’3’ to 5’

t- RNAt- RNA

Found in the cytoplasmFound in the cytoplasmAmino acyl t- RNA synthetase Amino acyl t- RNA synthetase is an enzyme that enables the is an enzyme that enables the amino acid to attach to t-RNAamino acid to attach to t-RNAAlso activates the t- RNAAlso activates the t- RNAClover leaf has a stem for Clover leaf has a stem for attachment to the amino acid attachment to the amino acid and an anticodon on the and an anticodon on the bottom of the clover leafbottom of the clover leaf

t- RNAt- RNA

Common FeaturesCommon Featuresa CCA a CCA trinucleotide at trinucleotide at the 3' end, the 3' end, unpairedunpairedfour base-paired four base-paired stems, and stems, and One loop One loop containing a T-containing a T-pseudoU-C pseudoU-C sequence and sequence and another another containing containing dihydroU. dihydroU.

tRNAtRNAtRNAs attach to a tRNAs attach to a specific amino specific amino acid and carry it to acid and carry it to the ribosomethe ribosomeThere are 20 There are 20 amino acids amino acids 61 different 61 different codons for these codons for these amino acids and amino acids and 61 tRNAs61 tRNAsThe anticodon is The anticodon is complementary to complementary to the codonthe codonBinds to the codon Binds to the codon with hydrogen with hydrogen bondsbonds

Ribosomal genesRibosomal genes

Very similar to the structure of Very similar to the structure of protein genesprotein genes

tRNA and rRNA genestRNA and rRNA genes

The genes for rRNA are also similar to the The genes for rRNA are also similar to the organization of genes coding for proteinsorganization of genes coding for proteins

All rRNA genes are transcribed as a large All rRNA genes are transcribed as a large precursor molecule that is edited by precursor molecule that is edited by ribonucleases after transcription to yield the ribonucleases after transcription to yield the final r RNA productsfinal r RNA products

Ribosomal RNARibosomal RNA

Combines with specific Combines with specific proteins to form ribosomesproteins to form ribosomes

Serves as a site for protein Serves as a site for protein synthesissynthesis

Associated enzymes and Associated enzymes and factors control the process of factors control the process of translationtranslation

Prokaryote ribosomesProkaryote ribosomesRibosomes are small, but Ribosomes are small, but complex structures, complex structures, roughly 20 to 30 nm in roughly 20 to 30 nm in diameter, consisting of diameter, consisting of two unequally sized two unequally sized subunits, referred to as subunits, referred to as largelarge and and small small which fit which fit closely together as seen closely together as seen below.below. A subunit is composed of A subunit is composed of a complex between RNA a complex between RNA molecules and proteins; molecules and proteins; each subunit contains at each subunit contains at least one ribosomal RNA least one ribosomal RNA (rRNA) subunit and a large (rRNA) subunit and a large quantity of ribosomal quantity of ribosomal proteins. proteins. The subunits together The subunits together contain up to 82 specific contain up to 82 specific proteins assembled in a proteins assembled in a precise sequence. precise sequence.        

Type of rRNA 

Approximate

number of nucleotide

s

Subunit Location

16s 1,542 30s

5s 120 50s

23s 2,904 50s

Prokaryote ribosomal RNA

Prokaryote ribosomes – Prokaryote ribosomes – polysomes- the process of polysomes- the process of

translationtranslation

Prokaryote transcriptionProkaryote transcriptionand translationand translation

Prokaryote transcription and Prokaryote transcription and translation take place in the translation take place in the cytoplasmcytoplasm

All necessary enzymes and All necessary enzymes and molecules are present for molecules are present for the transcription and the transcription and translation to take placetranslation to take place

TranslationTranslation

A molecule of messenger RNA A molecule of messenger RNA binds to the 30S ribosomebinds to the 30S ribosome

( small ribosomal unit) at the ( small ribosomal unit) at the Shine Dalgarno sequenceShine Dalgarno sequence

This insures the correct This insures the correct orientation for the moleculeorientation for the molecule

The large ribosomal sub unit The large ribosomal sub unit locks on toplocks on top

The RibosomeThe Ribosome

There are four significant There are four significant positions on the ribosomepositions on the ribosome

EPATEPAT

When the 5’ AUG 3’ of the When the 5’ AUG 3’ of the mRNA is on the P site the t-mRNA is on the P site the t-RNA with the anticodon, RNA with the anticodon, 5’UAG3’ forms a temporary 5’UAG3’ forms a temporary bond to begin translationbond to begin translation

From Gene to From Gene to polypeptidepolypeptide

E. ColiE. Coli Gene Map Gene Map

Mutations in DNAMutations in DNA

May be characterized by their May be characterized by their genotypic or phenotypic changegenotypic or phenotypic change

Mutations can alter the Mutations can alter the phenotype of a microorganisms phenotype of a microorganisms in different waysin different ways

Mutations can involve a change Mutations can involve a change in the cellular or colonial in the cellular or colonial morphologymorphology

Types of MutationsTypes of Mutations

Conditional mutations are those mutations Conditional mutations are those mutations that are expressed only under specific that are expressed only under specific environmental conditions ( temperature)environmental conditions ( temperature)

Biochemical mutations are those that can Biochemical mutations are those that can cause a change in the biochemistry of the cause a change in the biochemistry of the cellcell

( these may inactivate a biochemical ( these may inactivate a biochemical pathway)pathway)

These mutants are referred to as auxotrophs These mutants are referred to as auxotrophs because they cannot grow on minimal because they cannot grow on minimal mediamedia

Prototrophs are usually wild type strains Prototrophs are usually wild type strains capable of growing on minimal mediacapable of growing on minimal media

Two types of Two types of mutationsmutations

Spontaneous mutations – These Spontaneous mutations – These occur without a causative agent occur without a causative agent during replicationduring replication

Induced mutations are the result of Induced mutations are the result of a substance referred to as a a substance referred to as a mutagenmutagen

Cairns reports that a mutant E. coli Cairns reports that a mutant E. coli strain unable to use lactose is able strain unable to use lactose is able to regain its ability to use the sugar to regain its ability to use the sugar again – should this be referred to as again – should this be referred to as adaptive mutation?adaptive mutation?

HypermutationHypermutation

One possible explanation is One possible explanation is hypermutationhypermutationA starving bacterium has the A starving bacterium has the ability to generate multiple ability to generate multiple mutations with special mutations with special mutator genes that enable mutator genes that enable them to form bacteria with the them to form bacteria with the ability to metabolize lactoseability to metabolize lactoseThis is an interesting theory This is an interesting theory still under investigationstill under investigation

Spontaneous Spontaneous mutationsmutations

TypesTypes

1.1. A purine substitutes for a purine or a A purine substitutes for a purine or a pyrimidine substitutes of a pyrimidine. This pyrimidine substitutes of a pyrimidine. This type of mutation is referred ta as a type of mutation is referred ta as a transition. Most of these can be repaired by transition. Most of these can be repaired by proofreading mechanismsproofreading mechanisms

2.2. A pyrimidine substituted for by a purine is A pyrimidine substituted for by a purine is referred to as a transversion. These are referred to as a transversion. These are rarer due to steric problems in the DNA rarer due to steric problems in the DNA molecule such as pairing purines with molecule such as pairing purines with purines.purines.

3.3. Insertions or deletions cause frame shifts – Insertions or deletions cause frame shifts – the code shifts over the number of bases the code shifts over the number of bases inserted or deletedinserted or deleted

Mutation TypesMutation Types

Erors in replication Erors in replication due to base due to base tautomerizationtautomerization

AT and CG pairs are AT and CG pairs are formed when keto formed when keto groups participate in groups participate in hydrogen bondshydrogen bonds

In contrast enol In contrast enol tautomers produce tautomers produce AC and GT base AC and GT base pairingpairing

Spontaneous mutations Spontaneous mutations – another cause– another cause

DepurinationDepurination

A purine nucleotide can lose its A purine nucleotide can lose its basebase

It will not base pair normallyIt will not base pair normally

It will probably lead to a transition It will probably lead to a transition type mutation after the next type mutation after the next round of replication.round of replication.

Cytosine can be deaminated to Cytosine can be deaminated to uracil which can then create a uracil which can then create a problemproblem

Frame ShiftsFrame Shifts

Additions and Additions and deletions change deletions change the reading frame.the reading frame.The hypothetical The hypothetical origin of deletions origin of deletions and insertions may and insertions may occur during occur during replicationreplicationIf the new strand If the new strand slips an insertion or slips an insertion or addition may occuraddition may occurIf the parental slips If the parental slips a deletion may a deletion may occuroccur

MutagenesisMutagenesis

Any agent that Any agent that directly damages directly damages DNA, alters its DNA, alters its chemistry, or chemistry, or interferes with interferes with repair mechanisms repair mechanisms will induce will induce mutationsmutations

a.a. Base analogsBase analogs

b.b. Specific mispairingSpecific mispairing

c.c. Intercalating Intercalating agentsagents

d.d. Ionizing radiationIonizing radiation

Base analogs are structurally similar to normal nitrogenous bases and can be incorporated into the growing polynucleotide chain during replication.

The expression of The expression of mutationsmutations

Forward mutations – a mutation Forward mutations – a mutation from the wild type to a mutant from the wild type to a mutant form is called a forward mutationform is called a forward mutationReversion-If the organism regains Reversion-If the organism regains its wild type characteristics its wild type characteristics through a second mutationthrough a second mutationBack mutation – The actual Back mutation – The actual nucleotide sequence is converted nucleotide sequence is converted back to the originalback to the originalSuppressor mutation – overcomes Suppressor mutation – overcomes the effects of the first mutationthe effects of the first mutation

More on mutationsMore on mutations

Point mutations – caused by the Point mutations – caused by the change in one DNA basechange in one DNA baseSilent mutations – mutations can occur Silent mutations – mutations can occur which cause no effect – this is due to which cause no effect – this is due to the degeneracy of the code ( more the degeneracy of the code ( more than one base coding for the same than one base coding for the same amino acid)amino acid)Missense mutation – changes a codon Missense mutation – changes a codon for one amino acid into a codon for for one amino acid into a codon for another amino acidanother amino acidNonsense – In eukaryotes the Nonsense – In eukaryotes the substitution of a stop into the substitution of a stop into the sequence of a normal genesequence of a normal gene

Detection and isolation Detection and isolation of mutantsof mutants

Requires a sensitive systemRequires a sensitive system

Mutations are rareMutations are rare

One in about every 10One in about every 1077 – 10 – 101111

Replica plating is a technique that is Replica plating is a technique that is used to detect auxotrophsused to detect auxotrophs

It distinguishes between wild type and It distinguishes between wild type and mutants because of their ability to grow mutants because of their ability to grow in the absence of a particular in the absence of a particular biosynthetic end productbiosynthetic end product

Replica plating allows plating on Replica plating allows plating on minimal media and enriched media minimal media and enriched media from the same master platefrom the same master plate

The selection of The selection of auxotorph revertantsauxotorph revertants

The lysine The lysine auxotrophs ( Lys-) auxotrophs ( Lys-) are treated with a are treated with a mutagen such as mutagen such as nitroquanidine or uv nitroquanidine or uv light to produce light to produce revertantsrevertants

Ames TestAmes Test

Developed by Bruce AmesDeveloped by Bruce Ames

Used to test for carcinogensUsed to test for carcinogens

A mutational reversion assay A mutational reversion assay based upon mutants of based upon mutants of Salmonella typhimuriumSalmonella typhimurium

DNA repair DNA repair mechanismsmechanisms

Type I -Excision repair Type I -Excision repair

Corrects damage which causes distortions in the Corrects damage which causes distortions in the double helixdouble helix

A repair endonuclease or uvr ABC A repair endonuclease or uvr ABC endonuclease removes the damaged bases endonuclease removes the damaged bases along with some bases on either side of thee along with some bases on either side of thee lesionlesion

The usual gap is about 12 nucleotides long. It The usual gap is about 12 nucleotides long. It is filled by DNA polymerase and ligase joins is filled by DNA polymerase and ligase joins the fragments. the fragments.

This can remove Thymine-Thymine dimersThis can remove Thymine-Thymine dimers

A special type of repair utilizes glycosylases to A special type of repair utilizes glycosylases to remove damaged or unnatural bases yielding remove damaged or unnatural bases yielding the results discussed abovethe results discussed above

Mutations and repairMutations and repair

Type II – Type II – Removal of lesionRemoval of lesion

Thymine dimers and alkylated bases are often Thymine dimers and alkylated bases are often repaired directlyrepaired directly

PhotoreactivationPhotoreactivation is the repair of thymine is the repair of thymine dimers by splitting them apart into separate dimers by splitting them apart into separate thymines with the aid of visible light in a thymines with the aid of visible light in a photochemical reaction catalyzed by the photochemical reaction catalyzed by the enzyme photolyaseenzyme photolyase

Light repairLight repair--phrphr gene - codes for deoxyribodipyrimidine gene - codes for deoxyribodipyrimidine photolyase photolyase that, with cofactor folic acid, binds that, with cofactor folic acid, binds in dark to T dimer. When light shines on cell, in dark to T dimer. When light shines on cell, folic acid absorbs the light and uses the folic acid absorbs the light and uses the energy to break bond of T dimer; photolyase energy to break bond of T dimer; photolyase then falls off DNAthen falls off DNA

Dark repair of Dark repair of mutationsmutations

Dark repairDark repairThree typesThree types1) UV Damage Repair (also called NER - nucleotide 1) UV Damage Repair (also called NER - nucleotide excision repair)excision repair)Excinuclease (an endonuclease; also called Excinuclease (an endonuclease; also called correndonuclease [correction endo.]) that can correndonuclease [correction endo.]) that can detect T dimer, nicks DNA strand on 5' end of dimer detect T dimer, nicks DNA strand on 5' end of dimer (composed of subunits coded by (composed of subunits coded by uvrAuvrA, , uvrBuvrB and and uvrCuvrC genes). genes). UvrA protein and ATP bind to DNA at the distortion. UvrA protein and ATP bind to DNA at the distortion. UvrB binds to the UvrA-DNA complex and increases UvrB binds to the UvrA-DNA complex and increases specificity of UvrA-ATP complex for irradiated DNA. specificity of UvrA-ATP complex for irradiated DNA. UvrC nicks DNA 8 bases upstream and 4 or 5 bases UvrC nicks DNA 8 bases upstream and 4 or 5 bases downstream of dimer. downstream of dimer. UvrD (DNA helicase II; same as DnaB used during UvrD (DNA helicase II; same as DnaB used during replication initiation) separates strands to release replication initiation) separates strands to release 12-bp segment. 12-bp segment. DNA polymerase I now fills in gap in 5'>3' direction DNA polymerase I now fills in gap in 5'>3' direction and ligase seals.and ligase seals.

The Effects of uv lightThe Effects of uv light

Post replication repairPost replication repair

If T dimer not repaired, DNA Pol III can't make If T dimer not repaired, DNA Pol III can't make complementary strand during replication. complementary strand during replication. Postdimer initiation - skips over lesion and Postdimer initiation - skips over lesion and leaves large gap (800 bases). Gap may be leaves large gap (800 bases). Gap may be repaired by enzymes in recombination system - repaired by enzymes in recombination system - lesion remains but get intact double helix. lesion remains but get intact double helix.

Successful post replication depends upon the Successful post replication depends upon the ability to recognize the old and newly ability to recognize the old and newly replicated DNA strandsreplicated DNA strands

This is possible because the newly replicated This is possible because the newly replicated DNA strand lack methyl groups on their bases, DNA strand lack methyl groups on their bases, whereas the older DNA has methyl groups on whereas the older DNA has methyl groups on the bases of both strands. the bases of both strands.

The DNA repair system cuts out the mismatch The DNA repair system cuts out the mismatch from the non- methylated strandfrom the non- methylated strand

Recombination repairRecombination repair

The DNA repair for which there is no The DNA repair for which there is no remaining template is restoredremaining template is restored

RecARecA protein cuts a piece of template DNA protein cuts a piece of template DNA from a sister molecule and puts it into the gap from a sister molecule and puts it into the gap or uses it to replace a damaged strandor uses it to replace a damaged strand

Rec ARec A also participates in a type of inducible also participates in a type of inducible repair known as repair known as SOS SOS repair. repair.

If the DNA damage is so great that synthesis If the DNA damage is so great that synthesis stops completely leaving many gaps, the Rec stops completely leaving many gaps, the Rec A will bind to the gaps and initiate strand A will bind to the gaps and initiate strand exchange.exchange.

It takes on a proteolytic funtion that destroys It takes on a proteolytic funtion that destroys the lexA repressor protein which regulates the lexA repressor protein which regulates genes involved in DNA repair and synthesis genes involved in DNA repair and synthesis