Chapter 25 DNA metabolism

Problems: 3, 5, 10, 11, 13

25.0 IntroductionA. DNA metabolism includes:

Process that try to reproduce the informationreplication (faithful reproduction) - which must be incrediblyaccurate

Processes that try to preserve the current informationRepair and recombination

Processes to degrade DNA

Emphasis in this chapter is on the enzymes that perform these functions

Much of these discoveries were first found in E-coli

Figure 25-1 gives you a feel for how many enzymes we can potentiallystudy in even a simple organism like E coli

B. Terminologylook at 25-1 againby convention bacterial genes named using 3 lowercase, italicized letters

letters generally reflect apparent functionif several genes affect same process, then add A, B, ...

A, B, reflect order of discovery, not position in a pathwaysometimes have already isolated the protein corresponding to a gene socan refer to using either protein name or the gene name. Sometimeshaven’t isolated the protein yet, so continue to call by the gene name

to differentiate between the gene and the gene product Remove the italics and capitalize the first letter of the abbreviation

dnaA is the gene, DnaA, is the protein produced by thegene

Similar system used in eukaryotes, although not as systematically, so canget confusing

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25.05 DNA DegradationThis book talks about some of the DNA degrading enzymes (page 979) in thesection on replication. DNA degradation is a necessary part of several enzymesin this section, so I have pulled this part out and put it here so we known what weare talking about when we hit DNA degrading enzyme activities later in thischapter. A. DNA degraded by nucleases

Enzymes that degrade DNA called DNA nucleases or Dnases

Are specific for DNA not RNA

Two major classesExonucleases nibble in from end

May be 5' or 3' but not both

Endonucleases start somewhere in the middleEndonuclease that attack specific sequences are calledrestriction enzymes

A few endo and exo’s only work on single stranded DNA

Interestingly enough will see nuclease activity as a necessary and integralpart of many DNA synthesizing enzymes!

25.1 DNA ReplicationA. DNA replication governed by a set of fundamental rules

I. DNA replication is semi-conservativeEach strand of DNA is used to make new DNA so new DNAcontains one old strand and one new strand

This was one hypothesis of Watson Crick (1953) Proved 4 years later by Meselson and Stahl (1957)

Made heavy DNA using N15

Could then see one heavy strand passed on to offspringFigure 25-2

II. DNA replication begins at an origin and usually proceeds bidirectionallyFigure 25-3done by placing radioactive DNA on a photographic plate

Could see extra loop of replicated DNABy doing with a different DNA that had added denatured regions

Could observe that always used same origin and that wasbidirectional

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III. DNA Synthesis proceeds in 5'63' direction and is semi-discontinuous(Semidiscontinuous -means continuous on one strand,discontinuous on the other)

Nor only bidirectional, btu on both strandsAnd a bit amazing if your thinks of structure of NTP’s that can onlyadd to 3' end!

means are always attaching new nucleotide to free 3' of strandgo back to figure 8-7 to remind you what 3' and 5' means

Synthesis on 3' end makes sense - bringing in PPP-bases phosphorylated on 5'end so take 2 P ‘s of the 5' end as you attachand this gives you E and gets attached ONLY at the 3' end

Can’t get to work in any other orientation

If adding DNA in 5' 63' direction, then the template is being reading3'65' direction

If synthesis only in 1 direction how do your get replication forks andbubble growing on BOTH strands??

Figured out 1960's Okazaki

Figure 25-4

1 strand done continuously (called leading strand)Other strand goes in small pieces (called lagging strand)

Short pieces of DNA on lagging strand called Okazakifragments

DNA degraded by nucleases - this section was moved to 25.05

B. DNA synthesized by DNA polymerases1 polymerase isolated was by Kornberg in 1955 form E colist

called DNA polymerase I (E coli contains at least 4 other polymerases)

Single polypeptide MW 103,000

Will see in a bit, is not ‘THE’ polymerase, simply first one discovered

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Mechanism is common to all polymerasesFigure 25-53' OH on 3' end of DNA does a nuclephilic attack on áP of an nTPReleases PPi

Overall E should be about in equilibMade one PO bond, broke one PO bondAlso get some E from base stacking of new base in DNABut get major push (~19 kJ) from PPi 62Pi

Reaction requires a template DNAThat is obvious now, but when discovered that was the first time atemplate had ever been used in biologyRemember this is isolated 1955, two years after Watson CrickModel (1953), but 2 years before Messelson Stal (1957)1955 would be frist description of isolation, details we just looked atwould take years to come out!

Reaction requires a primer (a base already starting the new strand thatyou can attach to. Need someplace to start can - only add to a pre-existing stand)

3' end of primer called Primer Terminus

Will need to get a special enzyme to make primers (later)

Polymerases have varying degrees of processivityMay add a single base, fall off DNA then have to find it again, ormay stay attached to DNA was it adds thousands of bases. Thisvaries from enzyme to enzyme

C. Replication is very accurateE coli 1 mistake in 10 ro 10 nucleotides9 10

E coli chromosome 4.6x10 bp so makes a mistake once every 1000-6

10,000 replications

How do we achieve this accuracy?

Specificity not just in correct base pair, but in correct base pair geometryand P-P position

See figure 25-6Shows native base pairs and then several incorrect base pair thatcan occur.See how setting “box’ size and P position can rule out all incorrectbase pairs?

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Incorrect base pairs will not fit in active site

Specificity of active site not perfect, should still get errors once every 10 -4

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Most polymerase also have proofreading activityA 3'-5' exonuclease that can remove incorrect basesUsually if incorporate a bad base, the enzyme is slowed down(inhibited) so next base is added slowly. This added time givesexonuclease a chance to remove the bad base

PpiNot simply reverse of forward reaction, since can’t get backCan assay two polymerase and nuclease acivities separatelyCan have separate sites on the same enzymeHave 2 binding events so complimentary each other

And multiply selectivity togetherSay each binding is only selective to 1/1001/100 X 1/100 = 1/10,000 so greatly increase selectivity witha second binding event

Proofreading improves fidelity another 10 -102 3

Accuracy of E coli replication higher still

Has a mismatch repair mechanism that is applied to DNA after it issynthesized (will study later in chapter)

D. E coli has at least 5 polymerasesDNA polymerase I accounts for 90% of activity in E coli

But early evidence said wasn’t ‘the’ enzymes1. About 100 x to slow to keep up with replication forkmeasurements2. low processivity (falls off often, probably why so slow)3. Many other gene product known to be needed for replication4. 1969 discovered an E coli strain with nonfunctional DNA pol Ithat was viable

early 1970's discovered DNA pol II and DNA pol III (15-20 years later!)Pol II is a repair enzymePol III seems to be the principle replication enzymeProperties compared table 25-1

Pol IV and V identified 1999, seem to be involved in DNA repair

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Returning to Pol IThought to perform clean-up work in replication, recombination andrepairHas a 5'63' exonuclease

In addition to 3'65' proof reading nucleaseLocated on a separate domainThis activity allow it to remove or replace a segment of DNA

(or RNA it’s not fussy)In a process called nick translationFigure 25-9Most polymerases don’t have this activity

Pol I minus 5'63' nuclease domain called large or Klenow FragmentCan still polymerize and do proofreading

Pol IIILarger and more complex than pol I10 different subunits (table 25-2)

á subunit polymerizeså subunit proofreadsSeveral other units. Will come back for details when discusshow it works

E. DNA Replication requires many enzymes and protein factorsBesides the complicated DNA polymerase will need 20 more enzymesand proteinsentire complex called DNA replicase system or replisome

Won’t go over all details here, just the salient points

To replicate DNA need way to separate strands (unwind from each other)Need a helicase uses ATP energy to separate two strand of DNAfrom each other in a short region

Once have separate strand they want to fold back together, so needDNA-Binding Protein to stabilize separate strands

As you unwind, this puts in topological stressNeed topoisomerase to relieve this stress

Have already seen that DNA polymerases need a primer soPrimases synthesize short segments of RNA that polymerase thenextends

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RNA primers need to be removed. This is where DNA Pol I is thought tocome in

But doesn’t seal the nick so needDNA ligases to seal final gaps

All of the above must be coordinated and regulated

F. Replication of E coli chromosome proceeds in stagesinitiationelongationtermination

Different reactions and enzymes for each stage

I. InitiationOrigin of replication on DNA

Called oriC 245 bp of DNA with a sequence that is highly conserved among allbacteriaStructure indicated in figure 25-11

Key features on DNAR sites

5 repeats of 9 bpBinding site for key initiation protein DnaA

Region rich in AT pairsCalled DNA unwinding element (DUE)

I sitesAdditional binding sites for DnaA

IHF (Integration host factor) binding siteFIS (factor for inversion stimulation) binding siteLast two used in certain recombination events - Will

study later in chapter)

Process involves at least 10 different proteins (table 25-3)Open DNA at originEstablish pre-priming complex

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DnaA is key protein (figure 25-12)Is a AAA+ ATPase family

AAA+ stands for “ATPase associated with diverse cellularactivities”Typical AAA+ activity

form oligomershydrolyze ATP slowlySlow hydrolysis is switch between two statesFor DnaA

ATP bound for is activeHydrolyzed,-ADP bound form is inactive

Eight DnaA proteins (all with ATP bound) assemble to form helicalcomplex in oriC (figure 25-12)

This binding event uses both R and I sitesDnaA binds to R site in both ATP and ADP formsDnaA binds to I site only when ATP bound

Tight right hand wrap of DNA around structureMake + supercoilIn turn opens up AT rich DUE region

Several other DNA binding proteins join inHU (histone like protein binds non specificallyIHF and FIS at their specific sitesAlso serve to bend DNA

DnaC protein (another AAA+ ATPase) loads DnaB onto separatedDNA strands

A hexamer of DnaC (with ATP bound)Forms a tight complex with hexameric ring of DnaB

This opens up the hexameric DnaB ringNow interacts with DnaA2 rings of DnaB are loaded onto DNA in DUE region

1 ring on each strand of DNADnaC completes its slow hydrolysis of ATP

And this signals it to fall off complex

Loading of DnaB onto DNA is key eventDnaB is a helicaseMigrates along DNA in 5'63' directionUnwinds DNA as it goes

Each DnaB complex Is the start of a replication forkAll other proteins in replication complex will be linked to DnaB

ô subunit of DNA pol III binds to DnaB

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As strands are separatedMany molecules of SSB (Single strand binding protein) bind andstabilize separated strandsDNA Gyrase (DNA topoisomerase II)

Relieves unwinding stress

This is only phase of DNA replication that is regulatedWill only occur once each cell cycleRegulation mech not entirely clear yet, but here is what we know

End of initiation occurs when DNA pol III is loaded on DNAHda, another AAA+ ATPase

With bound ATP, binds to â subunit of DNA pol III atthis timeAlso binds to DnaA

Binding to DnaA make DnaA start its hydrolysisof ATP, and this makes DnaA complex fallapartBinding of Fresh ATP 20-40 minutes later ispart of signal for next round of replication

Other part of signal comes from DNA methylationEcoli DNA methylated by Dam methylaseMethyl on N of A in sequence GATC6

Chance of finding this sequence in 1 in 256 bpBut there are 11 GATC’s in 245 bp of orisequence

Since methyl group is added by Dam methylase, afterDNA is replicated, Newly synthesized DNA isHemimethylated, because only the old strand of DNAhas the methyl groups

After initiation the hemimethylated oriC sequence isbound by SeqA protein and sequestered in plasmamembrane (we don’t know how) After a time SeqAfalls off and it is released from membrane.

Now it must be methylated by Dam methylase beforeDnaA will bind again

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II. ElongationAll done on Pol III so lets look at the structural details of Pol III nowFigure 25-10, table 25-2

Assembled on siteá & å associate with è to form a core

á is polymerizing subunitå is proofreading subunit

Can polymerize but limited processivity (falls off DNAfast)2 cores associate with clamp loading complex

Called ã complex

2ô ãää’Add in ÷ and øAnd you have DNA polymerase III*

This has better processivity, but still not good enoughNow add 4â subunits that can encircle DNAAnd form complete DNA Pol IIICan’t fall off so very good processivity

Elongation process Figure 25-13DNA unwound by helicasesTopological stress relieved by topoisoerasesSingle strand DNA stabilized by SSB (single strand bindingprotein)

Different enzymes for leading and lagging strandsLeading strand

DnaG Primase synthesizes 10-60 nucleotides of RNA on theDNA template

Does this in conjunction with DnaB helicase that is onLagging strand!

Then DNA polymerase III takes over and start adding DNAProceeds down the replication fork as it open up the DNA

Lagging standDnaG Primase does its thingDNA polymerase III takes over to make DNAExtends until hits next primer

Seems pretty simple until realize that are doing BOTH AT ONCE INA SINGLE POLIII ENZYME COMPLEX

Accomplished by looping DNA as shown in figure 25-14DNA helices unwinding DNAPrimase occasionally binds to helices and initiates a primeron lagging strand

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DnaG Primase dissociates and DNA/RNA â-clamp is loadedonto DNA/RNA complex

When previous Okazaki fragment hits RNA of fragmentbefore it

Its clamp is discarded from coreNew clamp is added to coreNext fragment is polymerized

Clamp-loading complex consists of

2ô ãää’, and is another AAA+ ATPaseBinding of 3 ATP’s to complex opens up clamp soDNA can get inHydrolysis of ATP to ADP seals DNA into clamp

Rapid process about 1000 bp added to each strand /second

After RNA clear complex DNA PolI binds, edits out the RNAThen nick sealed by DNA ligase (25-16)

Summary of replisome proteins table 25-4

Ligase reaction shown figure 25-17Enzyme activated by attaching AMP

Viruses and eukaryotes use ATP as sourceBacteria use NAD as a source+

AMP transferred to 5'P of nick to reactivate that P3'OH can attack to seal nickAMP released

III. TerminationEventually 2 replicating forks meetNot a random eventMeet at a sequence called Ter

Multiple copies of a 20 bp sequenceTer sequence acts as binding site for protein Tus

(terminus utilization substance)Ter-Tus complex will halt a replication fork from onedirection but not the other

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Ordinarily replication forks stop when they meet, but this seems tobe a way to insure that both meet at the same place at the sametime

One fork halts when meets first complexOther fork stops when it meets the stalled forkDNA between complexes (a few hunderd bp) replicated(mechanism unknown)

Get two DNA molecules but are twisted around each otherCalled catenanes Figure 25-19

Separated by topoisomerase IV (a type II isomerase- ie breaksboth strand at once

Two molecules segregated into two daughter cells

G. Replication in Eukaryotic cells more complicatedEukaryotic DNA lots largerorganized into chromatin

So will be different

But essential steps seem to be the same

Origins - called autonomously replicating sequences (ARS) or replicatorsIdentified and studied in yeast150 bp several conserved sequences400 replicators in 16 chromosomes in haploid yeast~ 25/chromosome~Origins spaced out about 30,000-300,000 bp apartDoes replicate bidirectionally

RegulationCyclins and cyclin dependent kinases (CDK’s)

Cyclins destroyed after mitosisIn absence of cyclins, pre-replicatvie complexs form oninitiation sites, but don’t do anything

In bacteria key initiation step was loading DnaB/DnaCheterohexameric complex that was a helicase

Figure 25-20Similar complex in Eukariotes with minichromosomal

maintenence proteins (MCM) proteinsMCM2-7) for hexameric helicase like DnaB

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Loaded on DNA with hexamer origin replication complex (ORC)protein (equivalent to DnaC) also an AAA+ ATPase

Also needed are CDC6 and CDT1

Added controls - involve synthesis of cyclin CDK complexs thatbind to an phosphorylate several protein in the Pre-replicativecomplex to activate them

Replication fork moves 1/20 the speed of bacterial50 nucelotides/secIf single origin would take 500 hours to replicate genome(That’s why there are so many origins!)

Also several polymerases (á,â...)Several linked to different functionsReplication of nuclear chromosomes involved polymerase á and ä

á similar in all eukaryotic cellsHas a primase and a polymeraseNo 3'-5' exonuclease so no proofreading. Don’t think its ‘the’polymeraseThought to synthesize primers

Primers extended by ä ä associated and stimulated by PCNA (proliferating cellnuclear antigen)

PCNA heavily expressed in nuclei of replicating cells3D structure similar to â portion of Ecoli Pol IIIMake circular clamp of polymerase to stays on DNA

ä has 3'-5' exonuclease so can proofreadSeems to work on both leading and lagging strandsMay be ‘the’ nuclease

å polymerase replaces ä in DNA repairMay act to remove primers like E coli DNA pol I

Protein to that binds single stranded DNA is called RPA (replication protein A)

Clamp loader is called RFC (Replication Factor C)

Termination involved synthesis of special structures called telomeres atend of chromosomes

Will look at details next chapter(But nothing is said about termination within a chromosome)

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H. Viral DNA Polymerases provide targets for antiviral therapyMany DNA viruses encode their own DNA polymerase, so if you canspecifically inhibit this enzyme, you have killed the virus

25.2 DNA Repairif RNA or protein damaged, simply make a new copyif DNA damaged have a problemback in chapter 10 saw lots of ways DNA can be damagedHow do we repair this damage?

A. Mutations are linked to cancerdamage to DNA called a lesionif lesion leads to a change in sequence and

Bad sequence passed on to next generationnow have a mutation

MutationsSubstitution of one base for anotherInsertion of one or more new basesDeletions of one or more bases

If affect nonessential DNA or has negligible effect - called silentmutationOccasionally will offer advantage - evolution beginsOften are deleterious - damaging

B. All cells have multiple repair systemshave seen several different types of damage so several different repairmechanismsRepair mech can be extremely inefficient. Lots of ATP E is thrown awayyet want to be sure you have it right so need to do this

Repair mech relies on having two strand and assuming one is goodFiguring out the good one can e tricky

I. Mismatch repairCleanup synthesized DNA by a factor of 10 - 102 3

Assumes old strand is good and new strand is bad so need way torecognize old strand

Done in E coli by tagging old strand with methyl groupsMismatch repair involves at least 12 protein in e coli Table 25-5

Some for repair, some for strand identification

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Start with Dam methylase(DNA adenosine methylase)It has already methylated the N of all A in the sequence6

GATC on both strands(Already saw this guy as part of control of initiation)

It takes a few seconds up to a few minutes before it getsaround to methylating the new strand

During this time can tell old from newDo you need figure 25-22?

Mismatch near (within 1000 bp) a hemimethylated arearepaired using old strand as template Figure 25-23(Mismatch repair >1000 bp more difficult so not discussed)

If both strands methylated no repair occursIf neither strand methylated repair occurs but 50-50chance of getting it right

MutL and MutS proteins hydrolyze ATP to form complex atmismatched DNA (all except C-C mismatch)

Mut H bound to MutL/S complex and to a nearby GATC tomake a DNA loop

When Mut H finds a hemimethyated GATC It cleaves the DNA on the unmethylated side

Now depends on if nick is 5' or 3' from mismatchFigure 25-24

Mismatch on 5' sideUnwind and degrade DNA in 3'-5' direction untilgets to mismatchReplace with new DNANeed DNA helicase II, SSP, exoI or exoX,DNApol III, DNA ligase

Mismatch on 3' sideSame but use exoVII which can degrade either5'-3' or 3'-5'

Mismatch repair costs lots of EWill redo 1,000s of bases just to get 1 bad one

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This means costs 1000 of ATP’s

Eukaryotic cells have similar protein to Mut L and Mut SError in these genes associated with cancer-susceptibility (Box 25-1)Some details given in text, but there is still much we do not knowDon’t even know how identify old and new strand

II. Base-Excision RepairClass of enzymes that recognize common lesions

Let’s review lesion formed by spontaneous chemical reactions(Chapter 8 pages 289-291)Deamination (figure 8-30a)

C6U5mC6TA6HypoxanthineG6Xanthine

Depurination (figure 8-30b)UV dimerization (figure 8-31)DNA methylation (no figure)

Remove bad base by cutting base from sugarCleaving glycosidic linkage so called DNA Glycosylases

DNA has a apyrimidinic or apurinic siteShort called AP site

Each glycosylase specific for one type of lesion

Uracil glycosylase- removes C’s that deaminated to U’sBut will not remove U from RNABacteria a 1 U glycosylase

Humans have 4! Indicates how important it is

Another recognizes hypoxanthine (adenine deamination)3 methyl A7 methyl GPyrimidine dimersAP sites can also arise spontaneously

(Depurination)

Once AP site formed can’t simply attach a new base to the sugarNeed to replace the sugar and replace entire base

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Need AP endonuclease cleave DNAMay be either 3' or 5'

Segment of DNA removed (not just the one bad sugar)DNA replaced by DNA polymerase I and DNA ligase

Figure 25-25

III. Nucleotide-Excision RepairThe above lesions, methylations and demination, made minimaldistortions for the DNA helix so base excision was all that was needfor a first step

Lesions that cause larger distortion in DNA generally repaired byremoving entire region around a base and sugar in one step. hence the name nucleotide excision repair

Used for repair of pyrimidine/cyclobutane dimers, 6-4 photoproducts, and several other base adducts includingbenzo[á]pyrene-guanine from by exposure to cigarette smoke

In e coli. nucleotide excision repair done by a multienzyme complexcalled ABC exinuclease (figure 25-26)

Made up of UvrA (104,000) UvrB(78,000) and Uvr C(68,000)

2And A B unit scans DNA to find and bind to lesionA then dissociates and B tightly boundUvrC then bonds to BUvrB then clips 5 P 3' of lesionth

UvrC then clips 8 P 5'th

Total of 12-13 depending on size fo lesionUvrD (a helices) then removes the segmentDNA filled in with Pol ISealed with ligase

In humans and other eukaryotesSimilar actionBut requires 16 different polypeptidesNone of the peptides has any sequence similarities to E coli.enzyme

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IV. Direct RepairSome repairs can be made without removing base!Direct photoreactivations of pyrimidine dimer

Done by DNA photolyaseFigure 25-27Won’t go over mech, but in mammals required FAD andanother chromophore to help absorb light of the right E

Repair of O -methylguanine6

Common methylation site, highly mutagenicBecause G now wants to pair with T instead of C

Right margn page 999Repaired by O methyltransferase6

Pulls methyl group from G and puts on an protein’s Cys SHNot true enzyme because it suicides cannot regenerateSo used an entire protein to correct one mistakeInterestingly the dead enzyme is not simply discarded, but itacts as a signal to activate the synthesis of its own gene anda few other repair genes

1-methylA and 3-methylCThese amino groups sometimes methylated in single strand

DNAInterferes with proper base pairingIn Ecoli oxidatively removed by AlkB proteinFigure 25-29

C. More extreme damagedouble strand breaks, double strand cross-links, damage to singlestranded DNA during the replication or transcription process

All extremely harmful because there is no complementary strand to repairfrom

1 method recombinational DNA repairGo to the homologous chromosome for a copyWill study more later in chapterNote: this only works for diploid organisms ~ EukariotesUnder special circumstances can be used in haploid bacteria

Have to catch during DNA replication but before cell division

Since can’t generally use this method In E coli had a second methodcalled error-prone translesion DNA synthesis (TLS)

Much less accurate, a state of desperation repair systemTurned on when cell getting heavy UV damage or in extremecellular distressPart of the SOS response

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Some SOS response protein already expressed at low levelsfor DNA repair (UvrA & UvrB)

Under SOS,s level are boosted

Also start expressing other proteins (UmuC & UmuD)UmuD cleaved to UmuD’Makes complex with UmuC to make

DNA PolymeraseVMuch less finiky polymerase, can get aroundmany problems but error prone

Error can easily kill the cellOnly induced under extreme conditions

A few cells dieBut some survive

Will talk in more detail on SOS response in chapter 28

Also another error prone polymerase, polymerase IV

Error prone Translesion polymerases like IV and V are foundin ALL organisms

Lack proofreadingError rates 10-100x worseError rates as high a 1 in 1000!In Humans are used for some specific repair mechsAnd may only relace 1 or 2 bases at a time

25.3 DNA recombination Only works in diploid cellsrearrangement of genetic information within and among DNA moleculesthree general classes

Homologous genetic recombination (general recombination)Genetic exchanges between two DNA’s that share a large region ofnearly identical sequence, Actually sequence not important, justoverall similarity

Site specific recombinationRecombination occurs only at a specific sequence

DNA TranspositionShort segment of DNA that moves from one place to another

Functions and mechanisms are all different. Sometimes we don’t even

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know the function

In general seems to be a repair mechanism, and, as such, is integrated into DNA metabolism

A. Homologous Genetic RecombinationIn bacteria used for DNA repair hence name recombinational DNA repair

used to reconstruct DNA around a replication fork that stalled due to DNAdamage

Also used in conjugation (mating) when DNA from a donor is integratedinto recipient cell -a relatively rare event

In eukaryote generally associated with cell divisionOccurs most often during meiosis when diploid cell is dividinggenetic material into haploid sex cells (egg and sperm)

Figure 25-31

Cell starts in diploid state, 2 copies of each chromosome, one fromeach parent

Cell copies all DNA so now has 4 copies of each chromosome, 2from each parent

Cell divides If mitosis (normal cell division) a single copy of each of thepaired chromosomes is placed in the daughter cell.

In meiosis (cell division for sex) each cell gets one doubledcopy of only 1 of the paired chromosomes

Cell divided again and each cell gets a single copy of thechromosome

So have 4 cells each with a single copy of DNA of a single(not paired) chromosome

During prophase of first meiotic division have both copies of achromosome associated with a centromere holding them together(that is why the chromosomes look like X’s) at this point calledsister chromatids

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Before cell division have 2 pairs of sister chromatids, one fromeach parent

The sister chromatids from the homologous chromosomes areclosely associated

Breakage and reassociation can occur, resulting in crossingover

Where the genetic material from one chromosome crossesover and gets joined to the DNA on it’s homologous partner

Cross over points are called chiasmata (this is the plural)

Cross over points not entirely randomThere seems to be hot spotsBut for all practical purposes is randomUse to map genes

If 2 genes stay together often during crossingover then must by physically close onthe DNA

IF 2 gene often separated during crossingover, then must be far apart on the DNA

Homologous crossing over has at least 3 functions1. Contributes to repairs of some kinds of DNA damage - inparticular double strand breaks - next section2. Promotes orderly segregation of genes in meiotic process3. Enhances genetic diversityFigure 25-32

B. Recombination during meiosis is initiated at double strand breaksPossible mechanism figure 25-33See my diagram for product 2, its not obvious4 main features

1. Homologous chromosomes closely aligned (physically touching)2. Double strand break enlarged by exonucleases that nibble awaydifferent parts on two strands3. One strand invades homologous DNA, and in branch migrationDisplaces one strand and is extended to migrate the branch point4. end up with 2 interlinked DNA structures called a Hollidaystructure that can be observed with an electron microscope

As shown in figure Holliday structure can be unlinked in two ways,both are observed

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Details may be different from organism to orgnismSince the two strands involved came from different parents, theymay be the same in overall sequence, but there can be differencesin individual bases, that leads to small changes in new genome

C. Recombination requires specific EnzymesSeveral enzymes responsible in this process isolated in both prokaryote &eukaryote

`For now focus on E-coli systemNow we get into the hard to put together stuff. Many of these enzymeshave been identified genetically, this there is a series of nonsensicalnames like RecA, RuvB, etc. Some of these enzymes have beenisolated so we know what their activities are, some haven’t . Let’s see if Ican put these pieces together for you

RecBCD complex - is both nuclease and helicase, works in step 1clipping back the double stranded DNA to get some single strand stufffigure 25-35

Binds at a double strand breakUnwinds and removes BOTH strands of DNA using ATP for ERecB moves 3'65' on one strandRecD moves 5'63' on otherHits a chi sequence (GCTGGTGG)Binds tightly to RecCThen slows cutting 3' strand

Gets faster cutting 5' strand

There are about 1000 chi sequenced in E coli.Centers of recombinationSequences that promote recombination found in higher organisms

RecA active form is ordered helical filament of thousand of rec AStarts coating the single strand DNAThis coating can then be extended to the double strand DNA aswellAssembly and disassembly of recA filament controlled by

RecF, RecO, RecR,RecX and DinI proteins

RecA then mediates the pairing of the homologous DNA strand andcreation of Holliday structures Figure 25-38 with use of ATP

Exchange occurs ~ 6 bp/s and goes in 5'63' direction

Once Holiday structure formed a host of enzymes required to

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complete strand exchangeTopoisomerase, RuvAB branch migration protein, resolvase,other nucleases, DNA pol I or III, DNA ligase

Finally RuvC cleaves holiday intermediate to give unbranched, fulllength products

D. Everything comes together at stalled replication forksFigure 25-39Explained better in figure than in text itselfAll cells including E coli. have high levels of DNA damagemuch gets repaired in the double strand pathway have already studiedyet almost every replication fork in every replication will encounter anunrepaired lesion

DNA pol III cannot proceed properly through many of these lesions, sotend to leave single strand gaps or the replication fork just stall. Worseyet, If it hits a single strand break it give you a double strand break.

Under normal conditions there is an elaborate repair pathway to repair thelesions and restart replication. Virtually everything we have talked aboutin this chapter comes into play in this process

2 major paths to get things going both require recA Fig 25-39Lesion containing DNA gaps

Needs RecF, RecO, RecRDouble strand breaks

RecBCD (saw in recombination)

In both repair pathways first use recombination enzymes to getstrand transfers and recombine around the damaged parts (twopathway use different sets of enzymes)

Then need addition enzymes to process the recombinationintermediates and get back to a normal replication forkconfiguration (again different sets fo enzymes for differentpathways)Finally restart replication using a complex called replicationrestart primosome

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E. Site-specific Recombination - precise DNA rearrangementsjust looked at recombination that can occur anywhere between twohomologous strands

Now examine a different process recombination at specific sequences

Occurs in all cells May have different purposes in different cells

Regulation of expression of genes Promoting programed rearrangements during embryonic

developmentPart of life cycle of some plasmids and viruses

Each recombination system consists of an enzyme called a recombinase2 general types

Ser at active siteTyr at active site

And a DNA segment it recognizes, the recombination site usually 20-200bp

Also one or more auxiliary proteins for regulation

General pathway for Tyr type recombinase Figure 25-404 separate recombinases recognize 4 sites on DNA

(Book shows 2 sites on 2 different DNA’s, but can be 4 siteson 1 DNA)

Protein associates as a tetramer bringing 4 sites into near contactIn each pair of recombinases, 1 recombinase cleaves one strand of

DNA and get covalently bond at the cleavage site though aphospho-tyrosineThis linkage preserves energy of phosphate bond so canregenerate DNA linkage without ATP

Protein now interacts with opposite in other pair to link strands in aHolliday structure

Other half of pair now cleaves and binds and exchanges so get therecombination

In Serine type recombinase both strands of each stie are cut at the sametime and rejoined without going through Holliday structure

Can view recombinase as a site specific endonuclease and ligase.

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Unlike many of protein-DNA binding sites, the sites recognized byrecombinases are NOT symmetric. Thus the recombinase binds in aoriented manner and when sites on DNA pieces are aligned, the 2combining sites are in the same orientation

This has some interesting consequences, in the overall recombined DNAstructure

If we have a single piece of DNA with the sequence of the two sitesinvertedwhen we go through the recombination event we simply invert theintervening DNA(Figure 25-41 a)

However if we have a single piece of DNA with the sites in thesame orientationthe recombination event removes the intervening DNA and turns itinto a small circular loop!(Figure 25-41b)

If the sites are on different DNA and either one or both of the DNAs is acircular piece, then the recombination ends up inserting 1 DNA into theother

Figure 25-42

Various recombinases tend to be specific for each of these differentpathways

First recombinase system was isolated from bacteriophage ëë infects e coli.

Either replicates to produce more bacteriophage and kills hecellOr integrates into the E coli chromosome and waitsThus the recombinase allows to integrate

Or to clip out into a circle and reproduce

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F. Complete Chromosome replication can require site specific recombinationThinking back to the more general recombination method used to rescuestalled replication forks there is another problem we didn’t discuss.

When we do a cross over event on one part of the DNA, but not on theother we interconnect our two stand in what is called a dimeric genome(Figure 25-43)this interconnected DNA cannot be separated, into the two daughter cells

This is where a site specific recombinase (the XerCD system) is used toput a second recombination into the genome and separate the twostrands

G. Transposable genetic elements move from one location to anotherAnother use of recombination is in transposition - the movement oftransposable elements from one location to another

Transposons - segments of DNA found in all cells, that can hop from onelocation to another

Terminology - hop from a ‘donor’ site to a ‘target’ site

New location usually randomIf goes into a essential gene can kill

So very tightly regulated and not done too often

Transposon can be thought of as the simplest molecular parasitePassively reproduced by host cellIf caries a good gene, can be a simple symbiosis

2 classes of transposon in bacteriaInsertion sequences - simple transposons

Have the sequence required for transpositionAnd code for protein (transposases) that do the process

Complex transposonsCarry addition genesFor instance gene for antibiotic resistance thus making adrug resistant bacteria

bacterial transposons have different structures, but here is usual scenarioDNA sequence has short repeated sequences that is binding site oftranposasethese segments tend to be repeated in transposition process

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2 processes 25-451. Direct or simple

Cut at recognition sequences on both sides of transposon(Leaves behinds a double strand cut for the Repair enzymesto fix)Transposase makes a staggered cut at a new locationTransposon insertsDNA replicated to fill in gap

2. Replicative transpositionReplicate so leave copy behind at donor site

eukaryotic transposons same and differentsome involved RNA intermediates Will see next chapter

H. Immunoglobulin genes are assembled by recombinationan example of a programed developmental recombination events

Immunoglobulin your immune protein - binds antigens to fight infection

You are capable of expressing millions of different immunoglobulinsyet you only as about 100,000 immunoglobulin genes!

Use recombination event to mix and match different immunoglobulingenes together

May have evolved by early invasion of a tranposable element?

Look at immunoglobulin G (IgG)First review protein structure Figure 5-21 page 171

Now do gene structureFigure 25-46

Protein is a dimer of 2 light and 2 heavy chainsBoth chains have variable region, where sequences vary alot from one protein to the next. And a constant region,where sequence is nearly identical from one to the next

2 different families of light chains, kappa and lambda

In pictureHave a single constant DNALots of a short hypervariable DNAAnd several longer variable region

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Use recombination to mix and matchUse RNA splicing to get rid of unused DNAExpress protein

300 V segments4 J segments300x4 = 1,200 possible combosBut not nice clean recombination so 2.5 x more soabout 3000 combos

5000 C genes5000x3000 = 1.5x10 iGg’s7

Additionally high mutatiion rate in V sequences!

Each B lyphocyte cell will express only 1 IgG