Chapter 25 DNA metabolism

Transcription

1 Chapter 25 DNA metabolism 25.0 Introduction A. DNA metabolism includes: Process that try to reproduce the information replication (faithful reproduction) - which must be incredibly accurate Processes that try to preserve the current information Repair 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 potentially study in even a simple organism like E coli B. Terminology look at 25-1 again by convention bacterial genes named using 3 lowercase, italicized letters letters generally reflect apparent function if several genes affect same process, then add A, B,... A, B, reflect order of discovery, not position in a pathway sometimes have already isolated the protein corresponding to a gene so can refer to using either protein name or the gene name. Sometimes haven 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 the gene Similar system used in eukaryotes, although not as systematically, so can get confusing

2 25.05 DNA Degradation This book talks about some of the DNA degrading enzymes (page 1013) in the section on replication. DNA degradation is a necessary part of several enzymes in this section, so I have pulled this part out and put it here so we know what we are talking about when we hit DNA degrading enzyme activities later in this chapter. A. DNA degraded by nucleases Enzymes that degrade DNA called DNA nucleases or Dnases Are specific for DNA not RNA Two major classes Exonucleases nibble in from end May be 5' or 3' but not both Endonucleases start somewhere in the middle Endonuclease that attack specific sequences are called restriction enzymes A few endo and exo s only work on single stranded DNA Interestingly enough will see nuclease activity as a necessary and integral part of many DNA synthesizing enzymes! 25.1 DNA Replication A. DNA replication governed by a set of fundamental rules I. DNA replication is semi-conservative Each strand of DNA is used to make new DNA so new DNA contains 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 15 N Could then see one heavy strand passed on to offspring Figure 25-2 II. DNA replication begins at an origin and usually proceeds bidirectionally Figure 25-3 done by placing radioactive DNA on a photographic plate Could see extra loop of replicated DNA By doing with a different DNA that had added denatured regions Could observe that always used same origin and that was bidirectional 2

3 3 III. DNA Synthesis proceeds in 5' 3' direction and is semi-discontinuous (Semidiscontinuous -means continuous on one strand, discontinuous on the other) Not only bidirectional, but on both strands And a bit amazing if you think of structure of NTP s that can only add to 3' end! means are always attaching new nucleotide to free 3' of strand go 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 attach and 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' 3' direction, then the template is being reading 3' 5' direction If synthesis only in 1 direction how do your get replication forks and bubble growing on BOTH strands?? Figured out 1960's Okazaki Figure strand done continuously (called leading strand) Other strand goes in small pieces (called lagging strand) Short pieces of DNA on lagging strand called Okazaki fragments DNA degraded by nucleases - this section was moved to B. DNA synthesized by DNA polymerases st 1 polymerase isolated was by Kornberg in 1955 from E coli 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

4 4 Mechanism is common to all polymerases Figure ' OH on 3' end of DNA does a nuclephilic attack on áp of an ntp Releases PPi Overall E should be about in equilib Made one PO bond, broke one PO bond Also get some E from base stacking of new base in DNA But get major push (~19 kj) from PPi 2Pi Reaction requires a template DNA That is obvious now, but when discovered that was the first time a template had ever been used in biology Remember this is isolated 1955, two years after Watson Crick Model (1953), but 2 years before Messelson Stal (1957) 1955 would be first description of isolation, details we just looked at would take years to come out! Reaction requires a primer (a base already starting the new strand that you can attach to. Need someplace to start can - only add to a preexisting stand) 3' end of primer called Primer Terminus Primer may be DNA or RNA! Will need to get a special enzyme to make primers (later) Polymerase active site has two parts Insertion site - where incoming nucleotide is positioned Postinsertionsite - where nucleotide is located after polymerase translocates (moves 1 bp) After has added a base polymerase may either fall off, or may add another base Average number of bases added before falls off called processivity May add a single base, fall off DNA then have to find it again, or may stay attached to DNA was it adds thousands of bases. This varies from enzyme to enzyme C. Replication is very accurate 9 10 E coli 1 mistake in 10 or 10 nucleotides 6 E coli chromosome 4.6x10 bp so makes a mistake once every ,000 replications How do we achieve this accuracy?

5 5 Specificity not just in correct base pair, but in correct base pair geometry and P-P position See figure 25-6 Shows native base pairs and then several incorrect base pair that can occur. See how setting box size and P position can rule out all incorrect base pairs? Incorrect base pairs will not fit in active site Specificity of active site not perfect, should still get errors once every Most polymerase also have proofreading activity Figure 25-7 A 3'-5' exonuclease that can remove incorrect bases Usually if incorporate a bad base, the enzyme is slowed down (inhibited) so next base is added slowly. This added time gives exonuclease a chance to remove the bad base Not simply reverse of forward reaction, since can t get Ppi back Can assay two polymerase and nuclease activities separately Can have separate sites on the same enzyme Have 2 binding events so complimentary each other And multiply selectivity together Say each binding is only selective to 1/100 1/100 X 1/100 = 1/10,000 so greatly increase selectivity with a second binding event 2 3 Proofreading improves fidelity another Accuracy of E coli replication higher still Has a mismatch repair mechanism that is applied to DNA after it is synthesized (will study later in chapter) D. E coli has at least 5 polymerases DNA polymerase I accounts for 90% of activity in E coli But early evidence said wasn t the enzymes 1. About 100 x to slow to keep up with replication fork measurements 2. low processivity (falls off often, probably why so slow) 3. Many other gene product known to be needed for replication discovered an E coli strain with nonfunctional DNA pol I

6 6 that was viable early 1970's discovered DNA pol II and DNA pol III (15-20 years later!) Pol II is a repair enzyme Pol III seems to be the principle replication enzyme Properties compared table 25-1 Pol IV and V identified 1999, seem to be involved in DNA repair Returning to Pol I Thought to perform clean-up work in replication, recombination and repair Has a 5' 3' exonuclease In addition to 3' 5' proof reading nuclease Located on a separate domain This activity allow it to remove or replace a segment of DNA (or RNA it s not fussy) In a process called nick translation Figure 25-8 Most polymerases don t have this activity Pol III Pol I minus 5' 3' nuclease domain called large or Klenow Fragment Can still polymerize and do proofreading Larger and more complex than pol I 10 different subunits (table 25-2) á & å associate with è to form a core polymerase á is polymerizing subunit å is proofreading subunit Can polymerize but limited processivity (falls off DNA fast) 2 cores associate with clamp loading complex Called ã complex ô2ãää Add in and ø And you have DNA polymerase III * This has better processivity, but still not good enough Now add 4â subunits that can encircle DNA And form complete DNA Pol III Can t fall off so very good processivity

7 7 E. DNA Replication requires many enzymes and protein factors Besides the complicated DNA polymerase will need 20 more enzymes and proteins entire 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 DNA from each other in a short region Once have separate strand they want to fold back together, so need DNA-Binding Protein to stabilize separate strands As you unwind, this puts in topological stress Need topoisomerase to relieve this stress Have already seen that DNA polymerases need a primer so Primases synthesize short segments of RNA that polymerase then extends RNA primers need to be removed. This is where DNA Pol I is thought to come in But doesn t seal the nick so need DNA ligases to seal final gaps All of the above must be coordinated and regulated F. Replication of E coli chromosome proceeds in stages initiation elongation termination Different reactions and enzymes for each stage I. Initiation Origin of replication on DNA Called oric 245 bp of DNA with a sequence that is highly conserved among all bacteria Structure indicated in figure Key features on DNA

8 8 R sites 5 repeats of 9 bp Binding site for key initiation protein DnaA Region rich in AT pairs Called DNA unwinding element (DUE) I sites Additional binding sites for DnaA IHF (Integration host factor) binding site FIS (factor for inversion stimulation) binding site Last two used in certain recombination events - Will study later in chapter) Process involves at least 10 different proteins (table 25-3) Open DNA at origin Establish pre-priming complex DnaA is key protein (figure 25-11) Is a AAA+ ATPase family AAA+ stands for ATPase associated with diverse cellular activities Typical AAA+ activity form oligomers hydrolyze ATP slowly Slow hydrolysis is switch between two states For DnaA ATP bound for is active Hydrolyzed,-ADP bound form is inactive Eight DnaA proteins (all with ATP bound) assemble to form helical complex in oric (figure 25-11) This binding event uses both R and I sites DnaA binds to R site in both ATP and ADP forms DnaA binds to I site only when ATP bound Tight right hand wrap of DNA around structure Make + supercoil In turn opens up AT rich DUE region Several other DNA binding proteins join in HU (histone like protein binds non specifically IHF and FIS at their specific sites Also serve to bend DNA DnaC protein (another AAA+ ATPase) loads DnaB onto separated DNA strands A hexamer of DnaC (with ATP bound) Forms a tight complex with hexameric ring of DnaB This opens up the hexameric DnaB ring

9 9 Now interacts with DnaA 2 rings of DnaB are loaded onto DNA in DUE region 1 ring on each strand of DNA DnaC completes its slow hydrolysis of ATP And this signals it to fall off complex Loading of DnaB onto DNA is key event DnaB is a helicase Migrates along DNA in 5' 3' direction Unwinds DNA as it goes Each DnaB complex Is the start of a replication fork All other proteins in replication complex will be linked to DnaB ô subunit of DNA pol III binds to DnaB As strands are separated Many molecules of SSB (Single strand binding protein) bind and stabilize separated strands DNA Gyrase (DNA topoisomerase II) Relieves unwinding stress Initiation is only phase of DNA replication that is regulated Will only occur once each cell cycle Regulation mech not entirely clear yet, but here is what we know End of initiation occurs when DNA pol III is loaded on DNA Hda, another AAA+ ATPase With bound ATP, binds to â subunit of DNA pol III at this time Also binds to DnaA Binding to DnaA make DnaA start its hydrolysis of ATP, and this makes DnaA complex fall apart Binding of Fresh ATP minutes later is part of signal for next round of replication Other part of signal comes from DNA methylation Ecoli DNA methylated by Dam methylase 6 Methyl on N of A in sequence GATC Chance of finding this sequence in 1 in 256 bp But there are 11 GATC s in 245 bp of ori sequence Since methyl group is added by Dam methylase, after DNA is replicated, Newly synthesized DNA is Hemimethylated, because only the old strand of DNA has the methyl groups

10 After initiation the hemimethylated oric sequence is bound by SeqA protein and sequestered in plasma membrane (we don t know how) After a time SeqA falls off and it is released from membrane. Now it must be methylated by Dam methylase before DnaA will bind again 10 II. Elongation All done on Pol III so lets look at the structural details of Pol III now Figure 25-9, table 25-2 Assembled on site Elongation process Figure DNA unwound by helicases Topological stress relieved by topoisoerases Single strand DNA stabilized by SSB (single strand binding protein) Different enzymes for leading and lagging strands Leading strand DnaG Primase synthesizes nucleotides of RNA on the DNA template Does this in conjunction with DnaB helicase that is on Lagging strand! Then DNA polymerase III takes over and start adding DNA Proceeds down the replication fork as it open up the DNA Lagging stand DnaG Primase does its thing DNA polymerase III takes over to make DNA Extends until hits next primer Seems pretty simple until realize that are doing BOTH AT ONCE IN A SINGLE POLIII ENZYME COMPLEX Accomplished by looping DNA as shown in figure DNA helices unwinding DNA Primase occasionally binds to helices and initiates a primer on lagging strand DnaG Primase dissociates and DNA/RNA â-clamp is loaded onto DNA/RNA complex When previous Okazaki fragment hits RNA of fragment

11 11 before it Its clamp is discarded from core New clamp is added to core Next fragment is polymerized Clamp-loading complex consists of ô2ãää, and is another AAA+ ATPase Binding of 3 ATP s to complex opens up clamp so DNA can get in Hydrolysis of ATP to ADP seals DNA into clamp Figure Rapid process about 1000 bp added to each strand /second After RNA clear complex DNA PolI binds, edits out the RNA Then nick sealed by DNA ligase (25-15) Summary of replisome proteins table 25-4 Ligase reaction shown figure Enzyme activated by attaching AMP Viruses and eukaryotes use ATP as source + Bacteria use NAD as a source AMP transferred to 5'P of nick to reactivate that P 3'OH can attack to seal nick AMP released Now that you know the steps, watch an animation III. Termination Eventually 2 replicating forks meet Not a random event Figure Meet at a sequence called Ter Multiple copies of a 20 bp sequence Ter sequence acts as binding site for protein Tus (terminus utilization substance) Ter-Tus complex will halt a replication fork from one direction but not the other Ordinarily replication forks stop when they meet, but this seems to be a way to insure that both meet at the same place at the same time

12 12 One fork halts when meets first complex Other fork stops when it meets the stalled fork DNA between complexes (a few hunderd bp) replicated (mechanism unknown) Get two DNA molecules but are twisted around each other Called catenanes Figure Separated by topoisomerase IV (a type II isomerase- ie breaks both strand at once Two molecules segregated into two daughter cells G. Replication in Eukaryotic cells more complicated Eukaryotic DNA lots larger organized into chromatin So will be different But essential steps seem to be the same Origins AT rich sites Vary from organism to organism In Yeast called autonomously replicating sequences (ARS) or replicators 150 bp several conserved sequences 400 replicators in 16 chromosomes in haploid yeast ~ 25/chromosome ~Origins spaced out about 30, ,000 bp apart Does replicate bidirectionally Regulation Cyclins and cyclin dependent kinases (CDK s) Cyclins destroyed after mitosis In absence of cyclins, pre-replicatvie complexs form on initiation sites, but don t do anything In bacteria key initiation step was loading DnaB/DnaC heterohexameric complex that was a helicase Figure Similar complex in Eukariotes with minichromosomal maintenence proteins (MCM) proteins MCM2-7) for hexameric helicase like DnaB

13 13 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 that bind to and phosphorylate several protein in the Pre-replicative complex to activate them Replication fork moves 1/20 the speed of bacterial 50 nucelotides/sec If 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 functions Replication of nuclear chromosomes involved polymerase á and ä á similar in all eukaryotic cells Has a primase and a polymerase No 3'-5' exonuclease so no proofreading. Don t think its the polymerase Thought to synthesize primers Primers extended by ä ä associated and stimulated by PCNA (proliferating cell nuclear antigen) PCNA heavily expressed in nuclei of replicating cells 3D structure similar to â portion of Ecoli Pol III Make circular clamp of polymerase to stays on DNA ä has 3'-5' exonuclease so can proofread Seems to work on both leading and lagging strands May be the nuclease å polymerase replaces ä in DNA repair May 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 at end of chromosomes Will look at details next chapter (But nothing is said about termination within a chromosome)

14 14 H. Viral DNA Polymerases provide targets for antiviral therapy Many DNA viruses encode their own DNA polymerase, so if you can specifically inhibit this enzyme, you have killed the virus 25.2 DNA Repair if RNA or protein damaged, simply make a new copy if DNA damaged have a problem back in chapter 10 saw lots of ways DNA can be damaged How do we repair this damage? A. Mutations are linked to cancer damage to DNA called a lesion if lesion leads to a change in sequence and Bad sequence passed on to next generation now have a mutation Mutations Substitution of one base for another Insertion of one or more new bases Deletions of one or more bases If affect nonessential DNA or has negligible effect - called silent mutation Occasionally will offer advantage - evolution begins Often are deleterious - damaging In mammals - strong correlation between accumluation of mutations and cancer Lead to Ames test Add chemical to specialized bacterial strain Watch for easily detected mutations to occur Tie between bacterial mutations and cancer in humans? 90% of known carcinogens are mutagenic in Ames test So strong correlation B. All cells have multiple repair systems have seen several different types of damage so several different repair mechanisms Repair mech can be extremely inefficient. Lots of ATP E is thrown away yet want to be sure you have it right so need to do this Repair mech relies on having two strand and assuming one is good Figuring out the good one can be tricky

15 I. Mismatch repair 2 3 Cleanup synthesized DNA by a factor of Assumes old strand is good and new strand is bad so need way to recognize old strand Done in E coli by tagging old strand with methyl groups Mismatch repair involves at least 12 protein in e coli Table 25-5 Some for repair, some for strand identification Start with Dam methylase (DNA adenosine methylase) 6 It has already methylated the N of all A in the sequence 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 gets around to methylating the new strand During this time can tell old from new Do you need figure 25-21? Mismatch near (within 1000 bp) a hemimethylated area repaired using old strand as template Figure (Mismatch repair >1000 bp more difficult so not discussed) If both strands methylated no repair occurs If neither strand methylated repair occurs but chance of getting it right MutL and MutS proteins hydrolyze ATP to form complex at mismatched DNA (all except C-C mismatch) Mut H bound to MutL/S complex and to a nearby GATC to make 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 mismatch Figure Mismatch on 5' side Unwind and degrade DNA in 3'-5' direction until gets to mismatch Replace with new DNA

16 16 Need DNA helicase II, SSB, exoi or exox, DNApol III, DNA ligase Mismatch on 3' side Same but use exovii which can degrade either 5'-3' or 3'-5' Mismatch repair costs lots of E Will redo 1,000s of bases just to get 1 bad one This means costs 1000 of ATP s Eukaryotic cells have similar protein to Mut L and Mut S Error in these genes associated with cancer-susceptibility (Box 25-1) Some details given in text, but there is still much we do not know Don t even know how identify old and new strand II. Base-Excision Repair Class of enzymes that recognize common lesions Let s review lesion formed by spontaneous chemical reactions (Chapter 8 pages ) Deamination (figure 8-30a) C U 5mC T A Hypoxanthine G Xanthine Depurination (figure 8-30b) UV dimerization (figure 8-31) DNA methylation (no figure) Remove bad base by cutting base from sugar Cleaving glycosidic linkage so called DNA Glycosylases DNA has a apyrimidinic or apurinic site Short called AP site Each glycosylase specific for one type of lesion Uracil glycosylase- removes C s that deaminated to U s But will not remove U from RNA Bacteria a 1 U glycosylase Humans have 4! Indicates how important it is

17 17 Another recognizes hypoxanthine (adenine deamination) 3 methyl A 7 methyl G Pyrimidine dimers AP sites can also arise spontaneously (Depurination) Once AP site formed can t simply attach a new base to the sugar Need to replace the sugar and replace entire base Need AP endonuclease cleave DNA May 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 III. Nucleotide-Excision Repair The above lesions, methylations and demination, made minimal distortions for the DNA helix so base excision was all that was need for a first step Lesions that cause larger distortion in DNA generally repaired by removing 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 photo products, and several other base adducts including benzo[á]pyrene-guanine from by exposure to cigarette smoke In e coli. nucleotide excision repair done by a multienzyme complex called ABC exinuclease (figure 25-25) Made up of UvrA (104,000) UvrB(78,000) and Uvr C(68,000) And A2B unit scans DNA to find and bind to lesion A then dissociates and B tightly bound UvrC then bonds to B th UvrB then clips 5 P 3' of lesion th UvrC then clips 8 P 5' Total of depending on size fo lesion UvrD (a helices) then removes the segment DNA filled in with Pol I Sealed with ligase

18 In humans and other eukaryotes Similar action But requires 16 different polypeptides None of the peptides has any sequence similarities to E coli. enzyme 18 IV. Direct Repair Some repairs can be made without removing base! Direct photoreactivations of pyrimidine dimer Done by DNA photolyase Figure Won t go over mech, but in mammals required FAD and another chromophore to help absorb light of the right E 6 Repair of O -methylguanine Common methylation site, highly mutagenic Because G now wants to pair with T instead of C Right margin page Repaired by O methyltransferase Pulls methyl group from G and puts on an protein s Cys SH Not true enzyme because it suicides cannot regenerate So used an entire protein to correct one mistake Interestingly the dead enzyme is not simply discarded, but it acts as a signal to activate the synthesis of its own gene and a few other repair genes 1-methylA and 3-methylC These amino groups sometimes methylated in single strand DNA Interferes with proper base pairing In Ecoli oxidatively removed by AlkB protein Figure C. More extreme damage double strand breaks, double strand cross-links, damage to single stranded DNA during the replication or transcription process All extremely harmful because there is no complementary strand to repair from 1 method recombinational DNA repair Go to the homologous chromosome for a copy Will study more later in chapter Note: this only works for diploid organisms ~ Eukariotes

19 Under 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 method called error-prone translesion DNA synthesis (TLS) Much less accurate, a state of desperation repair system Turned on when cell getting heavy UV damage or in extreme cellular distress Part of the SOS response Some SOS response protein already expressed at low levels for 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 PolymeraseV Much less finiky polymerase, can get around many problems but error prone Error can easily kill the cell Only induced under extreme conditions A few cells die But 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 found in ALL organisms Lack proofreading Error rates x worse Error rates as high a 1 in 1000! In Humans are used for some specific repair mechs And may only replace 1 or 2 bases at a time 19

20 25.3 DNA recombination Only works in diploid cells rearrangement of genetic information within and among DNA molecules three general classes Homologous genetic recombination (general recombination) Genetic exchanges between two DNA s that share a large region of nearly identical sequence, Actually sequence not important, just overall similarity Site specific recombination Recombination occurs only at a specific sequence DNA Transposition Short segment of DNA that moves from one place to another Functions and mechanisms are all different. Sometimes we don t even know the function In general seems to be a repair mechanism, and, as such, is integrated in to DNA metabolism A. Bacterial Homologous Genetic Recombination - a repair mechanism In bacteria used for DNA repair hence name recombinational DNA repair used to reconstruct DNA around a replication fork that stalled due to DNA damage Also used in conjugation (mating) when DNA from a donor is integrated into recipient cell -a relatively rare event Figure Replication fork hits a DNA nick or extensive DNA damage and has to stop because can extend damaged strand First degrades 5' end of short (damaged) strand 20

21 A recombinase binds to exposed 3' end of short strand to invade the long (undamaged) double strand When gets to complement in other strand have created a branched DNA structure This branch point can then move forward or backward in a process called branch migration. These crossover structures are called Holliday intermediates Holliday structure then resolved with a special class of nuclease, and the replication fork is reconstructed Details RecBCD complex - is both nuclease and helicase, works in step 1 clipping back the double stranded DNA to get some single strand stuff figure Binds at a double strand break Unwinds and removes BOTH strands of DNA using ATP for E RecB moves 3' 5' on one strand RecD moves 5' 3' on other Hits a chi sequence (GCTGGTGG) Binds tightly to RecC Then slows cutting 3' strand Gets faster cutting 5' strand There are about 1000 chi sequenced in E coli. Centers of recombination Sequences that promote recombination found in higher organisms 21 Recombinase is the Rec A protein RecA active form is ordered helical filament of thousand of rec A Figure Starts coating the single strand DNA Not simple process, since single stranded DNA is coated with SSB The Rec BCD comples actually nucleates and starts the RecA filament growing, and many other proteins are involved. In growth and strand invasion. Branch migration promoted by RuvAB Figure 25-33a Cleaved by specialized nuclease called RuvC 25-33b Nicks are sealed with ligase,

22 22 Replication fork is restarted in a process called Origin-independent restart of replication Protein PriA, PriB, PriC, and DnaT act with DnaC to load DnaB helicase onto reconstructed replication fork. Primase synthesizes the primers and DNA polymerase reassembles on DnaB to restart DNA synthesis Complex of PriA,PriB, PriC DnaT DnaB and DnaC called replication restart primosome Restart also requires DNA pol II, but we don t know why B. Eukaryotic Recombination is required for proper Chromosome Segregation during Meisos Several roles for recomination in Eukaryotes Occurs with highest frequency during meiosis Meiosis Diploid germ cell haploid gametes Figure Diploid cell replicates DNA Get 4 copies of 2 pairs of sister chromatids Cell divides and two pairs of sister chromatids are separated Cell divides again and each gamete cell gets a single copy of each DNA Now remember our cohesins that we saw in previous chapter that provide physical links to guide chromosome segregation? They aren t around during the first meiotic division, so it is relatively easy for the chromosomes to get tangled, and for recombination to occur. This is called crossing over, and genetic material is exchanged between pairs of sister chromatids. Increases genetic diversity Crossing over is largely random, but here are some hot spots If assume total random then can use to calculate distance between genes Used to map genes If 2 genes stay together often during crossing over then must by physically close on the DNA

23 If 2 gene often separated during crossing over, then must be far apart on the DNA Can identify 3 functions for homologous recombination in Eukaryotes 1. Repair of several types of damage 2. Provides transient physical link between chromatids and st promotes orderly segregation in 1 meiotic division 3. Enhances genetic diversity C. Recombination during Meiosis is initiated at double strand breaks Possible mechanism figure 25-35a See my diagram for product 2, its not obvious 4 main features 1. Homologous chromosomes closely aligned (physically touching) 2. Double strand break enlarged by exonucleases that nibble away different parts on two strands 3. One strand invades homologous DNA, and in branch migration Displaces one strand and is extended to migrate the branch point 4. end up with 2 interlinked DNA structures called a Holliday structure that can be observed with an electron microscope Very similar to bacterial process This is called double-strand break repair model Details vary from species to species As shown in figure Holliday structure can be unlinked in two ways, both are observed 1. Flanking DNA not recombined 2. Flanking DNA recombined Since the two strands involved came from different parents, they may be the same in overall sequence, but there can be differences in individual bases, that leads to small changes in new genome D. Site-specific Recombination - precise DNA rearrangements just looked at recombination that can occur anywhere between two homologous 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 23

24 Promoting programed rearrangements during embryonic development Part of life cycle of some plasmids and viruses Each recombination system consists of an enzyme called a recombinase 2 general types Ser at active site Tyr at active site And a DNA segment it recognizes, the recombination site usually bp Also one or more auxiliary proteins for regulation General pathway for Tyr type recombinase Figure separate recombinases recognize 4 sites on DNA (Book shows 2 sites on 2 different DNA s, but can be 4 sites on 1 DNA) Protein associates as a tetramer bringing 4 sites into near contact In each pair of recombinases, 1 recombinase cleaves one strand of DNA and get covalently bond at the cleavage site though a phospho-tyrosine This linkage preserves energy of phosphate bond so can regenerate DNA linkage without ATP Protein now interacts with opposite in other pair to link strands in a Holliday structure Other half of pair now cleaves and binds and exchanges so get the recombination In Serine type recombinase both strands of each site are cut at the same time and rejoined without going through Holliday structure Can view recombinase as a site specific endonuclease and ligase. Unlike many of protein-dna binding sites, the sites recognized by recombinases are NOT symmetric. Thus the recombinase binds in a oriented manner and when sites on DNA pieces are aligned, the 2 combining sites are in the same orientation This has some interesting consequences, in the overall recombined DNA structure If we have a single piece of DNA with the sequence of the two sites inverted when we go through the recombination event we simply invert the intervening DNA 24

25 25 (Figure a) However if we have a single piece of DNA with the sites in the same orientation the recombination event removes the intervening DNA and turns it into a small circular loop! (Figure 25-38b) If the sites are on different DNA and either one or both of the DNAs is a circular piece, then the recombination ends up inserting 1 DNA into the other. We will explore this more in a minute. Various recombinases tend to be specific for each of these different pathways Site specific recombination is also used in e. coli in one additional step that sometimes has to be done after recombinational repair. Look at Figure Depending how you resolve the Holliday structure in recombination repair you either get a normal chromosome, or you get a dimeric genome. The dimeric chromosome cannot be segregated into two daughter cells, so it becomes a trap for the cell. To resolve this a specific recombination is performed by the XerCD system using a mechanism like Figure 25-38b E. Transposable genetic elements move from one location to another Another use of recombination is in transposition - the movement of transposable elements from one location to another Transposons - segments of DNA found in all cells, that can hop from one location to another Terminology - hop from a donor site to a target site New location usually random If 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 parasite Passively reproduced by host cell If caries a good gene, can be a simple symbiosis

26 26 2 classes of transposon in bacteria Insertion sequences - simple transposons Have the sequence required for transposition And code for protein (transposases) that do the process Complex transposons Carry addition genes For instance gene for antibiotic resistance thus making a drug resistant bacteria bacterial transposons have different structures, but here is usual scenario DNA sequence has short repeated sequences that is binding site of tranposase these segments tend to be repeated in transposition process Figure processes Figure Direct or simple Cut at recognition sequences on both sides of transposon (Leaves behinds a double strand cut for the Repair enzymes to fix) Transposase makes a staggered cut at a new location Transposon inserts DNA replicated to fill in gap 2. Replicative transposition Replicate so leave copy behind at donor site eukaryotic transposons same and different some involved RNA intermediates Will see next chapter H. Immunoglobulin genes are assembled by recombination an example of a programed developmental recombination events Immunoglobulin your immune protein - binds antigens to fight infection You are capable of expressing millions of different immunoglobulins yet you only as about ~29,000 immunoglobulin genes! Use recombination event to mix and match different immunoglobulin genes together May have evolved by early invasion of a tranposable element? Look at immunoglobulin G (IgG) First review protein structure Figure 5-21 page 176

27 27 Now do gene structure Figure this is just kappa light gene Protein is a dimer of 2 light and 2 heavy chains Both chains have variable region, where sequences vary a lot 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 picture Have a single constant DNA Lots of a short hypervariable DNA And several longer variable region Use recombination to mix and match Use RNA splicing to get rid of unused DNA Express protein 300 V segments (95 AA s) 4 J segments (12 AA s) 300x4 = 1,200 possible combos But not nice clean recombination so 2.5 x more so about 3000 combos 1 C genes 5000x1 = 5000 kappa light gene products Combining VJ to C done by RNA splicing (Next chapter) rather than recombination Combining V to J done by recombination sites (RSS- Recombination signal sequences) just after V and just before J Figure RAG1 and RAG2 (Recombination Activating Gene) perform double strand breaks between RSS sites then a second complex joins the DNA together Genes for heavy chains undergo similar processes Get about 5000 products for heavy chains x3000 = 1.5x10 complete IgG s

28 28 Additionally high mutation rate in V sequences! Each B lyphocyte cell will express only 1 IgG Is this a left-over transposon? Mech for double strand break by RAG1 & RAG2 does resemble several steps in transposition Deleted DNA with RSS sites has structure like a transposon It the test tube RAG1 and RAG2 can inset this DNA like a transposon into random places in DNA