MCB : Homologous Recombination

Transcription

1 MC : Homologous Recomintion Prt I. Definitions Chnges in DN re clled muttions. Muttions cn e chnges in one se, in severl ses, in mny ses. Recomintion is lso chnge is DN. How is it different from muttion? Def. #1 (simple): Recomintion is chnge in DN tht is not point muttion. Severl exmples: Point muttions: chnge or deletion or ddition of nucleotide. Recomintion: chnge or deletion or ddition of severl nucleotides. Def. #2 (simplistic): Recomintion is DN rerrngement (chnge in the order of elements). There re four sic types of enzymtic mechnisms for DN rerrngements: Type Frequency Requirements Ctlyzed y 1. Illegitimte extremely low Micro- or no vrious DN-processing homology enzymes 2. Trnsposition very low Ends of the jumping Trnsposse (regulted) element 3. Homologous low Extensive homology Rec / Rd / Rd51 (when DN dmge is low) 4. Site-specific high pir of short sites Site-specific recominse (integrse, invertse) Tody we will concentrte on Homologous Recomintion (HR). Homology is term for sequence identity. When two DNs hve exctly the sme sequence, we cll them homologous DN. We cn strt with defining HR s exchnge etween two homologous chromosomes (long DN molecules). How do we detect HR in live orgnisms? We need to ring two homologous chromosomes, residing initilly in different orgnisms, together in single orgnism. We lso need wy to seprte the two chromosomes into individul orgnisms, to detect the exchnge etween the chromosomes. Finlly, to distinguish the two chromosomes from one nother, we need to score some trits, ssocited with these chromosomes, on the orgnizml level, we need some phenotypes. In short, we need chromosomes crrying scorle lleles of the sme genes. These scorle lleles re clled mrkers. How mny mrkers do we need? One is not enough, we need t lest two: Do chromosomes need to e lmost completely homologous over their entire length for this exchnge? No, limited region of homology is enough: The miniml length of this limited region of homology fluctutes from one orgnism to nother nd from one recomintion system to nother. For the purpose of our discussion, it is enough to sy tht Rec-dependent recomintion, lthough quite low, is lredy detectle etween 50 se pir-long identicl sequences. For comprison, the E. coli genome is ~10 5 times longer, so, in theory, it cn undergo tht mny exchnges. Z Y Z Y

2 New Def. of HR: Exchnge etween two DN sequences in the region of homology. Now, trick question: if we flip one of the chromosomes 180, cn we still perform the exchnge? fter ll, this would e still n exchnge in the region of homology : Z ctully, such n exchnge is impossile, ecuse DN sequence hs polrity (unless it is n inverted plindrome), nd 180 flip produces different sequence: Therefore, we should drw: Y? Z Y 5 GGTG 3 3 TCCCT 5 FLIP: 5 TCCCT 3 3 GTGG 5 Z Y Z Y Z Y X Therefore: The finl Def. of HR: Exchnge etween two DN sequences in the region of ligned homology. Prt II. ppliction Now we cn continue with other properties of HR. Property #1: HR is n infrequent event. If we cross x chromosomes in cteri, we get out mostly prentl chromosomes nd only few recominnts ( nd ). Property #2: The proility tht n exchnge would hppen etween ny two se pirs in DN is minuscule, ut not zero. When there is significnt length of homology etween the two mrkers on the chromosomes, ll these tiny proilities re multiplied thousnds nd millions of times to result in detectle recomintion. Therefore, the frequency of HR etween the two mrkers is proportionl to (in fct, it is complex function of) the physicl distnce etween the two mrkers. Properties #1 nd #2 comined llow one to mp mrkers long the chromosomes, mesuring the frequency of their

3 segregtion from ech other. For exmple, if in the cross 1 1 c 1 x 2 2 c 2 the frequency of segregtion of from is 10%, from c is 5%, wheres from c is 13%, nd if we know tht the chromosomes in this orgnism re liner, we rrive t the gene order: - 10%- -5%-c (the lgorithm with plcing the second mrker on oth sides first). You should e fmilir with ll this from the eukryotic genetics. There is one helpful trit of the eukryotic life cycle tht mkes explining HR esy: it is clled syngmy. Syngmy is life stge when two entire genome complements re rought together in single nucleus of zygote in preprtion for meiosis. So, every chromosome in zygote hs its homolog, n essentilly identicl chromosome, with few differences (our mrkers, for exmple). Zygote cn multiply mitoticlly for some time. Meiosis is specil division of zygote, distinct from mitosis, tht regenertes gmetes, or cells with sinlge chromosoml sets. During meiosis, homologous chromosomes undergo from one to severl exchnges, so there re siclly no non-recominnt chromosomes in gmetes. Finlly, s you know, ll nturl eukryotic chromosomes re liner. n exchnge etween two homologous liner chromosomes lwys regenertes monomeric chromosomes. This ll sounds complicted, ut eukryotes re, ctully, geneticist hog heven. Clonl Growth n 2n n Syngmy & Meiosis Genetics is not so simple in cteri. The vst mjority of cteri grow s clones without detectle exchnge of genetic informtion. In the sence of syngmy s life stge, it is close to impossile to ring in the entire chromosome from nother cell. Wht is chievle is to ring liner chunk of nother chromosome into the cell. However, such n exchnge of whole chromosome with suchromosoml frgment cretes prolem (ONE-MINUTE WRITE: wht kind of prolem?): fter single exchnge, the chromosome ecomes frgmented nd, therefore, unstle (degrdtion, inility to complete repliction or to finish segregtion). To mke functionl, whole chromosome, two exchnges re required in cteri:

4 nother common trit of cteril chromosomes tht complictes cteril genetics is their circulrity. For exmple, imgine tht there were wy to crete cteril zygote, cell tht would contin two whole, geneticlly mrked chromosomes. If the prticipting chromosomes re circulr, single exchnge etween them would crete dimer chromosome, which is eqully non-functionl: The solution to this compliction, gin, lies in the doule exchnge, which preserves the originl, monomer stte of the chromosome: Therefore, in cteri with their mostly circulr chromosomes nd with the mjority of recomintionl events tht involve exchnges with suchromosoml frgments, recominnts re lwys the result of two (or ny even numer of) exchnges. In these conditions, it is more convenient to look for the simultneous incorportion of two mrkers from the suchromosoml frgment, the so-clled co-inheritnce of the two mrkers: The higher the frequency of co-inheritnce, the closer the two genes on the chromosome re, opposite to wht we surmise from recomintion frequencies! For exmple, if mong the progeny tht inherited the gene, 60% inherited nd 30% c, while mong those tht inherited only 10% inherited c, we cn conclude tht the order of genes is --c. The importnt concept to rememer is tht, if our genetic cross involves one complete nd one incomplete chromosome, or two complete circulr chromosomes, it will require two (or ny even numer of) exchnges to generte vile progeny. Prt III. The Genes nd the Pthwys There re quite few genes implicted in ctlysis of HR in eucteri. In E. coli, for exmple, these re rec,, C, D, E, F, G, J, L, N, O, Q, R, T; ruv,, C, this is jokingly clled the recomintionl lphet. Mutnts, identifying these genes, were isolted for their defect nd sometimes even deficiency in homologous recomintion. ut do they work in single pthwy or in severl pthwys? nd wht re the possile

5 differences etween these pthwys? Two mjor genetic pproches cn e used to elucidte pthwys of ny process: 1) sustrte nlysis; 2) episttic nlysis. Sustrte nlysis involves vrying the physicl nture dimer of recomintionl sustrtes nd detecting the resulting recomintion frequencies in vrious rec mutnts. One populr sustrte configurtion is liner piece of donor chromosome, which is introduced into the recipient y either conjugtion or generlized trnsduction, with susequent selection for its integrtion into the recipient chromosome. nother populr recomintionl setup includes two circulr plsmids (extrchromosoml elements), which cn recomine y regions of homology: For exmple, these re the reltive (normlized to WT) frequencies of conjugtionl nd plsmid recomintion in vrious E. coli mutnts: Conjugtionl rec. Plsmid rec. Rec rec rec recc recf recg reco recr ruv Such sustrte nlysis shows tht: 1) Rec protein ctlyzes oth conjugtionl nd plsmid recomintion. 2) Rec nd RecC proteins ctlyze conjugtionl, ut not plsmid recomintion. 3) RecF, RecO nd RecR proteins ctlyze plsmid, ut not conjugtionl recomintion. 4) RecG nd Ruv proteins re importnt, ut not criticl, for oth conjugtionl nd plsmid recomintion. Since we hve n ide out the nture of the sustrtes, we cn conclude tht the Rec-RecC pthwy ctlyzes exchnges etween two DNs if t lest one of them hs free ends (like during conjugtion), while the Rec-RecFOR pthwy ctlyzes exchnges etween chromosomes without ends, for exmple, etween two circulr plsmids. We cn lso sy tht oth RecG nd Ruv functions help recomintion, ut the specificity of their ction is uncler. Episttic nlysis involves comining two muttions in single orgnism nd monitoring the resulting phenotype. Epistsis mens covering over, nd originlly episttic nlysis ws designed to find genes cting together y looking for doule mutnts with phenotype of one of the single mutnts. Remrkly, episttic nlysis turned out to hve more distinguishing power thn t first thought, ecuse it cn revel distinct pthwys within one mechnism. Suppose there re two mutnts, ech showing 30% decrese of some mesurle phenotype. The three kinds of episttic interctions re: 1) the doule mutnt possesses the phenotype of the single mutnts (still shows only 30% drop), this clssic epistsis suggests tht the two genes work in the sme pthwy; 2) the doule mutnt shows n dditive (ctully, multiplictive) Donor Recipient

6 effect (60% decrese), the two genes must e working in seprte pthwys, nd there re more functionl pthwys left; 3) the doule mutnt shows synergistic effect (99% down) there re only two pthwys, nd the two muttions inctivte oth. To see how episttic nlysis works, let us consider specific exmple. Homologous recomintion is implicted in the cellulr resistnce to DN dmging tretments, for exmple, resistnce to ultrviolet light (UV). If we determine UV resistnce of doule mutnts nd compre it to UV resistnce of single mutnts, we cn generte mtrix. In the following mtrix, the vlues show how mny times mutnts re more UV-sensitive thn WT cells: WT rec rec recc recf recg reco recr ruv WT 1 rec rec recc recf recg reco recr ruv Such episttic nlysis of UV-resistnce due to recomintionl repir in E. coli shows tht: 1) y themselves, most rec muttions decrese the resistnce ~10-fold (the first column). Only rec muttions mkes cells very sensitive to UV. This suggests severl pthwys of recomintionl repir, with Rec protein plying the key role. 2) To confirm the key role of Rec, we comine rec muttion with other mutnts pirwise (the second column): rec mutnt sensitivity is otined for ll doule mutnts, => therefore, rec must control the limiting step common to ll pthwys of recomintionl repir. Comining ll other mutnts pirwise produces the following picture: 3) rec mutnt does not chnge UV sensitivity when comined with recc, recg nd ruv mutnts, => Rec protein works together with the corresponding proteins. 4) rec mutnt ecomes very UV-sensitive if comined with recf, reco nd recr mutnts, => RecFOR group my define pthwy lterntive to RecC-G-ruv pthwy. 5) recc mutnt comintions confirm our rec nlysis. 6) recf mutnt does not chnge UV sensitivity when comined with recg, reco, recr or ruv, ut it mkes cells very UV sensitive when comined with rec or recc, => RecFGORruv group works together nd is seprte from RecC group.

7 Now we strt to see the pttern of RecC versus RecFOR pthwys, ut we re still confused with RecG nd Ruv, ecuse they group with oth the RecC nd RecFOR pthwys! However, when we comine recg with ruv mutnts together, we get very sensitive doule mutnt gin! ll these results with recg nd ruv mutnts suggest tht RecG nd RUV proteins 1) work t the stge of recomintionl repir tht is different from the stge t which RecC nd RecFOR work; 2) define the two lterntive pthwys of the stge. If we comine the results of episttic nlysis with the previous results of the sustrte nlysis ove, we rrive t the following scheme of homologous recomintion in E. coli: 1) rec is the control gene of HR, ctlyzing the centrl rection of the process. 2) RecC nd RecFOR complexes define the two lterntive erly pthwys, working efore the Recpromoted stge. 3) RuvC nd RecG enzymes represent the two lterntive lte pthwys, working fter the Rec-promoted stge. X X RecC RuvC Rec RecFOR RecG Thus, the two genetic pproches, the sustrte nlysis nd the episttic nlysis, llows one to ssemle the following scheme of pthwys of homologous recomintion in E. coli: Prt IV. The Mechnisms In the center of mny models of HR lies the unique structure clled the Hollidy junction. ck in 1964 Roin Hollidy sked himself how HR cn strt, nd cme up with seemingly simple mechnism (oth strnds of the DN duplex re shown here): This recomintion intermedite, in which the prentl chromosomes re held y DN junction, turned out to e rekthrough in understnding HR. Nowdys it is widely known s the Hollidy junction, proly ecuse Hollidy not only suggested it s n intermedite of HR, ut lso proposed the two wys to resolve the junction: X

8 oth wys regenerte pir of chromosomes, ut the left pir comprises the recominnt chromosomes, wheres the right pir comprises the prentl chromosomes. Hollidy s intermedites in the junctions were mjority of HR events. The process of their susequently confirmed clevge is clled the resolution ; in E. coli, it is ctlyzed y the RuvC resolvsome. However, the mechnism of Hollidy does not provide the reson to exchnge strnds in the first plce. How to find this reson? The esiest wy is to look for conditions tht stimulte homologous recomintion. It hs een known for long time tht homologous recomintion is strongly induced y DN dmge. Specificlly, rek in oth DN strnds, clled doule-strnd rek, in one of the two homologous chromosomes is one of the strongest inducers of exchnge (lso clled cross-over ) etween these homologs: Interestingly, if there is third mrker ner the rek site, it revels the second type of recomintion events, clled gene conversion : the mrker on the roken chromosome is lost nd replced with mrker from the intct chromosome. Remrkly, gene conversions re independent of crossing-over: hlf of them re ssocited with crossing-over, while the other hlf is not:

9 So, the mechnism of doule-strnd rek-stimulted homologous recomintion should explin: 1) formtion of cross-overs; 2) formtion of gene conversion; 3) their independence from ech other. We do not hve time to discuss how the underlying mechnism ws estlished, ut we still hve time to drw the mechnism: Rec Gene conversion without chrossing-over Gene conversion with crossing-over It explins oth crossing-over (through specific configurtions of the Hollidy junction resolution) nd gene conversion (through degrdtion of the roken ends in preprtion for the Rec-ctlyzed rection nd their susequent resynthesis using the homolog s the templte). The iochemicl detils of this mechnism, s well s its iologicl implictions for repir of DN dmge re eyond the scope of this course.

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