Patent Application: US-22717106-A

Abstract:
this invention relates to chromosomal engineering via dna repair process . the process of the invention comprises the steps of : 1 ) submitting at least one source of biological activity , e . g . deinococcus radiodurans , to radiation , desiccation and / or chemical treatment liable to damage the dna , so as to substantially shatter its chromosomes into short fragments ; 2 ) annealing complementary single strand tails extended by the synthesis templated on partially overlapping dna fragments of said shattered chromosomes ; 4 ) converting the resulting long linear dna intermediates into intact circular chromosomes , by means of a reca dependent homologous recombination ; whereas at least one foreign source of genetic material , e . g . dna , can be introduced during steps 2 and / or 3 ; and 4 ) optionally separating and collecting the recombined chromosomes thus obtained .

Description:
along with starvation , cold and heat , dehydration or desiccation is one of the most frequent and severe challenges to living cells 9 . therefore , resistance to desiccation is important part of life &# 39 ; s robustness . bacterium deinococcus radiodurans is the best known extremophile among a handful of organisms found to resist extremely high exposures to ionizing radiation and desiccation 10 , 11 . it appears that the evolution of d . radiodurans &# 39 ; extreme radiation resistance is a by - product of natural selection for its desiccation resistance 12 . selection for desiccation resistance in bacteria co - selects radiation resistance 13 , and all of 41 tested deinococcus radiodurans radiation sensitive mutants are also desiccation sensitive proportionally to their radiation sensitivity 12 . both treatments have in common that they cause extensive intracellular dna double strand breakage ( dsb ) 12 which is the most severe form of genomic damage because a single unrepaired dsb is usually lethal 14 . standard vegetative prokaryotic and eukaryotic cells can repair less than a dozen simultaneous dsbs 15 . d . radiodurans survives ionizing radiation breaking its genome into several hundred fragments ( up to 2000 dsbs per cell containing 4 to 10 copies of its genome ) due to a dna repair process accomplishing an efficient and precise fragment assembly 16 , 10 , 11 that is a physical equivalent of the computer - assisted contig assembly of shotgun sequenced random genomic fragments . d . radiodurans is a small non - sporulating and non - pathogenic bacterium whose sequenced genome is composed of two circular chromosomes ( 2 . 65 and 0 . 41 mega bp ) and two circular plasmids ( 0 . 177 and 0 . 046 mega bp ) 17 . because the reca and pola mutants of d . radiodurans are its most radiation sensitive mutants 10 , 11 , the reca recombinase and dna polymerase i ( pola ) clearly play key roles in the repair of shattered chromosomal dna . with few if any exceptions 18 , this dna repair process in d . radiodurans is much more efficient than are the reca and pola dependent repair processes in other biological species , but its molecular mechanism remained a mystery 19 . neither the comparative amounts , nor the enzymatic properties of the reca and pola proteins gave any clear hint 19 , 20 . the chromosomal copy number 21 and special forms of chromatin 14 , 15 have been evoked , but it turns out that neither can account for d . radiodurans &# 39 ; resistance 19 , 20 . this invention reveals and makes use of a new form of dna repair involving mutually dependent dna replication and recombination processes , one of which is reminiscent of a single - round multiplex pcr ( fig4 ), reconstituting functional d . radiodurans chromosomes shattered by 7 kgy ( 0 . 7 megarad ) gamma ray radiation to 25 - 30 kb dna fragments and causing only about 10 % lethality . in this respect , the efficiency of uv light in producing dsbs by photolysis of 5 - brdu , suggests that such blocks of newly synthesized double stranded dna in repaired chromosomes are large , in agreement with density gradient analysis ( fig4 ). the fragmented chromosomes are probably not homologously paired because in such a case ( e . g ., a yeast meiosis ) the sdsa repair synthesis is limited to the short region between recessed ends of the individuals dsbs 4 , 6 . the structure of chromosomes repaired by esdsa is shown in fig1 . the distinct feature of the esdsa model is that it involves an “ ends - out ” multiplex pcr - like step ( steps 2 and 3 in fig1 ) resulting in long newly synthesized single strand overhangs that allow an accurate annealing process . alternatives to the esdsa mechanism , e . g ., nhej or hr working exclusively on newly synthesized dna ends , are highly unlikely . a ssa mechanism with an extensive nick translation synthesis is conceivable ; however the e . coli klenow fragment pola complements d . radiodurans pola mutants for resistance to gamma radiation 22 showing no requirement of nick translation for dna repair . the pattern of dna repair seen in pfge ( fig2 e ) and in optical mapping experiments 23 suggests that the majority of dna fragments “ grow ” progressively and that most fragments are being used early in the chromosomal assembly process ( fig2 ). one can therefore envisage esdsa “ chain reactions ” involving numerous “ nucleation ” points in the cell producing long linear dna repair intermediates . such long linear dna , if shorter than the chromosome size , would not be likely to find the exact two - end matching chromosomal segment to complete the circular chromosome structure by esdsa or ssa . such shorter - than - chromosome linear intermediates , as well as those longer than chromosome size , would require homologous , typically reca - dependent , recombination 24 to produce mature unit circular chromosomes ( fig3 ). this is consistent with pfge experiments with a reca mutant ( fig2 e ). both dna synthesis and repair are less efficient in the reca mutant ( fig2 e , f ) than in wild type , so that it is therefore possible that deinococcal reca , through its preferential double - strand binding activity 20 brings together dna fragments with overlapping homology that should facilitate an esdsa mechanism . however , the key involvement of reca occurs late in the repair process ( fig3 ). pola , but not reca , mutant is clearly deficient in esdsa repair ( fig2 ). however , dna base and sugar damage by radiation - induced oxygen free radicals , which can cause single - strand breaks directly and , more frequently , by proximal base excision repair ( ber ) events , are at least 10 times more frequent than dsbs 25 and typically require pola for the completion of repair 26 . therefore , it is expected that pola protects the integrity of the dna fragments ( see the loss of fragments in fig2 c ) produced by gamma radiation by repairing single nucleotide gaps created by the ber enzymes , a prerequisite for their assembly where pola is presumably directly involved in the esdsa process ( fig1 ). however , before the isolation of a conditional mutant of dna polymerase iii , one cannot exclude the possibility that pola only initiates a dna polymerase iii - catalyzed single strand elongation or even contributes only to the maintenance of fragments . unlike other bacteria , all radiation resistant bacterial species show condensed nucleoids even after ionizing radiation 27 . it was suggested that a special ring - like chromatin holds broken dna ends in register and facilitates repair by nhej 3 . however , the high concentration of dna fragments in the condensed nucleoid is expected to facilitate any bimolecular homologous interaction required for dna repair , e . g ., in sdsa / esdsa , ssa and hr . perhaps , in addition to the peculiarity of the reca 20 , the condensed chromatin is another evolutionary innovation of d . radiodurans assuring high efficiency of esdsa and , therefore , radiation resistance . as to the question whether esdsa can account for the apparent fidelity of dna repair in d . radiodurans , one can generally say that the larger the number of dna fragments , the higher the precision required for avoiding their incorrect assembly and the longer must be the required homology . if there are unusually many dsbs , unusually long homology — and hence single strand exposure — is required . such unusually long overhangs are newly synthesized in the course of the esdsa ( fig1 ). however , any homology - based repair of shattered dna raises the question of the accuracy of assembly of chromosomal fragments containing repetitive sequences . the fact that all long repeats , e . g ., transposons , have been completely counter - selected from d . radiodurans genome 17 , 28 may be due to their deleterious effect on the precision of the esdsa . the problem of an incorrect fragment assembly via repetitive sequences could be circumvented in esdsa if the single - stranded overhangs were much longer than the longest repetitive sequences . in that case , annealing only a repeated sequence block within a long single - stranded overhang could not readily link the two fragments . such partially annealed structures are the preferred substrates for dna helicases and , therefore , these aberrant dna associations should be readily aborted . the avoidance of lethal non - contiguous dna fragment assembly may have provided sufficient selective pressure for the evolution of the esdsa mechanism ( with long single stranded overhangs ) rather than a ssa ( with shorter overhangs ) ( fig1 ). the inventors &# 39 ; experiments suggest that the size of esdsa overhangs is similar to the fragment size , i . e ., about 20 to 30 kb — much longer that the longest d . radiodurans &# 39 ; repetitive sequences ( is of about 1 kb ) 17 , 28 . as the precision of fragment assembly on the esdsa process becomes high due to long overhangs , the fidelity of making such overhangs by strand extension ( the bracketed intermediates in fig1 ) becomes limiting for the global fidelity dna repair . it is therefore suggested by the inventors that one can think of four strategies for assuring the fidelity of both priming and annealing : ( 1 ) homologous pairing of double - stranded dna fragments before initiation of d - loops , perhaps by the deinococcal reca 20 , ( 3 ) repeat - binding proteins 17 preventing sequence repeats from becoming single - stranded or from annealing , and ( 4 ) stable secondary structures ( in practice hairpins ) of repetitive sequences 28 preventing their annealing . provided that one could learn what are the intermediate steps in deinococcal dna repair , then reproducing , in silico , the fragment assembly process could become an alternative to the current contig programs . finally , given the high transformability of d . radiodurans by exogenous dna as shown according to this invention , it may be that , in the evolutionary past of d . radiodurans , an esdsa - like repair after desiccation periods has led to the incorporation of foreign dna . this would account for the extraordinary diversity of genetic lineages represented in the d . radiodurans chromosome ( bacillus , thermus and multiple eukaryotic genes ) 17 , 28 . the experiments on which the present invention is based are summarized as follows . dna synthesis and repair of d . radiodurans chromosomes shattered by radiation . to discriminate between these hypothetical mechanisms , we explored in parallel the kinetics of the joining of dna fragments by pulsed - field gel electrophoresis ( pfge ) and the rate of dna synthesis by 15 min 3h - tdr pulse labelling following 7 kgy gamma radiation . there is clearly a temporal coincidence of the fragment assembly and the observed massive dna synthesis occurring without cell division at much higher rate than in the growing non - irradiated cell culture ( fig1 a and b ). this dna synthesis is absent in a pola strain ( fig1 d ) that also shows no evidence of dna repair ( fig1 c ). along with high radiation sensitivity of the pola mutant , these observations cannot be easily accommodated by mechanisms ( i ) and ( ii ). they generally agree with all mechanisms ( iii - v ) involving extensive dna synthesis . sdsa ( iv ), and to a lesser degree ssa ( iii ) mechanisms are favored if the pola dependence is due to pola ( with or without participation of dna pol iii ) catalyzed single - strand elongation ( fig4 ), but the cc mechanism ( v ) cannot be a priori excluded because pola could initiate or otherwise facilitate a pol iii - catalyzed semi - conservative two - strand synthesis ( replication forks ). we confirm the already observed 5 largely reca - independent fragment assembly ( fig1 e ) which is paralleled by a substantial dna synthesis during the time of effective repair ( fig1 f ). these experiments establish a correlation between the pola - dependent dna synthesis and the recombination repair of highly fragmented d . radiodurans chromosomes . the strict requirement of the reca for the appearance of full - size chromosomes , a prerequisite for cell survival , suggests reca &# 39 ; s key involvement in the maturation of functional circular chromosomes ( fig5 ). the density analysis of dna from unirradiated d . radiodurans cultures ( fig2 c ) agrees with the meselson - stahl experiment with e . coli , i . e ., the replication of all chromosomal elements appears semi - conservative : the h / l ( heavy / light ) density appears after the first replication cycle , h / h during the second . furthermore , upon dna denaturation and centrifugation in alkaline cscl gradients , all single - stranded material segregates into either h or l densities ( fig2 a 1 , b 1 , c 1 ). because of the pattern of radioactive ( l 3h - tdr )* (* denotes radioactive label ) and density ( h 5 - brdu ) labelling , the non - irradiated growing d . radiodurans cultures show , in the pre - labelling regime , only h / l * ( fig2 a ) ( and only l * in denaturing gradients , fig2 a 1 ) material ; in the post - labelling predominantly the h */ l and — to the extent of second replication round — also the h */ h * ( fig2 b ) ( and only * h in denaturing gradients , fig2 b 1 ). we shall call “ old ” the light dna synthesized before irradiation and “ new ” the heavy dna synthesized after irradiation . because only old dna is damaged in radiation experiments , and the repair takes place in the heavy medium , the two strands of the dna repaired by nhej and hr are expected to be essentially old / old and those made by cc fully new / new . the density patterns of dna repaired during 3 hours after gamma radiation do not resemble semi - conservative replication and do not support either nhej , hr or cc as the predominant repair mechanism ( text above and fig2 a - c ). in neutral density gradients , the old “ pre - labeled ” dna is spread towards intermediate densities by the 5 - brdu incorporation after irradiation ( fig2 a ). on the other hand , the “ post - labeled ” new dna synthesized after irradiation is shifted towards heavy densities ( fig2 b ). both results are compatible with the sdsa ( and , to a lesser degree , ssa ) mechanism but only if , after denaturation , single strands also show intermediate densities because of the expected patchwork of the blocks of old and new material ( fig4 ). indeed , in alkaline cscl density gradients , the densities of pre - labeled single strands are shifted substantially from light ( old ) towards heavy ( new ) ( fig2 c 1 ). because the size of the dna isolated by the method described is in the range of 15 to 20 kb , i . e ., generally smaller than the in vivo size of fragments following 7 kgy irradiation ( about 30 kb ), the detection of fragments containing old and new material is diminished . these results support the hypothesized “ distributive ” mode 31 of dna ( repair ) replication predicted by sdsa and , to a lesser degree , ssa mechanism . what is the relative size of newly synthesized patches ? fig2 a 1 shows that , in the “ pre - labelling ” regime , the density of the majority of single stranded material is heavier than light ( old ) but lighter than heavy ( new ). the reciprocal is not true : in the “ post - labelling ” regime , the single - stranded dna is under the heavy peak and skewed towards lighter densities ( fig2 b 1 ). in other words , during the repair in the presence of 5 - brdu of initially light fragments , the old light strands are made heavier ( by association with heavy material ) than are the new heavy strands made lighter ( by association with light material ). this suggests that , in the analyzed dna fragments , the tract of newly synthesized ( density and radioactively labeled ) material is often longer than the tract of original light ( radioactively but not density labeled ) material . this agrees well with the sdsa model . if the ssa model were correct , it would require that the recession of fragment ends be very extensive to explain the massive pola - dependent dna synthesis associated with repair ( fig1 a , b ). on the single strand level , the repaired dna appears as a patchwork of old and new material , expected from both sdsa and ssa ) mechanisms ( fig4 ). if one mechanism is predominant , can we discriminate between the two ? ssa mechanism predicts that — upon dna breakage by sonication ( to about 3 kb fragments , not shown )— the double - stranded dna should be segregating towards the new / old and old / old , whereas the sdsa mechanism predicts ( in the post - labelling regime ) a significant segregation towards the new / new densities in neutral gradients ( see fig4 ). fig2 a 2 - c 2 show a very large density distribution of sonicated dna fragments obscuring the detection of any minority distinct density molecular species . the amount of l / l material in the pre - labelling experiment ( fig2 a ) shows that a large fraction of molecules contains no significant tract of dna repair synthesis . because the size of isolated dna is even smaller than the fragment size of radiation - broken dna , it could be that the l / l material represents ( a ) dna fragments dissociated from repair synthesis , ( b ) the unrepaired dna fragments from dead cells and / or ( c ) the unused fragments from surviving cells . given that we see no evidence of unused dna fragments in pfge experiments ( fig1 a , e ), option ( a ) remains most likely . 5 - brdu substitution sensitizes dna strands to breakage by photolysis using uv or short wavelength visible light 32 - 34 . we confirm that , also under our experimental conditions , one - strand 5 - brdu substitution ( during 1 . 5 h , i . e ., less than one generation time ) of non - irradiated d . radiodurans chromosomes does not significantly sensitize dna to the double - strand breakage by the uv induced photolysis . two - strand 5 - brdu substitution causes extensive dna breakdown ( results not shown ). the intracellular photolysis decomposes dna repaired in 5 - brdu by double - strand breakage almost reproducing the pattern of the initial dna breakage by gamma rays seen before the onset of repair ( fig3 ). uv light induced dna fragmentation does not increase beyond the saturating dose of 250 - 500 j / m 2 , whereas it does for the fully substituted dna ( not shown ). this pattern of 5 - brdu - dependent photolysis suggests that most , perhaps all , reassembled fragments are linked together via double - stranded blocks of newly synthesized dna ( red colored in fig4 ) as predicted by the sdsa . bacterial strains , growth conditions , and gamma irradiation . the following d . radiodurans strains were used : r1 ( atcc 13939 ) wild type 35 , gy10922 d ( cina - reca ) 1 :: kan 36 , and irs501 pola ( j . r . battista ). a thymine - requiring ( thy -) derivative of r1 strain was isolated by selection on solid minimal medium containing thymine ( 50 mg / ml ) and trimethoprim ( 100 mg / ml ) 37 . bacteria were grown in tgy broth ( 0 . 5 % tryptone , 0 . 1 % glucose , 0 . 15 % yeast extract ) at 30 ° c . to the late exponential phase ( od650 = 0 . 6 - 0 . 8 ). cultures were washed in 10 mm sodium phosphate buffer , concentrated 10 times in the same buffer , and irradiated on ice with a 60co gamma ray source at a dose rate of 11 gy / s . the dose of 7 kgy was applied to the cells in all irradiation experiments . the number of viable cells was estimated by plating serial dilutions onto tgy plates . the plates were incubated for 34 days at 30 ° c . before the colonies were counted . kinetics of dna repair measured by pulsed - field gel electrophoresis . irradiated cultures were diluted in tgy to an od650 = 0 . 2 and incubated at 30 ° c . at intervals during post - irradiation incubation , 5 - ml samples were taken to prepare dna plugs as described by mattimore and battista4 . the dna contained in the plugs was digested with 60 units of noti restriction enzyme ( roche ) for 16 h at 37 ° c . after digestion , the plugs were subjected to pulsed - field gel electrophoresis in 0 . 5 × tbe using a chef - dr iii electrophoresis system ( bio - rad ) at 6 v / cm2 for 20 h at 14 ° c ., with a linear pulse ramp of 50 - 90 s and a switching angle of 120 °. rate of dna synthesis measured by dna pulse labelling . at intervals during exponential growth of both non - irradiated and irradiated cultures , 0 . 5 ml samples were taken and mixed with 0 . 1 ml pre - warmed tgy medium containing 6 mci 3h - thymidine ( amersham ; specific activity 86 ci / mmol ). pulses were terminated after 15 min by addition of 2 ml ice - cold 10 % tca . samples were kept on ice for at least 1 h , and then collected by suction onto whatman gf / c filters followed by washing with 5 % tca and 96 % ethanol . filters were dried overnight at room temperature , and placed in 5 ml scintillation liquid . the precipitated counts were measured in a liquid scintillation counter ( wallac ). radioactive and density dna labelling . d . radiodurans thy - cells were radioactively and density labeled during growth in the presence of 3h - thymidine and 5 - bromo - 2 ′- deoxyuridine ( 5 - brdu ), respectively . density labelling was performed in all experiments by adding 5 - brdu to the medium only after irradiation . the radioactive labelling was performed in three different regimes . ( a ) “ pre - labelling ”: cells were grown overnight in tgy supplemented with 5 mci / ml 3h - thymidine . they were collected by centrifugation , washed twice with phosphate buffer , concentrated 10 times in the same buffer , and exposed to 7 kgy gamma radiation . both irradiated and non - irradiated cultures were diluted to an od650 = 0 . 2 in tgy containing 20 mg / ml 5 - brdu . the non - irradiated culture was grown in 5 - brdu - supplemented tgy for 2 . 5 h ( corresponding to one mass - doubling ), while the irradiated culture was grown for 3 h ( the time required for dsb repair to be completed ) at 30 ° c . ( b ) “ post - labelling ”: cells were grown overnight in non - radioactive tgy , and radioactivity ( 20 mci / ml 3h - thymidine ) was added to the 5 - brdu - supplemented tgy only after irradiation . the experimental procedure was otherwise the same as described for “ pre - labelling ”. ( c ) “ pre - and post - labelling ”: the procedure was a combination of ( a ) and ( b ), i . e . the cells were radioactively labeled before and after irradiation . dna preparation and density gradient analysis . radioactively and density labeled dna was isolated from d . radiodurans by the use of qiagen dneasy tissue kit according to the supplier &# 39 ; s instructions . in some experiments , the dna was additionally fragmented by sonication . for that purpose , 100 - ml dna samples were subjected to sonication ( vibracell 72434 , bioblock ) with increasing number of 5 sec bursts . optimal sonication conditions producing 3 - 5 kb fragments were determined by gel electrophoresis . the dna was centrifuged to equilibrium in neutral or alkaline cesium chloride ( cscl ) solution ( 1 . 7246 g / ml ) in a vti90 rotor ( beckman ) for 24 h at 40 . 000 rpm and 20 ° c . to obtain the desired cscl concentration , the refractive index of cscl solution was adjusted ( by adding water ) to 1 . 4030 for neutral gradients , and to 1 . 4050 for alkaline gradients . for alkaline gradients , the dna was denatured by 10 min heating in boiling water , chilling in ice water , and the cscl solution was adjusted to ph 11 . 8 . gradients were collected from the bottom of pierced tubes ( optiseal 4 . 9 ml , beckman ) in about 25 ( 12 - drop ) fractions . 100 - ml aliquots of fractions were applied on round filters , dried under the lamp for several hours , and the radioactivity was measured by scintillation counter . uv - induced photolysis of bu - substituted dna . d . radiodurans thy - culture was grown and irradiated with 7 kgy gamma radiation as described above . the irradiated culture was diluted to an od650 = 0 . 2 and grown in 5 - brdu - supplemented tgy for 3 h . the cells were collected by centrifugation , resuspended in the phosphate buffer and incubated ( starved ) in buffer for one hour at 30 ° c . cell suspension was cooled in ice and exposed in thin layer to 1000 j / m2 of 254 - nm uv light . both uv - irradiated and non - irradiated cells were embedded in agarose plugs for dna analysis by pfge ( see above ). 1 lees - miller , s . p . & amp ; meek , k . repair of dna double strand breaks by non - homologous end joining . biochimie 85 , 1161 - 1173 ( 2003 ). 2 wilson , t . e ., topper , l . m . & amp ; palbos , p . l . non - homologous end - joining : bacteria join the chromosome breakdance . trends biochem . sci . 28 , 62 - 66 ( 2003 ). 3 levin - zaidman s . et al . ringlike structure of the deinococcus radiodurans genome : a key to radioresistance ? 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