2.7. Non Homologous End Joining :
Broken end recognize by Ku70/80 heterodimer because this dimer act as sensor and Ku70/80 heterodimer binds to those ends, then the bridge between those end form by MRN complex (in yeast MRX) contain Rad 50, Mre II and Nbsi (in yeast called Xrs2). Broken end also come together because individual Ku protein has affinity towards each other. Then Artemis protein get activated by phosphorylation through DNA dependent protein kinase (DNA-PKcs), and now in activated form act as endonuclease and exonuclease and responsible for trimming the overhangs. Both DNA-PKcs and artemis start to act by the permission of Ku protein. After that the gaps get fill by DNA polymerase and the end join finally by the DNA ligase IV in alliance of XRCC4. Non homologous end joining takes place throughout the cell cycle but specifically occur in GI and G0. For example this mechanism visualize in the case of recombination of immunoglobin gene. Due to this pathway change occur in DNA strands.
Homologous end joining:
It is carried out by general homologous recombination mechanism, this mechanism takes place throughout the cell cycle but specifically occur in G2 and S. Due to this pathway not any kind of change occur in DNA strands.
Recombination is define as process that involve rearrangement of genetic material which takes place by the means of breaking of genetic material and then rejoining of that genetic material. In eukaryotes, crossing over which is known as physical exchange of genetic material and occur in prophase I of meiosis, also govern by mechanics of recombination. In eukaryotes recombination also takes place during repair. In prokaryotes recombination occur in conjugation, transformation and transduction during the integration of transferred genetic material in to the bacterial chromosome. Here we mention three classes of genetic recombination, first – homologous recombination, second – site specific recombination and third – DNA transposition.
2.8. Homologous recombination or General recombination
Exchange of segments of genetic material between the homologous gene sequences (minimum size length of homologous sequence is up to100 base pair or ?75 base pair which is necessary for recombination process) or homologous chromosome. During meiosis crossing over (occur in pachytene of prophase I) and accurate chromosomal segregation govern by homologous recombination, thus responsible genetic diversity which occur between resulting gametes. In prokaryotes, homologous recombination occur during conjugation, transformation and transduction and in those processes genetic material get exchange reciprocally.
There are certain models which explain the mechanism of homologous recombination:
1. Holiday model:
It is given by Robin Holiday in 1964. This model is also known as heteroduplex model because this model also explains the recombination between short homologous sequences between two heteroduplex.
Firstly two homologous DNA molecule get align with each other after that nick is introduce in both dsDNA at same position, in turn strand next to nick get unpaired to its complementary strand and free to invade the near align dsDNA at the same location where nick form and after invasion base pair with its homologous region within that dsDNA, as a result, this invasion create holiday junction (a cross) which is the key recombination intermediate, this move is known as strand invasion.
After this step movement of holiday junction takes place along the DNA which is called as branch migration, during the migration of holiday junction base pair within parental DNA strand get broken at the same time as identical base pair produce within the recombination intermediate. After this step holiday junction get cleave, which occur by cleaving the DNA strand within the holiday junction, this event is known as resolution of holiday junction and it takes place by two cleaving pattern occurs in different orientation, those pattern get visualize by study the chi form of Holiday structure or three dimensional structure of Holiday structure.
In one pattern cut takes place in those strand which does not get nicked during initiation of recombination process or cut takes place up-down and then join by DNA ligase, in turn resulted products called splice recombination product or crossover product, here reassortment of genes that next to the recombination site also takes place. In second pattern cut takes place in those strand which get nicked during the initiation reaction or cut takes place left-right and then join by DNA ligase, in turn resulted products called patch or non cross over product.
One modification which is called Meselson – Radding modification which explain that the nick produce in one of two dsDNA (hetroduplex DNA) and the nicked strand had free end which invade the dsDNA at the homologous position and complementary base pair with its homologous region as a result form D loop by displacing one strand of the non nicked dsDNA and displace strand get cleaved at the junction, between base –paired regions and its single-stranded region which result in the production of the hetroduplex. This modification explain the formation of two nick in two dsDNA at the same position and the formation of heteroduplex by two dsDNA molecule which interact at the beginning of recombination procsess.
Double-strand break model:
Firstly double stranded break is created in one of the two dsDNA called recipient by endonuclease, in meiosis Spo11 protein act as endonuclease. Spo11 (DNA topoisomerase like enzyme) binds to 5’ end of the break. DBSs in mitotic cell get generate by DNA damage, mating switch in yeast and V(D)J recombination. In both cases 5’ attack by an exonuclease and in case of meiotic cell Spo11 get dissociate by the action of exonuclease , in turn create free 3’ overhang in the assistance of helicase and this process is known as 5’-end resection. 3’ end invade the non nicked dsDNA called donor at the homologous region, in turn form complementary base pairing, as a result form D loop by displacing the non complementary strand and form heteroduplex, the free 3’ end act as primer for the DNA synthesis and cause the extension of D loop.
The point is known as recombinant point or branch point where the individual DNA strand of one dsDNA crosses the other dsDNA. After this process branch migration takes place by the movement of recombinant joint or Holiday junction or branch point in either direction and its depend upon the direction of strand displacement. Capture of second double strand break done when other side of the break get pair by D loop due to the extension of D loop. Thus here also a holiday junction get form after the DNA synthesis along with break filing and gap sealing by DNA ligase, as a result two holiday junction get form, which resolve by cutting within the holiday junction, If both cross over resolvation takes place in opposite way, result in the formation of cross over product and If both cross over resolvation takes place in same way, result in the formation of non cross over product.
2.9. Homologous recombination in becteria
In bacteria homologous recombination require a free 3’ end, which is provided by various mean but for homologous recombination this end processing done by three recombination system name RecBCD nuclease/helicase, RecE, RecF system, from all these RecBCD nuclease/helicase also called Exonuclease V having highest importance. As the name shows RecBCD has three subunit along with both nuclease and helicase activity. RecBCD is bipolar helicase because RecB is 3’to5’ helicase and RecD is 5’ to 3’ helicase. RecB also has nuclease activity. RecC recognise Chi site.
Firstly binding of RecBCD takes place at nicked or broken (free) end after that unwinding of dsDNA occur by helicase action in ATP dependent manner through the movement of RecB, a slow helicase in 3’to5’ direction and RecD in 5’ to 3’ faster than RecB in presence of SSB protein, as a result , in front of RecB accumulation of ssDNA occur. Distortion within the activity of enzyme takes place when it get interact with crossover Hotspot Instigator (Chi) sequence (Chi 1,009 in no within E.coli chromosome), which posses consensus sequence 5’GCTGGTGG3’ and takes place one time each 5-10 kbp and get recognise by RecC and get bind on RecC, after interaction and recognition with Chi, RecC signal RecD for stopping of unwinding and simultaneously RecD signal RecB to cut DNA.
RecB cut the strand with Chi, in turn release the 3’ end having strand, after cutting strand continuous unwinding cause by the enzyme, as a result a free 3’ and with terminal chi sequence get generated, which act as a site to which ReaA (homologous to meotic Dmc1 protein and Rad51 of eukaryotes) loaded by RecBCD enzyme (act as RecA loader), RecA assemble on that strand in cooperative manner as six RecA monomer per turn of the strand by the disassembly of SSB protein, this RecA protein coated strand capable to cause invasion within the intact dsDNA, in turn form D loop which leads to the formation of Holiday junction by successive cleavage of the strand which get displaced.
E.coli ruv genes product identify the holiday junction, RuvA firstly recognise the holiday junction and bind at there with all four strand of DNA. RuvA sandwich the DNA through binding, in a tetrameric form on both side of DNA. A hexameric helicase RuvB bind to both DNA strand upstream to crossover and supply motor for branch migration, in turn specifically catalyze branch migration, both RuvA/B complex mediate branch migration with the rate of 10 to 20 bp/sec by the displacement of RecA, RecG also a helicase function in association with A/B. After branch migration get completed two RuvC protein replace the RuvA/B and resolve the holiday junction by its endonucleleolytic activity. ATTG is an asymmetric tetranucleotide act as hotspot for RuvC for resolvation of holiday junction and responsible to mediate the resolution by consideration of nick production takes place in which pair of strands, as a result formation of cross over or non crossover product formation.
2.10. Site specific recombination :
Recombination can also takes place when there is a short region of homology is present known as specific region of homology responsible for recombination , this type of recombination called site-specific recombination and visualize during the λ becteriophage integration within E.coli genome. This recombination occur due to the presence of att (attachment site) site on both λ bacteriophage called attP contain 250 bp along with POP’ and in E.coli called attB contain 23 bp along with BOB’. Both att site has 15 base pair central homologous sequence called ‘O’on to which recombination occur in case of integration but incase of exsition those att site known as attL and allR.
Firstly integration is carried out by lambda integrase which is related to IB topoisomerase family and possess a conserve tyrosine residue thus also called tyrosine recombinase. Integrase require the assistance of IHF (integration host factor), which is a hetrodimer of 20 kDa and responsible to cause bend in DNA by binding the ~20 bp sequence of attP site,in turn integrase binding site come close, which present on the DNA arm, as a result integration of lambda genome into E.coli genome takes place. Through integration the attachment (att) site generate called attL or BOP’ and attR or POB’ and excision takes place due to reciprocal recombination takes place between these two att site by the function of integrase, IHF, and Xis which act as excisionase. Xis inhibit Integation but promote excision.
Site specific recombination done by recombinase enzyme family and this event require for the integration of phage genome sequence into E.coli genome and during the excision of phage genome sequence from the E.coli genome. For integration integrase enzyme is required which is known as Int in phage, Cre in phage P1 and FLP in yeast (cause inversion of chromosome). Here we explain Site specific recombination between lambda phage and E.coli. lambda phage possess two type of life cycle mode , first, lytic in which it reside within E.coli as independent chromosomal molecule and second, lysogenic, in which lambda phage DNA integrate in E.coli chromosome called integration as well as release from the E.coli chromosome called excision and thus site specific recombination require to carry out both event.
There are two families of conservative site-specific recombinases: The serine recombinases and the tyrosine recombinases. Fundamental to the mechanism used by both families is that when they cleave the DNA, a covalent protein–DNA intermediate is generated. For the serine recombinases, the side chain of a serine residue within the protein’s active site attacks a specific phosphodiester bond in the recombination site. This reaction introduces a single-strand break in the DNA and simultaneously generates a covalent linkage between the serine and a phosphate at this DNA cleavage site. Likewise, for the tyrosine recombinases, it is the side chain of the activesite tyrosine that attacks and then becomes joined to the DNA.