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.