2.4. SOS response
When DNA get damage severely then it require immediate excision repair mechanism which carried out by the product of SOS responsive gene, whose product also important for cell survival because play important role in repair, recombination, cell division, mutagenesis and replication.
Activation of RecA protein occur by the signal generate due to DNA damage, in turn replication halt and that activated RecA cause cleavage of LexA repressor at Ala-Gly peptide bond to inactivate LexA, because LexA is a repressor of SOS responsive gene and by bind to SOS box (20 bp) at SOS responsive gene promoter cause repression of these gene, as LexA inactivate, activation of SOS responsive gene takes place and further repair process takes place. This whole process collectively called SOS response.
In prokaryotes due to severe DNA damage, DNA strand cannot be replicate by DNA polymerase III and then it recruit the DNA polymerase V (UmuD’2C). Cleavage of UmuD done by RecA during sever DNA damage and production of UmuD’ occur, when two UmuD’ subunit combine with UmuC form (polymerase V) UmuD’2C. When polymerase V bind to the lesion then add one correct nucleotide but not able to read second nucleotide thus add incorrect one and after that release, which leads to recruitment of DNA polymerase III and further replication continue.
In Eukaryotes, during this type of damage DNA polymerase δ/ε replace by DNA polymerase η, which add on it first nucleotide of dimer correct then release after that recruitment any of ( i/τ/μ/rev-1/k) one polymerase takes place which add wrong nucleotide and after that release, which leads to recruitment of DNA polymerase III and further replication continue.
Due to the addition of wrong nucleotide called error prone repair or trans lesion repair.
2.5. Direct repair :
DNA damage can directly be reverse by changing the damage base in to normal or damage base simply remove and replace by normal one, this type of damage repair known as direct repair. One of these repair systems is known as photoreactivation (a nonmutagenic repair system), in this repair pyrimidine dimer get repair by a light dependent enzyme that bind to dimer and cause reversal of inappropriate covalent bond between adjacent pyrimidine residue and convert into monomeric pyrimidine containing nucleotide.
In E.coli phr gene product name DNA photolyase, an enzyme. DNA photolyase get stimulated by wavelength of 300 to 500 nm (blue light) and at this wavelenght DNA photolyase binds to pyrimidine dimer and cause reversal of inappropriate covalent bond between adjacent pyrimidine residue and convert into monomeric pyrimidine containing nucleotide. This repair is not universal but widespread, specifically present in bacteria and plant. Interstrand pyrimidine dimer able to take the form of 6,4 photoproduct or CPD (cyclobutane pyrimidine dimer), but 6,4 photoproduct more mutagenic then CPD. Least common form of pyrimidine dimer is cytosine-cytosine dimer and most common is thymine-thymine dimer.
Conversion of one type of base into other result in distortion of DNA structure and it is due to replication error, for example conversion of cytosine to uracil by deamination, cause by chemical mutagen form mismatch and this mismatch visualize in future generation. Alkglating agent convert guanine into O6-methylguanine, which is highly mutagenic lesion, in turn form mismatch because now O6-methylguanine pair with thymine in spite of cytosine. An alkyl transferase called O6-methylguanine DNA methyltransferase mediate transfer of methyl group of O6-methylguanine to a specific cytosine residue , which is present within same protein, as a result repair of O6-methylguanine into guanine takes place.
2.6. Recombination repair :
This repair also known as post replication repair because gap remain in daughter strand due to presence of dimer in the parent strand, at this situation, replication cannot be stop, thus in this condition we need to bypass the damage and synthesis the gap containing daughter strand and that gap in daughter strand fill by the help of normal duplex by taken it single strand or mediate single strand exchange, which act as template for gap filling. The gap create in normal strand get fill by the repair process, in turn form normal duplex strand.
Replication fork stalls due to two reason, first if the lesion present in template strand and second nick present in parent strand, restoration of fork in both case done by separate method which explain below:
If replication fork get stalls due to damage in the parent strand at the damage site, at that situation, stall fork (look like Holiday junction) get revert up to short distance that allow the pairing between two daughter strand and the first daughter strand get form by DNA polymerase by the acting of second daughter strand act as template for the first daughter strand synthesis because we know one daughter strand has long than other in this case and after the synthesis of the daughter strand to which lesion containing parent strand act as template, After that restoration of fork done by forward branch migration of homologous recombination, which is done by the action of helicase. Some protein name RecA, RecF, RecR and RecO help in this process.
If replication fork stall due to presence of nick in one parent strand, in turn, double strand break present in one double strand daughter, as a result, replication fork collapse occur (lost of replication fork). Here homologous recombination carried out to fill the break in both duplex by the help of RecBCD, RecA and RuvABC. In this process free 3’ end get generated by RecBCD, then it become able for invasion by the binding of RecA on it, in turn cause strand invasion of free 3’ into donor duplex and forming D loop which is homologous and act as template for DNA synthesis by DNA polymerase, as a result formation of Holiday junction takes place and branch migration done by RuvAB then Holiday junction resolve by RuvC and the restoration of replication fork done by cleavage of the Holiday junction.
Repair of Double strand break:
Double strand DNA break get arise due to replication error, ionizing radiation and oxidizing agent, those type of break repair by homologous or non homologous end joining when there is not any kind of temple present to fill the gap. If this gap not get fill cause fragmentation of chromosome.
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.