DEOXYRIBONUCLEIC ACID (DNA):DNAis the chief component of any living organism.it is responsible for thetransfer of genetic information from one generation to the next; this traitmakes DNA hereditary material. Monomer of DNA isnucleotide that takes up sugar, phosphate group and a base from the fouruniversal bases: · Adenine(A)· Guanine(G)· Cytosine(C)· Thymine(T) Ø “A” pairs with “T” (A=T)Ø “C” pairs with “G” (C=T) DNA DAMAGE:Ø DNA exists in double helical form inwhich strands of DNA are anti-parallel to each other. Ø Errors in DNA strings are possible& very frequent in case of prokaryotes & called DNA DAMAGE.
Ø This damage could be in the loss ofnucleotides or strand breakage etc.iii RECOMBINATION:”Atype of genetic recombination in which exchange of nucleotide sequences occurbetween two similar or identical molecules of DNA is called recombination”.Ø Forthe exchange of genetic material in different species of bacteria and virusesthe process of recombination is used.Ø Thethree main processes involved in recombination of bacteria:1) Conjugation2) Transformation3) Transduction 1) CONJUGATION:”Theprocess through which by the direct contact transfer of genetic material fromone bacterium to the other takes place contact is called conjugation.”o In E. coli this process was postulatedby Joshua Lederberg and Edward Tatum (1946).o In 1958, they were also awarded Nobel priceon the work of salmonella, vibrio and pseudomonas.MECHANISM:Ø Inthe process of conjugation there are two bacteria one donates the geneticmaterial and other act as recipient which receives the DNA.
Ø Fertilityfactor or F factor is present in the donor cell. The F factor allows theformation of pilus in the donor cell. With the help of pillus the donor cellcontact the recipient cell.
Ø Bythe pilus two bacteria come together and donor transfer genetic material to therecipient.Ø Inlast step, both cells containing single stranded DNA, replicate it and formdouble stranded F plasmid which is identical to the original one. Now bothcells are donors or F+ because F-plasmid has ability for the piliformation.Ø Usuallygenetic material is in the form of plasmid, or a small, circular DNA piece.Ø Plasmidthat carries antibiotic resistance genes can also transferred by theconjugation process.iiiiv 2) TRANSFORMATION:”In molecular biology, transformation is the genetic modification of a cell by the direct uptake and expressionof DNA from its surroundings usually through the liquid medium.
“Ø It was described by FREDERICK GRIFFITH in 1928TYPES:Thereare two types of transformation: i. Natural transformation ii. Artificial or forced transformation i) NATURAL TRANSFORMATION:”Thetype of transformation in which no force is required for the transfer ofgenetic material is natural transformation.”MECHANISM:The transfer of isolated fragment is transformation Ø The transformation takes place in the mixed population. Thebreak in the DNA of donor bacteria take apart and then the release andfragmentation of isolated DNA take place.Ø Then this fragmented DNA gets attached with the recipientDNA.Ø During passage one strand dissolved by enzymes and single strand ofdouble stranded get attached with the recipient single stranded DNA.
Ø Thisincorporated DNA transformed new character in the bacteria.Ø ATPand Rec A a special type of protein which is required in this process. It formcoating around the single stranded DNA and provide protection ii) ARTIFICALTRANSFORMATION:”Inthe laboratory, by applying high voltageelectric field and by high concentration of CaCI2.
Under suchcondition, the bacterial cells are forced to take up foreign DNA. This type oftransformation is called artificial”.MECHANISM:Ø Inthis process recipient cells are able to take up double stranded DNA by thephysical and chemical processes.Ø RecA protein, mediated in the process of integration of foreign DNA with thehomologous chromosome.
Ø Electroporationis another process of promoting competence. By this method, the cells areshocked for the short time with an electric field of 10-20 kV/cm which willcreate holes in the cell membrane for the entry of plasmid DNA. After theelectric shock, the holes are rapidly closed by the cell’s membrane-repairmechanisms.Ø Divalentcation in cold conditions also weakens the cell surface structure so thattransformation becomes easy.
3) TRANSDUCTION:Ø Theprocess in which genetic material is exchanged from one living organism toanotherØ Thismechanism is discovered by two scientists namely Joshua Leaderberg & NortorZinder.Ø Thismechanism occurs in bacteriophageTYPES:There are two types oftransductionØ SpecializedtransductionØ Generalizedtransduction A. SPECIALIZEDTRANSDUCTION: Ø Tothe bacterial cell wall, a receptor site is present.Ø Atthis receptor site bacteriophage gets attached to the virus to which it attacksand it transfers its nucleic Acid to the host cell.Ø Thistransferred nucleic acid encodes specialized protein in host cells.Ø Theseproteins may include repressor protein.
Ø Thesetypes of proteins inhibit the replication in bacteria by this repressorprotein.Ø Whileviral chromosomes carry with it the part of bacteriophage nucleic acid PROPHAGE.Ø Thevirus with prophage step is called LYSOGENIC and the peaceful collaboration ofbacteriophage & virus is called LYSOGENY.Ø Thislysogenic phase continuous many generations and the time till both remainstogether both types of genetic material transfers to the progeny.
Ø Sometimesthis phage genetic material gets separate from the other and startssynthesizing its own genetic material via replication & statsrecombination.Ø Thiskind of transduction is named as specialized transduction.B. GENRALIZEDTRANSDUCTION:Ø Morefrequently occur.Ø Atstart the mechanism of both type of transduction is same.
Ø Theonly difference is that after bursting of host cell the bacteriophage containsome fragments of the viral genome.REASONSOF DNA DAMAGE: There are numerous reasons to damageto the DNA. Some of which are given below: Ø DNA damage by oxidationØ Hydrolytic attack on DNAØ Radiation/UV-damageØ Exposure Ø Free radicals from normal metabolicprocessesØ Errors in DNA synthesisØ Mutagens exposureØ Breakage of strandsØ Loss of baseØ Damage to sugarØ Damage of base Ø adduct TYPESOF REPAIR MECHANISMS IN PROKARYOTES:Following are some repair mechanismsin prokaryotes.1. Recombination repair (a) Broken fork repair(b) Double strand break repair(c) Gap filling recombination repair2.
Excision repair(a) Mismatch repair(b) Base excision repair(c) Nucleotide excision repair3. Single strand breaks4. Error prone repair5. Direct repair6.
Double strand breaksRELATIONSHIPOF RECOMBINATION AND REPAIR MECHANISMRecombination is called homologousif the recombination occurs in identical type of chromosomes.If we talk about mechanism ofrepairing in prokaryotes here recombination reactions are used and three typesare used in repairing. The name of this useful mechanism is following:Ø Broken fork repairØ Double-strand break repairØ Recombination gap-?lling repair 1. BROKENFORK REPAIR: Collapsedfork which is damaged in replication is getting back. This damaged fork issubjected to restoration. After regaining its activity this replication fork isable to perform its activity. On stolen template with the help of replicationthese forks appear.
Endonucleases may present this type of forks.Mutation is the mainreason which arise this forks for replication. Nicked or damaged chromosome istarnished if recombination does not repair damaged forks.MECHANISM:Ø Here3′ end is exposed, of the damaged chromosome by consuming the other strand. Ø Thendisplacement loops or D-loop is synthesized. Ø Thisloop created by entering the unprotected 3′ end in to the duplex of homologous.
Ø Bythis way one strand from duplex is get displaced & an intermediate branchshape is produced. Here branch migration occursby the movement of exposed end into the duplex and the outside movement of thebranch of duplex. Both of these move opposite to each other.Ø HereHolliday Junction is made bythis movement. Ø Thisholiday junction is basically a branch of four strands. Damaged fork activityis regained by ligation of strands from D-loop & Holliday junction.
Ø Thismechanism is detailed & extensively studied in prokaryote i.e. E.
coli. In E. coli, this mechanism is alsohaving many names. Ø RecombinationDependent Replication” (RDR) Ø DoubleStrand End Repair (DSE repair)Ø Abovethese are the names of this mechanism.Ø Nowrepairing of the damaged fork is completed. Ø Hereanother important step, the arise of replisome occurs. Replicationis restarted by these replisome.
in prokaryotes unique features are required toinitiate replisome.v2. EXCSIONREPAIR: Excisionrepair have further types. All of which have same mechanism of following steps:1. Cut 2.
Copy 3. PasteØ Cutting:enzyme and molecules are used to cut the damaged part of DNA.Ø Copy:here DNA polymerase helps to remove the damage. Ø Itcopies the damaged part. It helps in synthesis at 3’OH. Ø Thissynthesis starts at site of nick or gap.
Ø Paste:here ligases are used to join the DNA.Ø These3 steps are involved in all three-excision repair mechanism namely A. Miss-match repair (MMR)B. Base-excision repair (BER)C.
Nucleotide Excision Repair (NER)A. DNAMISSMATCH REPAIR OR INSERTION-DELETION LOOP (MMR):Ø Thissystem is used for the repairing of bases which are wrongly inserted, or anyerror related to the bases in DNA.Ø Base’stautomerization is the main cause of miss-match. Ø Thisis helpful in repairing, recombination & replication in case of any error. Ø Strandspecificity is one of the characteristic of this type of repair.
Ø Inreplication process, usually errors are usually present in daughter strand.sofor repair initiation the first step is to recognize the daughter strand.Ø Herehemi-methylation plays the main role in recognition of parental strand fromdaughter strand.Ø Damageto DNA by different sources like internal or external could also be removed bythis method.viMECHANISM:Ø Herejust recognition of the daughter or newly synthesized is done & thenwrongly inserted base or damage is repaired. Ø Themain work is to identification of daughter strand.
in eukaryotes &prokaryotes the recognition mechanism is different. Ø Onlyin gram-negative bacteria this mechanism is still known while in otherorganisms it is still unknown.EXAMPLES:G & T, C & G both type of pairing fall in the category of miss-matchrepair. By this methodthousands of repairs could be done.B. BASEEXCISION REPAIR (BER): Ø Thismechanism is also used in repairing the damage of DNA.
Ø Duringthe whole cycle of cell this mechanism is useful.Ø Usuallyhelpful in removing the small lesions of damage.Ø Thisrepair differs from nucleotide excision repairs.Ø Themaindifference is in the recognition steps where substrate varies.1. This system also removes that type ofdamage even which is not involved in distortion of DNA.
2. Although G, U is not involved in thedistortion of DNA but still uridine is removed damage base.3. The other difference removal of purinesor pyrimidines from DNA. This is done by the breakdown of glycosidic bond.
MECHANISMØ Glycosylasesare used here. This helps to identify the damaged bases in DNA.Ø Byresolving the N-glycosidic bond glycosylases done their work.1. Removal of defected bases is thefunction of glycosylases.2. Function of DNA polymerase is done bythe help of AP endonuclease by facilitating with primer.
3. This DNA polymerase removes bases &fills these gaps via exonuclease & polymerase activity respectively.vii C. NUCLEOTIDEEXCISION REPAIR: Ø Thebasic principle of this mechanism is same like other excision mechanisms.
Ø Thisis used to remove the damage that is caused by UV-light & chemical adductsthat areØ Usuallybulky.Ø Thismechanism occurs both in eukaryotes as well as prokaryotes.Ø Inprokaryotes, this system uses usually UvrABC exonuclease & UvrD helicase. viiiGAP-FILLINGRECOMBINATIONAL REPAIR:Inhomologous recombination third type of repair is single-strand DNA gaps forfilling of this gap the reaction between homologous chromosomes occurs in whichstrands transfer.Ø Agap due to incomplete replication can also be produced in daughter-strand. Theincomplete replication can occur due to damage in template strand, DNAstructure or protein that play role in binding.
Ø Oneof the first mechanisms for recombinational repair (Rupp et al., 1971) was gaprepairing in daughter strand (DSGR) it is the most poorly understood.Ø Theprocess has been best studied in E. coli after UV irradiation, where it hasbeen called “post-replication repair.” Ø Thedaughter strands with gaps are having low molecular weight and that waspreviously assayed by using thymidine labeling and denaturing sucrose gradientcentrifugation. By this, new strand become complemented with the parentalstrand. This signifies the mechanism of Recombinational gap filling.
MECHANISM:Ø Thegap in the single-strand is complement with the intact DNA molecule. Ø Thestrand invaded and invasion may be aided by displacement by one or both of thestrands that flank the break. Ø Becausethe synapsis does not involve a free end, to overcome this problemtopoisomerases are used in this process.
Ø DNAsynthesis starts from the ends by replacing the transferred strand, setting upa branched molecule, consisting of one or more crossed strands. Ø Twoduplex molecules are restoring by cleavage and the gap is filled. Ø Crossoveror non-crossover products are produced by the cleavage of these junctions, asin DSB model.ix DOUBLE-STRAND BREAK REPAIR:Break in double-strand arerepaired by 2 different types of mechanism. 1. Homologousrecombination (HR) 2. Non-homologous endjoining (NHEJ).
HOMOLOGOUS RECOMBINATION:o Interactions in homologues chromosomesrepair the breaks in the double strand. o E. coli lacking the non-homologous endjoining pathway, so homologous recombination is the only way to recover brokenchromosomes.
o Yeast mitotic recombination process can alsounderstand by DSB repair.MECHANISM:Ø D-loopor Holliday junction is formed by the invasion of broken end; in thisintermediate displaced strand can recruit and pair with the second resectedbroken end. Ø Theinformation can be restored by DNA synthesis in paired intermediate which hadlost from any broken end. Ø TwoHolliday junctions are created by this process (a feature noticeably confirmedfor yeast meiotic recombination intermediates) Ø Twointact chromosomes are produced by the resolution of Holliday junction, whichare “crossover” or “non-crossover” products (may or may not have exchanged theflanking information).Ø Becausethe shape of E.coli chromosome is circular, dimeric circular chromosome willform by crossing over between sisters chromosome.
Ø ProteinXerCD is present at various sites near the terminus which is important in thesite-specific recombination system, this protein converted dimers into monomerfor appropriate separation to daughter cells.Ø Inan alternative version of double-strand break repair is also known as “synthesis-dependent strand annealing”(SDSA), capture of the twobroken ends occurs sequentially. Ø Thesynapsis of the second end is dependent on the successful synapsis of the firstend. After the invasion of first strand the DNA synthesis happens from theinvading strand which extends it away from the point of the original break.Ø Whenthe process ended the invading strands are separated from the intermediates forthe annealing of other processed broken ends. Ø Theprocess of break repair accomplishes, without the cleavage of any branchedstructure, and so crossover products are not yielded.Ø InE.
coli, DSB repair has been studied by introducing double-strand breaks fromsequence-specific endonucleases and excision of transposable elements. xCAUSES OF BREAKS:Ionizing radiation causes:Ø Doublestrand break also produces DSBs Ø Lethallesion to bacteria.Ø Lesionin sugar oxidation and base.Ø Single-strandbreaks. It is necessary to have intactsister chromosome for the repairing of broken strand although homologoussequence recombination with other chromosome can also be detected.xi COMPARISION:Ø Inpractice, it is difficult to distinguish between the one-ended replication forkbreak repair and two-ended double-strand break repair.
Ø Theseboth (double-stranded and replication fork) are not associated with each other.But an agent that causes the DSB also produces broken forks. Ø Theyalso differ from each other in the aspect that repair of two ends correspondeach other, so it may require particular functions which is not required forsingle-end break repair. Ø Onthe other hand, uncoordinated and independent one-ended repair is involved inrepair of two broken end and with each process establishing a replication fork.Ø Gapfilled recombination differs from the DSB recombination process due to theinitiating substrate.Ø Differentproteins are involved in this mechanism as compare to the DSB mechanism. Ø Inaddition, due to incomplete replication single-strand gaps are produced whichwill bind by single stranded DNA binding protein. In the complete process,recombination protein will remove single stranded break.
Ø Themechanism of gap filling recombination was not studied earlier in E.coli. onlyDSB-mediated processes were reported after conjugation and transduction.
Ø InDNA damage repair, it can be difficult to distinguish between DSB repairs andgap filled recombination repair. Ø DSBare also produced by the agents that produce gaps e.g., UV irradiation.
Ø Inthe process of DNA damage repair, gap repair are usually underestimated andthat gaps which are not repaired converted to the DSB and which will furtherrepaired by the DSB repair mechanism. NON-HOMLOGOUSEND JOINING:Ø This mechanism occurs occasionally inprokaryotes.Ø Especially in prokaryotes this mechanism doesnot occur.Ø While some prokaryotes also still use thismechanism of homologous end joining.Ø Here in prokaryotes just in little number ofproteins all mechanism of non-homologous end joining is done.Ø Here this mechanism occurs in its simplestform.xiixiii