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DEOXYRIBONUCLEIC ACID (DNA):

DNA
is the chief component of any living organism.it is responsible for the
transfer of genetic information from one generation to the next; this trait
makes DNA hereditary material. Monomer of DNA is
nucleotide that takes up sugar, phosphate group and a base from the four
universal bases:

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·        
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 in
which 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 of
nucleotides or strand breakage etc.iii

 

 

RECOMBINATION:

“A
type of genetic recombination in which exchange of nucleotide sequences occur
between two similar or identical molecules of DNA is called recombination”.

Ø  For
the exchange of genetic material in different species of bacteria and viruses
the process of recombination is used.

Ø  The
three main processes involved in recombination of bacteria:

1)      Conjugation

2)      Transformation

3)      Transduction

 

1)     
CONJUGATION:

“The
process through which by the direct contact transfer of genetic material from
one bacterium to the other takes place contact is called conjugation.”

o  
In E. coli this process was postulated
by Joshua Lederberg and Edward Tatum (1946).

o  
In 1958, they were also awarded Nobel price
on the work of salmonella, vibrio and pseudomonas.

MECHANISM:

Ø  In
the process of conjugation there are two bacteria one donates the genetic
material and other act as recipient which receives the DNA.

Ø  Fertility
factor or F factor is present in the donor cell. The F factor allows the
formation of pilus in the donor cell. With the help of pillus the donor cell
contact the recipient cell.

Ø  By
the pilus two bacteria come together and donor transfer genetic material to the
recipient.

Ø  In
last step, both cells containing single stranded DNA, replicate it and form
double stranded F plasmid which is identical to the original one. Now both
cells are donors or F+ because F-plasmid has ability for the pili
formation.

Ø  Usually
genetic material is in the form of plasmid, or a small, circular DNA piece.

Ø  Plasmid
that carries antibiotic resistance genes can also transferred by the
conjugation process.iiiiv

 

2)     
TRANSFORMATION:

“In molecular biology, transformation is the genetic modification of a cell by the direct uptake and expression
of DNA from its surroundings usually through the liquid medium.”

Ø  It was described by FREDERICK GRIFFITH in 1928

TYPES:

There
are two types of transformation:

       
i.           
Natural transformation

      ii.           
Artificial or forced transformation

i)       
NATURAL TRANSFORMATION:

“The
type of transformation in which no force is required for the transfer of
genetic material is natural transformation.”

MECHANISM:

The transfer of isolated fragment is transformation

Ø  The transformation takes place in the mixed population. The
break in the DNA of donor bacteria take apart and then the release and
fragmentation of isolated DNA take place.

Ø  Then this fragmented DNA gets attached with the recipient
DNA.

Ø  During passage one strand dissolved by enzymes and single strand of
double stranded get attached with the recipient single stranded DNA.

Ø  This
incorporated DNA transformed new character in the bacteria.

Ø  ATP
and Rec A a special type of protein which is required in this process. It form
coating around the single stranded DNA and provide protection

 

ii)      ARTIFICAL
TRANSFORMATION:

“In
the laboratory,  by applying high voltage
electric field and by high concentration of CaCI2. Under such
condition, the bacterial cells are forced to take up foreign DNA. This type of
transformation is called artificial”.

MECHANISM:

Ø  In
this process recipient cells are able to take up double stranded DNA by the
physical and chemical processes.

Ø  Rec
A protein, mediated in the process of integration of foreign DNA with the
homologous chromosome.

Ø  Electroporation
is another process of promoting competence. By this method, the cells are
shocked for the short time with an electric field of 10-20 kV/cm which will
create holes in the cell membrane for the entry of plasmid DNA. After the
electric shock, the holes are rapidly closed by the cell’s membrane-repair
mechanisms.

Ø  Divalent
cation in cold conditions also weakens the cell surface structure so that
transformation becomes easy.

3)      TRANSDUCTION:

Ø  The
process in which genetic material is exchanged from one living organism to
another

Ø  This
mechanism is discovered by two scientists namely Joshua Leaderberg & Nortor
Zinder.

Ø  This
mechanism occurs in bacteriophage

TYPES:

There are two types of
transduction

Ø  Specialized
transduction

Ø  Generalized
transduction

 

A.     SPECIALIZED
TRANSDUCTION:

 

Ø  To
the bacterial cell wall, a receptor site is present.

Ø  At
this receptor site bacteriophage gets attached to the virus to which it attacks
and it transfers its nucleic Acid to the host cell.

Ø  This
transferred nucleic acid encodes specialized protein in host cells.

Ø  These
proteins may include repressor protein.

Ø  These
types of proteins inhibit the replication in bacteria by this repressor
protein.

Ø  While
viral chromosomes carry with it the part of bacteriophage nucleic acid &
called PROPHAGE.

Ø  The
virus with prophage step is called LYSOGENIC and the peaceful collaboration of
bacteriophage & virus is called LYSOGENY.

Ø  This
lysogenic phase continuous many generations and the time till both remains
together both types of genetic material transfers to the progeny. 

Ø  Sometimes
this phage genetic material gets separate from the other and starts
synthesizing its own genetic material via replication & stats
recombination.

Ø  This
kind of transduction is named as specialized transduction.

B.     GENRALIZED
TRANSDUCTION:

Ø  More
frequently occur.

Ø  At
start the mechanism of both type of transduction is same.

Ø  The
only difference is that after bursting of host cell the bacteriophage contain
some fragments of the viral genome.

REASONS
OF DNA DAMAGE:

 

There are numerous reasons to damage
to the DNA. Some of which are given below:

Ø  DNA damage by oxidation

Ø  Hydrolytic attack on DNA

Ø  Radiation/UV-damage

Ø  Exposure

Ø  Free radicals from normal metabolic
processes

Ø  Errors in DNA synthesis

Ø  Mutagens exposure

Ø  Breakage of strands

Ø  Loss of base

Ø  Damage to sugar

Ø  Damage of base

Ø  adduct

 

 

TYPES
OF REPAIR MECHANISMS IN PROKARYOTES:

Following are some repair mechanisms
in prokaryotes.

1.     
 Recombination repair

(a)    Broken fork repair

(b)   Double strand break repair

(c)    Gap filling recombination repair

2.     
Excision repair

(a)    Mismatch repair

(b)   Base excision repair

(c)    Nucleotide excision repair

3.     
Single strand breaks

4.     
Error prone repair

5.     
Direct repair

6.     
Double strand breaks

RELATIONSHIP
OF RECOMBINATION AND REPAIR MECHANISM

Recombination is called homologous
if the recombination occurs in identical type of chromosomes.

If we talk about mechanism of
repairing in prokaryotes here recombination reactions are used and three types
are used in repairing. The name of this useful mechanism is following:

Ø  Broken fork repair

Ø  Double-strand break repair

Ø  Recombination gap-?lling repair

 

1.      BROKEN
FORK REPAIR:

Collapsed
fork which is damaged in replication is getting back. This damaged fork is
subjected to restoration. After regaining its activity this replication fork is
able to perform its activity. On stolen template with the help of replication
these forks appear. Endonucleases may present this type of forks.

Mutation is the main
reason which arise this forks for replication. Nicked or damaged chromosome is
tarnished if recombination does not repair damaged forks.

MECHANISM:

Ø  Here
3′ end is exposed, of the damaged chromosome by consuming the other strand.

Ø  Then
displacement loops or D-loop is synthesized.

Ø  This
loop created by entering the unprotected 3′ end in to the duplex of homologous.

Ø  By
this way one strand from duplex is get displaced & an intermediate branch
shape is produced. Here branch migration occurs
by the movement of exposed end into the duplex and the outside movement of the
branch of duplex. Both of these move opposite to each other.

Ø  Here
Holliday Junction is made by
this movement.

Ø  This
holiday junction is basically a branch of four strands. Damaged fork activity
is regained by ligation of strands from D-loop & Holliday junction.

Ø  This
mechanism is detailed & extensively studied in prokaryote i.e.  E. coli. In E. coli, this mechanism is also
having many names.

Ø  Recombination
Dependent Replication” (RDR)

Ø  Double
Strand End Repair (DSE repair)

Ø  Above
these are the names of this mechanism.

Ø  Now
repairing of the damaged fork is completed.

Ø  Here
another important step, the arise of replisome occurs. Replication
is restarted by these replisome.in prokaryotes unique features are required to
initiate replisome.v

2.      EXCSION
REPAIR:

Excision
repair have further types. All of which have same mechanism of following steps:

1.     
Cut

2.     
Copy

3.     
Paste

Ø  Cutting:
enzyme and molecules are used to cut the damaged part of DNA.

Ø  Copy:
here DNA polymerase helps to remove the damage.

Ø  It
copies the damaged part. It helps in synthesis at 3’OH.

Ø  This
synthesis starts at site of nick or gap.

Ø  Paste:
here ligases are used to join the DNA.

Ø  These
3 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.     DNA
MISSMATCH REPAIR OR INSERTION-DELETION LOOP (MMR):

Ø  This
system is used for the repairing of bases which are wrongly inserted, or any
error related to the bases in DNA.

Ø  Base’s
tautomerization is the main cause of miss-match.

Ø  This
is helpful in repairing, recombination & replication in case of any error.

Ø  Strand
specificity is one of the characteristic of this type of repair.

Ø  In
replication process, usually errors are usually present in daughter strand.so
for repair initiation the first step is to recognize the daughter strand.

Ø  Here
hemi-methylation plays the main role in recognition of parental strand from
daughter strand.

Ø  Damage
to DNA by different sources like internal or external could also be removed by
this method.vi

MECHANISM:

Ø  Here
just recognition of the daughter or newly synthesized is done & then
wrongly inserted base or damage is repaired.

Ø  The
main work is to identification of daughter strand.in eukaryotes &
prokaryotes the recognition mechanism is different.

Ø  Only
in gram-negative bacteria this mechanism is still known while in other
organisms it is still unknown.

EXAMPLES:
G & T, C & G both type of pairing fall in the category of miss-match
repair.

By this method
thousands of repairs could be done.

B.     BASE
EXCISION REPAIR (BER):

 

Ø  This
mechanism is also used in repairing the damage of DNA.

Ø  During
the whole cycle of cell this mechanism is useful.

Ø  Usually
helpful in removing the small lesions of damage.

Ø  This
repair differs from nucleotide excision repairs.

Ø  The
main
difference is in the recognition steps where substrate varies.

1.     
This system also removes that type of
damage even which is not involved in distortion of DNA.

2.     
Although G, U is not involved in the
distortion of DNA but still uridine is removed damage base.

3.     
The other difference removal of purines
or pyrimidines from DNA. This is done by the breakdown of glycosidic bond.

MECHANISM

Ø  Glycosylases
are used here. This helps to identify the damaged bases in DNA.

Ø  By
resolving the N-glycosidic bond glycosylases done their work.

1.     
Removal of defected bases is the
function of glycosylases.

2.     
Function of DNA polymerase is done by
the help of AP endonuclease by facilitating with primer.

3.     
This DNA polymerase removes bases &
fills these gaps via exonuclease & polymerase activity respectively.vii

 

C.     NUCLEOTIDE
EXCISION REPAIR:

 

Ø  The
basic principle of this mechanism is same like other excision mechanisms.

Ø  This
is used to remove the damage that is caused by UV-light & chemical adducts
that are

Ø  Usually
bulky.

Ø  This
mechanism occurs both in eukaryotes as well as prokaryotes.

Ø  In
prokaryotes, this system uses usually UvrABC exonuclease & UvrD helicase. viii

GAP-FILLING
RECOMBINATIONAL REPAIR:

In
homologous recombination third type of repair is single-strand DNA gaps for
filling of this gap the reaction between homologous chromosomes occurs in which
strands transfer.

Ø  A
gap due to incomplete replication can also be produced in daughter-strand. The
incomplete replication can occur due to damage in template strand, DNA
structure or protein that play role in binding.

Ø  One
of the first mechanisms for recombinational repair (Rupp et al., 1971) was gap
repairing in daughter strand (DSGR) it is the most poorly understood.

Ø  The
process has been best studied in E. coli after UV irradiation, where it has
been called “post-replication repair.”

Ø  The
daughter strands with gaps are having low molecular weight and that was
previously assayed by using thymidine labeling and denaturing sucrose gradient
centrifugation. By this, new strand become complemented with the parental
strand. This signifies the mechanism of Recombinational gap filling.

MECHANISM:

Ø  The
gap in the single-strand is complement with the intact DNA molecule.

Ø  The
strand invaded and invasion may be aided by displacement by one or both of the
strands that flank the break.

Ø  Because
the synapsis does not involve a free end, to overcome this problem
topoisomerases are used in this process.

Ø  DNA
synthesis starts from the ends by replacing the transferred strand, setting up
a branched molecule, consisting of one or more crossed strands.

Ø  Two
duplex molecules are restoring by cleavage and the gap is filled.

Ø  Crossover
or non-crossover products are produced by the cleavage of these junctions, as
in DSB model.ix

DOUBLE-STRAND BREAK REPAIR:

Break in double-strand are
repaired by 2 different types of mechanism.

1.     
Homologous
recombination (HR)

2.     
Non-homologous end
joining (NHEJ).

HOMOLOGOUS RECOMBINATION:

o  
Interactions in homologues chromosomes
repair the breaks in the double strand.

o  
E. coli lacking the non-homologous end
joining pathway, so homologous recombination is the only way to recover broken
chromosomes.

o  
Yeast mitotic recombination process can also
understand by DSB repair.

MECHANISM:

Ø  D-loop
or Holliday junction is formed by the invasion of broken end; in this
intermediate displaced strand can recruit and pair with the second resected
broken end.

Ø  The
information can be restored by DNA synthesis in paired intermediate which had
lost from any broken end.

Ø  Two
Holliday junctions are created by this process (a feature noticeably confirmed
for yeast meiotic recombination intermediates)

Ø  Two
intact chromosomes are produced by the resolution of Holliday junction, which
are “crossover” or “non-crossover” products (may or may not have exchanged the
flanking information).

Ø  Because
the shape of E.coli chromosome is circular, dimeric circular chromosome will
form by crossing over between sisters chromosome.

Ø  Protein
XerCD is present at various sites near the terminus which is important in the
site-specific recombination system, this protein converted dimers into monomer
for appropriate separation to daughter cells.

Ø  In
an alternative version of double-strand break repair is also known as “synthesis-dependent strand annealing”
(SDSA), capture of the two
broken ends occurs sequentially.

Ø  The
synapsis of the second end is dependent on the successful synapsis of the first
end. After the invasion of first strand the DNA synthesis happens from the
invading strand which extends it away from the point of the original break.

Ø  When
the process ended the invading strands are separated from the intermediates for
the annealing of other processed broken ends.  

Ø  The
process of break repair accomplishes, without the cleavage of any branched
structure, and so crossover products are not yielded.

Ø  In
E. coli, DSB repair has been studied by introducing double-strand breaks from
sequence-specific endonucleases and excision of transposable elements. x

CAUSES OF BREAKS:

Ionizing radiation causes:

Ø  Double
strand break also produces DSBs

Ø  Lethal
lesion to bacteria.

Ø  Lesion
in sugar oxidation and base.

Ø  Single-strand
breaks.

It is necessary to have intact
sister chromosome for the repairing of broken strand although homologous
sequence recombination with other chromosome can also be detected.xi

 

COMPARISION:

Ø  In
practice, it is difficult to distinguish between the one-ended replication fork
break repair and two-ended double-strand break repair.

Ø  These
both (double-stranded and replication fork) are not associated with each other.
But an agent that causes the DSB also produces broken forks.

Ø  They
also differ from each other in the aspect that repair of two ends correspond
each other, so it may require particular functions which is not required for
single-end break repair.

Ø  On
the other hand, uncoordinated and independent one-ended repair is involved in
repair of two broken end and with each process establishing a replication fork.

Ø  Gap
filled recombination differs from the DSB recombination process due to the
initiating substrate.

Ø  Different
proteins are involved in this mechanism as compare to the DSB mechanism.

Ø  In
addition, due to incomplete replication single-strand gaps are produced which
will bind by single stranded DNA binding protein. In the complete process,
recombination protein will remove single stranded break.

Ø  The
mechanism of gap filling recombination was not studied earlier in E.coli. only
DSB-mediated processes were reported after conjugation and transduction.

Ø  In
DNA damage repair, it can be difficult to distinguish between DSB repairs and
gap filled recombination repair.

Ø  DSB
are also produced by the agents that produce gaps e.g., UV irradiation.

Ø  In
the process of DNA damage repair, gap repair are usually underestimated and
that gaps which are not repaired converted to the DSB and which will further
repaired by the DSB repair mechanism.

 

 

 

NON-HOMLOGOUS
END JOINING:

Ø  This mechanism occurs occasionally in
prokaryotes.

Ø  Especially in prokaryotes this mechanism does
not occur.

Ø  While some prokaryotes also still use this
mechanism of homologous end joining.

Ø  Here in prokaryotes just in little number of
proteins all mechanism of non-homologous end joining is done.

Ø  Here this mechanism occurs in its simplest
form.xiixiii

                                                                                     

 

 

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