➢ Template: In terms of dna is a stretch of sequence onto which molecules would bind in a order

to create a macromolecule with a unique sequence and function

➢ one strand is the complement of the other and each strand provides the template for a new
strand with a complementary sequence
➢ DNA Replication Follows a Set of Fundamental Rules
➢ DNA Replication Is Semiconservative (Meams half conserved) Each DNA strand selves as a
template for the synthesis of a new strand, producing two new DNA molecules, each with one
new strand and one old strand. This is semiconservative replication.
➢ Meselson & Stahl carried out an experiment where Cells were Grown for many generations in a
medium containing only heavy nitrogen, 15N where all the bases were heavy N15, later when
the cells were transferred to a medium containing only light nitrogen 14N when cells replicated it
was found that the cells contain both light 14N & Heavy 15N strand, when a second cycle of
replication was carried out then a hybrid DNA band containing 14N DNA was observed this
confirms the g semiconservative replication
➢
➢ Replication Begins
at an Origin and Usually
proceeds Bidirectionally
➢ chromosome of E. coLi is
a single huge circle, 1.2 mm long
➢ where parent DNA is
being unwound and the
separated strands quickly
replicated
➢ replication of bacterial
chromosomes is bidirectional:
both ends of the loop have active
replication forks
➢ In this system the
replication loops always initiate
at a unique point, which is
termed an origin.
➢ replication is usually
bidirectional. For circular DNA
molecules, the two replication
forks meet at a point on the side
of the circle opposite to the
origin
➢
➢ DNA Synthesis Proceeds in a E,-->S,Direction and Is Semi Discontinuous
➢ A new strand of DNA is always synthesized in the 5'->3' direction
➢ with the free 3, OH as the point at which the DNA is elongated
➢ the strand serving as the template is read from its 3' end toward its 5’ end
➢ that one of the new DNA strands is synthesized in short pieces called Okazaki fragments
➢ This work ultimately led to the conclusion that one strand is synthesized continuously (leading
strand) and the other discontinuously (lagging strand)
➢ The continuous strand, or leading strand, is the one in which 5'->3' synthesis proceeds in the
same direction as replication fork movement
➢ The discontinuous strand, or lagging strand, is the one in which 5'->3' synthesis proceeds in the
direction opposite to the direction of fork movement.
➢ Okazaki fragments range in length from a few hundred to a few thousand nucleotides
➢
➢
➢
➢
➢ DNA ls Degraded by Nucleases
➢ nucleases are the enzyme which degrade the DNA they are also known as DNases which are
specific for DNA
➢ cells contain two types of nucleases exonucleases and endonucleases,
➢ Exonucleases they degrade nucleic acid from one end of the molecule 5' → 3' or the 3' → 5' by
removing nucleotides from 5’ OR 3’ end
➢ Endonucleases can degrade at degrade at specific internal sites in a nucleic acid strand reducing
it to smaller strands
➢
➢ DNA is Synthesized by DNA Polymerases
➢ DNA polymerase 1 features are not common to other DNA polymerase
➢ in this a phosphoryl group transfer occurs at 3rd -hydroxyl group of the nucleotide ay at the 3’
end of the growing strand
➢ the Nucleophilic attack occurs at alpha phosphorus of the 5th carbon inorganic phosphate is
released in this reaction
➢ However, noncovalent base-stacking and base-pairing interactions provide additional
stabiLization to the lengthened DNA

➢
➢ all DNA polymerase requires a template. The polymerization reaction is guided by a template
DNA strand
➢ polymerase requires a primer.(A primer is a strand segment (complementary to the template)
with a free 3' hydroxyl group to which a nucleotide can be added)
➢ primers are oligonucleotides of RNA which are synthesized by primase
➢ after addition of a nucleotide the polymerase either dissociates or continues further along the
template by adding other nucleotides
➢ processivity: is defined as . The average number of nucleotides added before a polymerase
dissociate, they vary in their processivity some add few nucleotide while others add thousands
➢ Replication ls Very Accurate
➢ In E. coli,, a mistake is macle only once for every 109 to 1010 nucleotides added, an error occurs
only once per 1,000 to 10,000 replication
➢ during polymerization addition of nucleotide depends both on the addition of the hydrogen
bond and the geometry of the strand, incorrect nucleotide can make hydrogen bond but will not
fit in the active site
➢ DNA polymerase insert one incorrect nucleotide for every 104 to 105 correct one
➢ All DNA polymerase has an 3’ —> 5’ exonuclease activity it double checks nucleotide after it
is added
➢ This nuclease activity of the polymerase helps it to remove wrongly added nucleotide and is
specific for mismatch base pair
➢ if polymerase adds a wrong nucleotide then the polymerase enzyme does not moves forward for
the addition of new nucleotide
➢ This kinetic pause provides the opportunity for a correction
➢ Proofreading The 3'->5' exonuclease activity removes the mispaired nucleotide, and the
polymerase begins again.,
➢ Proofreading improves the accuracy of the polymerization to reaction 102- to 103-fold , c DNA
polymerase I, the polymerizing and proofreading activities have separate active sites within the
same polypeptide
➢ When base selection and proofreading are combined, DNA polymerase leaves behind one net
error for every 106 to 108 bases added

➢
➢ E.coli Has at Least Five DNA Polymerase
➢ More than 90% of the DNA polymerase activity is contributes by DNA polymerase I
➢ it was found that (1. First, the rate at which it adds nucleotides is too slow, 2. d, DNA polymerase
I has a relatively low processivity, 3. DNA polymerase I clearly does not act alone )
➢ E. coli, DNA polymerase II and DNA polymerase III s. DNA polymerase II is an enz).rne involved
in one type of DNA repair DNA polymerase III is the principal replication enzyme in E. coli
➢ DNA polymerase I, then, is not the primary enzyme of replication; instead it performs a host of
clean-up functions during replication, recombination, and repair
➢ Nick translation. In this process, an RNA or DNA strand paired to a DNA template is
simultaneously degraded by the 5'---> 3' exonuclease activity of DNA polymerase I and replaced
by the polymerase activity of the same enzyme
➢ DNA polymerase III is much more complex than DNA polymerase I
➢ polymerase and proofreading activities of DNA polymerase reside in its α (alpha) & ε (epsilon)
submit of protein
➢ The θ (Theta) subunit associates with α & ε to form polymerase with low processivity, another
complex called γ (gamma) complex has 5 different subunit τ2γδδ (2 Tou, 1 gamma, 2 delta)
➢ the polymerase activity of DNA Polymerase I is less but enhance with adding of β subunit
➢ The B subunits associate in pairs to form donut-shaped structures that encircle the DNA and act
like clamp
➢ The B sliding clamp prevents the dissociation of DNA polymerase III from DNA, dramatically
increasing processivity-to greater than 500,000
➢ Each dimer associates with a core subassembly of polymerase III and slides along the DNA as
replication proceeds
➢ RNA primers are removed and replaced by DNA; in E coli, this is one of the many functions of
DNA polymerase I
➢ DNA Replication Requires Many Enzymes and Protein factors
➢ helicases separation of the two parent strands. This is accomplished by helicases is the enzymes
that move along the DNA and separate the strands using chemical energy from ATP
➢ topoisomerases Strand separation creates topological stress in the helical DNA structure which
is relieved by the action of
➢ The separated strands are stabilized by DNA binding proteins (SSB)
➢ primases short segments of RNA synthesized by enzymes known as primases
➢ RNA primers are removed and replaced by DNA; in E coli, this is one of the many functions of
DNA polymerase I
➢ DNA ligases after removal of primer the gap is filled, a nick is remained in the DNA, these nicks
are filled by DNA ligases
 ➢
➢

Replication occurs in three stages initiation, elongation, and termination,

Initiation:

⮚ In e.coli the origin of replication has 245 Bp , the conserved sequence has 5 repeats of 9 Bp
sequence called R site . region rich in A-T sequence called the DNA unwinding element (DUE).
And has additional binding site called I site, IHF & FIS
⮚ At least 10 different types of protein/enzymes participate in initiation phase
⮚ They open DNA at origin and establish a prepriming complex (Place where DNA replication can
begin)
 ⮚ DnaA protein forms oligomers and hydrolyze ATP

⮚ The ATP-bound form is active and the ADP-bound form is inactive

⮚ 8 DnaA protein bound to ATP assemble to form a helical complex binding at R&I site , has higher
affinity towards R site

⮚ The binding of DNA around the complex induces a positive super coil this leads to the break of
A-T Rich DUE region
⮚ DnaC protein (an atp bound protein) loads DnaB protein on the separated strand
⮚ DnaC a hexamer binds to the hexamer of DnaB (is Helicase protein)
⮚ Two DnaB proteins are loaded in each strand of the Dna
⮚ The ATP present in DnaC is hydrolysed which releases DnaC
⮚ DnaB migrates along the DNA strand unwinding the DNA in opposite direction of each creating
two replication forks
⮚ As the strands get separated SSB proteins come and bind to the strand
⮚ DNA gyrase reduces the stress caused by unwinding of the strands

Elongation
➢ elongation includes synthesis of leading and lagging strand synthesis it involves many enzymes
➢ it begins with unwinding of the parent DNA strand by helicase , topoisomerases helps in
reducing the physical stress.SSB protein then bind to separated strands
➢ LEADING STRAND synthesis begins with the synthesis by primase (DnaG Protein) which adds a
RNA primer at the replication origin, primase interact with helicase to start the reaction
➢ This leads to the synthesis of primer, primer is synthesized opposite to that in which the DnaB
helicase is moving
➢ DnaB helicase moves along the strand that becomes the lagging strand in DNA synthesis
➢ Deoxyribonucleotides are added to this primer by a DNA polymerase III, leading strand
continuous with the unwinding of DNA
➢ LAGGING STRAND synthesis in short Okazaki fragments, strats by RNA primer synthesis followed
by , DNA polymerase III binds to the RNA primer and adds deoxyribonucleotides
➢ both the leading and lagging strand are synthesized by DNA polymerase III, primosome, a protein
complex containing DNA primase and DNA helicase
➢ DNA polymerase III uses one set of its core subunits to synthesize the leading strand
continuously, while the other set of core subunits to from one Okazaki fragment to the next on
the looped lagging strand
➢ DnaG primase occasionally associates with DnaB helicase and synthesizes a short RNA primer as
the unwinding of the DNA occurs
➢ The complete replication apparatus moving along the DNA molecule at a replication fork is called
the replisome. includes all the proteins
➢ The replication proteins are thus linked together into a single large unit enabling DNA to be
synthesized on both sides of the replication fork in a coordinated and efficient manner
➢ Beta sliding clamp is positioned at the primer which helps to keep the polymerase intact
➢ Beta sliding clamp gets dissociate from the polymerase When synthesis of an Okazaki fragment
has been completed this marks the complete synthesis of a single Okazaki fragment
➢ the beta loading complex binds to ATP and to the new Beta sliding clamp this binding opens the
ring hear enters the new lagging strand which has a primer to it
➢ the clamp loading complex hydrolyse the ATP
➢ The replisome promotes rapid DNA synthesis, adding -1,000 nucleotides per second in both the
leading and the lagging strands
➢ RNA primer are removed by 5’ —> 3’ exonuclease activity of DNA polymerase I and the nick are
sealed by DNA ligase
➢
➢
➢ Termination when the replication fork meets the region containing Ter sequence the replication

stops
➢ the replication fork can enter but cannot leave the Ter factor
➢ A complex called Ter-Tus can stop the replication in only one direction
➢ out of the two replication fork when one of the fork meets the Tus-Ter complex it Halts the
replication the other fork halts when it meets the first halted replication fork
➢ catenanes are called the place where two circular DNA are linked to each other as a chain
➢ separation of catenanes requires s topoisomerase IV these separated chromosomes are the new
daughter cells

➢
➢

⮚ Replication in Eukaryotic cells ls Both Similar And More complex

⮚ The DNA molecules in eukaryotic cells are considerably Larger than those in bacteria and are
organized into complex nucleoprotein structure
⮚ eukaryotic replication is regulated and coordinated with the cell cycle
⮚ Yeast has defined replication origins called autonomously replicating sequences (ARS), or
replicators.
⮚ Much of this regulation involves proteins called cyclins and the cyclin-dependent kinases
(CDKs)
⮚ yeast replicator is 150 bp & has about 400 replicators are distributed among the 16 chromosome
⮚ Regulation ensures that all cellular DNA is replicated once per cell cycle
⮚ the main event in initiation of replication in eukaryotes is loading of the Helix a heterohexamer
complex of minichromosome maintenance (MCM) proteins (ring shaped MCM2-7 helicase)
⮚ MCM2-7 helicase functions like a bacterial DnaB helicase, this MCM2-7 helicase is loaded by
another protein called ORC (origin recognition complex)
⮚ ORC has five AAA+ ATPase domains and is analogous to the bacterial DnaA
⮚ other proteins CDC6 (cell division cycle), CDTI these both proteins are required to load the
MCM2-7 complex
⮚ The rate of movement of the replication fork in eukaryotes (-50 nucleotides/s) is only
one-twentieth that observed in E. coli
⮚ DNA polymerase α a multisubunit enzyme of which one subunit has primase activity and the
largest subunit contains the polymerase activity, but has no proofreading 3’---> 5’ exonuclease
activity
⮚ DNA polymerase α is believed to function only in the synthesis of short primers for Okazaki
fragments on the lagging strand
⮚ the primers added by DNA polymerase α are extended by DNA polymerase δ
⮚ This enzyme is associated and stimulated by proliferating cell nuclear antigen PCNA, PCNA is
found in nuclei of proliferating cells
⮚ The three-dimensional structure of PCNA is similar to DNA polymerase III PCNA formins a
circular clamp that greatly enhances the processivity of the polymerase
⮚ DNA polymerase δ has 3’---> 5’ proofreading exonuclease activity and seems to carry out both
leading and lagging strand synthesis
⮚ DNA polymerase ε replaces DNA polymerase δ in DNA repair mechanism, function as replication
fork & removes primers
⮚ RPA (replication protein A) is a eukaryotic single-stranded DNA-binding protein, equivalent in
function to the E. coli, SSB protein
⮚ RFC (replication factor C) is a clamp loader for PCNA, has similarity to bacterial clamp loading γ
of bacterial cell
⮚ telomeres involves the termination of replication in eukaryotes
⮚ DNA Repair System
⮚ Mismatch Repair
⮚ inefficient energetically
⮚ Due to Deamination Cytosine is replaced with Uracil
⮚ The mismatches are nearly always corrected to reflect the information in the old (template)
strand
⮚ The cell accomplishes this by tagging the template DNA with methyl groups to distinguish it from
newly synthesized strands
⮚ this is distinguish by methylated & non-methylated strand, the parent strand is methylated and
newly synthesized daughter strand is not methylated, methylation takes place only at GATC
sequence and only the Adenine is methylated by DAMmethylase
⮚ Immediately after passage of the replication fork, there is a short period during which the
template strand is methylated but the newly synthesized strand is not
⮚ MutS has the ability to identify the mismatch, then it recruits MutL which has the ability to
distinguish b/w methylated and non-methylated strand, MutL binds to methylated strand
⮚ MutL protein forms a complex with MutS protein and the complex binds to all mismatched base
pairs
⮚ MutH cleaves the mismatch strand
⮚ MutH has a site-specific endonuclease activity that is inactive until the complex encounters a
hemimethylated GATC sequence
⮚ another protein Uvrd is incorporated Basically a helicase, polymerase synthesis the strand a nick
is left which is sealed by ligase
⮚ Mismatch repair is a particularly expensive process for E coli in terms of energy expended
⮚ Base-Excision Repair
⮚ Every cell has a class of enzymes called DNA glycosylases that recognize particularly common
DNA lesion
⮚ cytosine and adenine deamination; see Fig. 8-30a) and remove the affected base by cleaving the
N-glycosyl bond. This cleavage creates an apurinic or apyrimidinic commonly referred to as an AP
site
⮚ Uracil DNA glycosylase remove that results from spontaneous deamination of cytosine
⮚ The most abundant human uracil glycosylase, UNGe it eliminates the U residue inserted in place
of a T
⮚ Once an AP site has been formed by a DNA glycosylase, another type of enzyme must repair it
⮚ The repair is not made by simply inserting a new base and re-forming the N-glycosyl bond.
Instead, the deoxyribose 5'-phosphate left behind is removed and replaced with a new
nucleotide
⮚ Nucleotide-Excision Repair
⮚ DNA lesions that cause large distortions in the helical structure of DNA generally are repaired by
the nucleotide-excision system
⮚ In nucleotide-excision repair a enzyme hydrolyzes two phosphodiester bonds, one on either side
of the distortion caused by the lesion
⮚ in e coli enzyme hydrolyse 5th phosphodiester bond on the 3' side and the eighth
phosphodiester bond on the 5' side to generate a fragment of 12 to 13 nucleotides
⮚ In human the enzyme hydrolyzes the sixth phosphodiester bond on the 3' side and the
twenty-second phosphodiester bond on the 5' side producing a fragment of 27 to 29
nucleotides
⮚ resulting gap is filled-by DNA polymerase I in e coli
⮚ e.coli enzyme complex has 3 subunits UvrA, UvrB, UvrC, UvrA, UvrB, scans the DNA and binds to
the site of lesion, the UvrA proteins leaves the complex forming an UvrB - DNA complex UvrC
binds to UvrB, UvrB makes a lesion followed by UvrC incision
⮚ Direct Repair
⮚ Several types of damage are repaired without removing a base or nucleotide it includes
pyrimidine dimers