Lecture 45 of 223: DNA Replication in Prokaryotes & Eukaryotes- Elongation (31 mins) | CUET (Common University Entrance Test) PG Botany (SCQP07) | Complete Video Course 223 Lectures [156 hrs : 16 mins]

Details

• DNA polymerases catalyze addition of deoxyribonucleotide to growing DNA chain.
• hydroxyl group at 3՚ end of the primer attacks the α-phosphoryl group of the incoming nucleoside triphosphate.
• The leaving group for the reaction is pyrophosphate (β- and γ-phosphates)
• phosphodiester bond forms with concomitant release of pyrophosphate.
• Two divalent metal ions (typically Mg or Zn) bound to DNA polymerase catalyze nucleotide addition.
• The two metal ions (metal ion A and metal ion B) are held in place by interactions with two highly conserved aspartate residues.
• One metal ion primarily interacts with the 3-OH, resulting in a reduced association between the O and the H. This leaves a nucleophilic 3՚0.
• Second metal ion interacts with the β- and γ-phosphates of the triphosphates of the incoming dNTP to neutralize their negative charge.
• After catalysis, the pyrophosphate product is stabilized through similar interactions with a metal ion.
• The subsequent hydrolysis of pyrophosphate by pyrophosphatase, a ubiquitous enzyme, helps to drive the polymerization forward.

Replication Fork

• Replication starts from the origin of replication to the terminus and is accompanied by the movement of the replicating point, called the replication fork.
• In the case of bidirectional replication, two replication forks form that move in opposite directions.
• The entire 4.6 Mb E. coli genome is replicated by two replication forks
• In E. coli, the replication fork proceeds at about 1000 bp/sec/fork.
• In eukaryotic cells, the rate of fork movement is slow, 100 bp/sec/fork.
• The velocity of fork movement has been estimated from the size of a replicon and the duration of its replication period.
• DNA polymerases can distinguish between ribonucleoside and deoxyribonucleoside triphosphates (NTPs and dNTPs).
• This discrimination is mediated by the steric exclusion of rNTPs from the DNA polymerase active site.
• In DNA polymerase, the nucleotide-binding pocket cannot accommodate a 2՚-OH on the incoming nucleotide.
• This space is occupied by two amino acids (the discriminator amino acids) that make van der Waals contacts with the sugar ring
• leading strand synthesized continuously from a single primer and grows in the 5‘ … 3’ direction.
• Synthesis of the lagging strand is more complicated because DNA polymerases can add nucleotides only to the 3՚ end of a primer or growing DNA strand.
• lagging strand synthesized discontinuously from multiple primers
• Short pieces of DNA, called Okazaki fragments, are repeatedly synthesized on the lagging-strand template.
• The sizes of the Okazaki fragments are 1000 to 2000 nucleotides in bacterial cells and 100 to 200 nucleotides in eukaryotic cells.
• Mode of replication in which one new strand is synthesized continuously while the other is synthesized discontinuously is known as semi-discontinuous replication.
• Shortly after being synthesized, Okazaki fragments are covalently joined together to generate a continuous, intact strand of new DNA.
• Okazaki fragments are therefore transient intermediates in DNA replication.
• The combination of all of the proteins that function at the replication fork is referred to as the replisome.
• It consists of the DNA polymerase III holoenzyme complex and associated proteins, primase and helicase, necessary for replication function.

Primer Removal in Prokaryotes

• As each Okazaki fragment formation completes, the RNA primer of the previous fragment is removed by the 5‘ … 3’ exonuclease activity of DNA polymerase I.
• Removal of the RNA primer leaves a gap in the dsDNA.
• Enzyme DNA polymerase I also fills in the gaps, which then are ligated by DNA ligase.
• Enzyme DNA ligase catalyzes the formation of a phosphodiester bond
• In eukaryotes, ATP is the energy source, In E. coli NAD typically plays this role
• Primer Removal in Eukaryotes

RNase model

• There appears to be no eukaryotic DNA polymerase with the 5 to 3՚ exonuclease activity.
• In eukaryotes, most of the RNA component of the primer is removed by RNase H
• RNase H can degrade the RNA part of a base-paired RNA-DNA hybrid but cannot cleave the phosphodiester bond between the last ribonucleotide and the first deoxyribonucleotide.
• The last ribonucleotide is removed by flap endonuclease (FEN1)

Flap model

• for removal of primer
• Synthesis of an Okazaki fragment displaces the RNA primer of the preceding fragment in the form of a ‘flap’.
• The base of the flap is cleaved by the enzyme FEN1.
• In this reaction, FEN 1 functions as an endonuclease, but it also has a 5‘-3’ exonuclease activity.

Supercoiling

• As the strands of DNA are separated at the replication fork due to DNA helicase, the dsDNA in front of the fork becomes increasingly positively supercoiled.
• If there were no mechanism to relieve the accumulation of these supercoils, the replication forks would halt.
• Enzyme topoisomerases are required to relieve the positive supercoilling
• In E. coli, this role is typically fulfilled by DNA gyrase.
• Eukaryotes rely primarily on Topo 18 for relaxation of positive supercoils.
• DNA gyrase use free energy from ATP hydrolysis.
• It is a tetramer of two different subunits.
• The GyrA subunit cuts and rejoins the DNA and the GyrB subunit is responsible for providing energy by ATP hydrolysis.
• DNA gyrase is inhibited by quihalone antibiotics, such as nalidixic acid and their fluorinated derivatives such as norfloxacin and ciprofloxacin, which bind to the GyrA protein.
• Novobiocin also inhibits gyrase by binding to the GyrB protein and preventing it from binding ATP