Question

Describe the various steps involved in the initiation of replication in eukaryotes. What proteins are involved in each of the steps, and when in the cell cycle does each of them occur? How does their temporal segregation contribute to the limitation of origin firing to at most once per cell cycle?

Answer 1

REPLICATION

Replication is a process of duplication of DNA .the replication had specifc time in S phase.All the cellur molecules had timing programe inside the cells,the time depends on species.replication occur during the S phase of eukaryotic cell cycle.it is highly accurate process and involved the faithfull transmission of the genetical information from parental DNA to two daughter DNA duplixes.

Replication involves the polymerization of precursor of d NTPs ( d ATP, dGTP, dCTP, dTTP).

A.semi conservative mechanism:

in this the 2 strands of DNA seperated from each other and each strand act as template for synthesis of a new complimentary strand ,such daughter duplex contain 1 parental and 1 newly synthesize strand.

Replication always began from fixed point called origin.as unwinding DNA produced a Y shaped junction called REPLICATIONPORK.that represents origin of replication.

As the replication pork moved in one direction the dna copies continuosly in both strands.then the replication missionary should be able to synthasize in both 5'-3' and 3'-5' direction.

But the DNA polymerase involved in replication can added a new nucleotide only to 3'end of growing chain.so DNA always synthasized in 5'-3' direction,template strand should red in 3'-5' direction.

1. LEADING STRAND: replication occur in the same direction,continous as the replication fork movement.

2.LAGGING STRAND:  replication occur in the opposite direction,dis continous as the replication fork movement.

Replication is continous in leading strand,dis continous in lagging strand so reinitation is needed for laggingstrand.therefore shorter fragments produced later called OKAZAKI fragments.

B. ENZYMOLOGY IN REPLICATION:

Replication is a complex process the various enzymes/proteins are involved in this.

1.DNA polymerase:

It can polymarised the dNTPs directed by template strand of DNA so called DNA DEPENDENT DNA POLYMARASE.

In eukaryotes 5 different DNA polymerase are there (pol ${\color{Red} \alpha}$ ,pol ${\color{Red} \beta }$ ,pol ${\color{Red} \gamma }$ ,pol ${\color{Red} \delta }$ ,pol ${\color{Red} \epsilon }$ ).

DNA pol ${\color{Red} \alpha}$ ,can intiate replication in both leading and lagging strands,it dual activity so oftenly called pol ${\color{Red} \alpha}$ / primase enzyme.

DNA pol ${\color{Red} \delta }$ is a major replication enzyme involved in the replication of both leading and lagging strands.

2.DNA A protein: also called initiation protein,it involved in recognation & binding to origin.

3. DNA HELICASE: 1st component in replisoma,it posses an ATPase activity,it seperates the 2 parental strands of DNA,It always binds to lagging strand.

4.PRIMASE:Involved in synthesis of RNA primer,always binds to helicase results the formation of primosome.

5.SSB protein: It prevents reanneling of DNA,it removes the secondary structure prevent in DNA.

6.DNA Topoisomerase 1: Relaxes tension due to un coiling of dna.it binds down stream of replication fork.

7.DNA Gyrase: It binds up stream of replication fork and introduce -ve supercoiling.

8.DNA LIGASE:It covalently seal the nick between the adjucent okazaki fragments.

9.Termination protein: Also called TUS protein,binds the TER sites results termination.

10.DNA Topoisomerase 4: Involved in decatination of the 2 circular daughter DNA duplexes obtained after replication.

C.MECHANISM OF REPLICATION:

Replication contain 3 steps they are

1.INITIATION:

Initiation always begins from fixed points called ORIGIN, in eukaryotes there are multiple points of origin.

these origins are staggerd throughout the S Phase early,middle,late S phase.every replicon replicates only once during one cellcycle.the initiation is mainly involved in 4 major events.

a. Recognition of origin.

b.Melting of DNA

C.Stablization of SS DNA.

d.Assembly of primosome to synthesis RNA primer.

2.ELONGATION:

It begans with the loading the DNA pol 3 on to DNA.initially 2 beta clamps are loadded,one to each strands of DNA by the clamp loader gama in an ATP dependent process.

The catalytic cores binds to beta clamps finally dimerised by the addition of "T" subunit.the holo enzyme of polymerase 3 continuos replication in both strands in 3'end of growing chain.

DNA Always synthasis in 5'-3' direction.in this 1 Strand called Leading strand another is Lagging strand.

the RNA polymerase synthesised to initate the replication is removed by DNA pol 1.

during the Nick translation in okazaki fragments DNA pol 1 exibits 5'-3' exo activity& 5'-3' pol activity simultaniously.the nick finally covalently sealed by DNA Ligase to continuous strand of DNA.

3.TERMINATION:

Termination occurs at specifoc sites sequences called TER sites,there are 6 ter sites, 3 arrest replication in clock wise & 3 in anti clock wise direction.

TER C & TER A are major terminator sites.TER sites are recognised by TUS protein.Forming TER-TUS complex prevent helicase activity there results in terminationof replication.

TELOMERASE a reverse transcriptase that RNA dependent DNA polymerase prevent END REPLICATION problem in eukarotes replication.

Answer 2

REPLICATION

Replication is a process of duplication of DNA .the replication had specifc time in S phase.All the cellur molecules had timing programe inside the cells,the time depends on species.replication occur during the S phase of eukaryotic cell cycle.it is highly accurate process and involved the faithfull transmission of the genetical information from parental DNA to two daughter DNA duplixes.

Replication involves the polymerization of precursor of d NTPs ( d ATP, dGTP, dCTP, dTTP).

A.semi conservative mechanism:

in this the 2 strands of DNA seperated from each other and each strand act as template for synthesis of a new complimentary strand ,such daughter duplex contain 1 parental and 1 newly synthesize strand.

Replication always began from fixed point called origin.as unwinding DNA produced a Y shaped junction called REPLICATIONPORK.that represents origin of replication.

As the replication pork moved in one direction the dna copies continuosly in both strands.then the replication missionary should be able to synthasize in both 5'-3' and 3'-5' direction.

But the DNA polymerase involved in replication can added a new nucleotide only to 3'end of growing chain.so DNA always synthasized in 5'-3' direction,template strand should red in 3'-5' direction.

1. LEADING STRAND: replication occur in the same direction,continous as the replication fork movement.

2.LAGGING STRAND:  replication occur in the opposite direction,dis continous as the replication fork movement.

Replication is continous in leading strand,dis continous in lagging strand so reinitation is needed for laggingstrand.therefore shorter fragments produced later called OKAZAKI fragments.

B. ENZYMOLOGY IN REPLICATION:

Replication is a complex process the various enzymes/proteins are involved in this.

1.DNA polymerase:

It can polymarised the dNTPs directed by template strand of DNA so called DNA DEPENDENT DNA POLYMARASE.

In eukaryotes 5 different DNA polymerase are there (pol ${\color{Red} \alpha}$ ,pol ${\color{Red} \beta }$ ,pol ${\color{Red} \gamma }$ ,pol ${\color{Red} \delta }$ ,pol ${\color{Red} \epsilon }$ ).

DNA pol ${\color{Red} \alpha}$ ,can intiate replication in both leading and lagging strands,it dual activity so oftenly called pol ${\color{Red} \alpha}$ / primase enzyme.

DNA pol ${\color{Red} \delta }$ is a major replication enzyme involved in the replication of both leading and lagging strands.

2.DNA A protein: also called initiation protein,it involved in recognation & binding to origin.

3. DNA HELICASE: 1st component in replisoma,it posses an ATPase activity,it seperates the 2 parental strands of DNA,It always binds to lagging strand.

4.PRIMASE:Involved in synthesis of RNA primer,always binds to helicase results the formation of primosome.

5.SSB protein: It prevents reanneling of DNA,it removes the secondary structure prevent in DNA.

6.DNA Topoisomerase 1: Relaxes tension due to un coiling of dna.it binds down stream of replication fork.

7.DNA Gyrase: It binds up stream of replication fork and introduce -ve supercoiling.

8.DNA LIGASE:It covalently seal the nick between the adjucent okazaki fragments.

9.Termination protein: Also called TUS protein,binds the TER sites results termination.

10.DNA Topoisomerase 4: Involved in decatination of the 2 circular daughter DNA duplexes obtained after replication.

C.MECHANISM OF REPLICATION:

Replication contain 3 steps they are

1.INITIATION:

Initiation always begins from fixed points called ORIGIN, in eukaryotes there are multiple points of origin.

these origins are staggerd throughout the S Phase early,middle,late S phase.every replicon replicates only once during one cellcycle.the initiation is mainly involved in 4 major events.

a. Recognition of origin.

b.Melting of DNA

C.Stablization of SS DNA.

d.Assembly of primosome to synthesis RNA primer.

2.ELONGATION:

It begans with the loading the DNA pol 3 on to DNA.initially 2 beta clamps are loadded,one to each strands of DNA by the clamp loader gama in an ATP dependent process.

The catalytic cores binds to beta clamps finally dimerised by the addition of "T" subunit.the holo enzyme of polymerase 3 continuos replication in both strands in 3'end of growing chain.

DNA Always synthasis in 5'-3' direction.in this 1 Strand called Leading strand another is Lagging strand.

the RNA polymerase synthesised to initate the replication is removed by DNA pol 1.

during the Nick translation in okazaki fragments DNA pol 1 exibits 5'-3' exo activity& 5'-3' pol activity simultaniously.the nick finally covalently sealed by DNA Ligase to continuous strand of DNA.

3.TERMINATION:

Termination occurs at specifoc sites sequences called TER sites,there are 6 ter sites, 3 arrest replication in clock wise & 3 in anti clock wise direction.

TER C & TER A are major terminator sites.TER sites are recognised by TUS protein.Forming TER-TUS complex prevent helicase activity there results in terminationof replication.

TELOMERASE a reverse transcriptase that RNA dependent DNA polymerase prevent END REPLICATION problem in eukarotes replication.