DNA Replication

Introduction to DNA Replication

  • Every time a cell divides the entire content of its chromosomal DNA must be duplicated so that a complete complement can be given to each daughter cell.
  • In this way the genetic information is transmitted to new generation. 
  • Replication may be defined as synthesis of daughter DNA from mother DNA.

Important aspects of replication

  • Semi conservative.
  • Starts at an A=T rich region called origin of replication or ORI.
  • Runs in 5’ to 3’ direction.
  • Semi discontinuous

Semi conservative replication

  • In replication two parental strands separate and each strand serves as a template on which a new strand is synthesized , so that each daughter molecule contains one parental strand and a new daughter strand.
  • The above process is called Semi conservative

Messelson & Stahl Experiment

  • The hypothesis of  semi conservative replication was proposed by Watson & Crick  and proved by Messelson & Stahl by an experiment. 
  • This experiment distinguishes between two types of replication mechanisms

The experiment

  • Cells were grown for many generations in a medium containing only 15N. So that all nitrogen in the DNA were 15N.
  • The cells were then transferred in a medium containing only 14N for many generations.
  • Cellular DNA was isolated and centrifuged to equilibrium in cscl medium.
  • 15N DNA came to equilibrium at a lower position than the 14N DNA.
  • Hybrid DNA equilibrated at an intermediate position.
  • Light DNA settled at higher position
  • Conservative replication would yield 15N DNA and 14n DNA and no hybrid DNA.

The prokaryotic replication process

  • The enzyme involved is DNA polymerase that can synthesize the complementary sequence of each strand with extra ordinary fidelity.
  • Prokaryotes contain DNAP I, II, & III. 
  • Eukaryotes have DNAP alpha, beta, gamma, delta & epsilon

Steps

  • Initiation ->
  • Separation of 2 strands & formation of replication forks and replication bubble.
  • RNA primer.  Topoisomerase.
  • Elongation ->
  • Direction of replication.
  • Excision of RNA primer & its replacement by DNA. 
  • Termination

Proteins required for strand separation

dna A protein -> 20 – 50 monomers bind to origin of replication which is an A=T rich region. ATP required. This protein melts the d/s DNA.

SSB binds to s/s of DNA and keep them separated.

  • Helicases -> force the two strands apart. Needs ATP

Problem of super coiling

  • As the two strands separate the entire chromosome ahead would accumulate +ve super coils which will interfere with further unwinding.
  • Topoisomerases relax super coils during replication and transcription. They perform their role by cleaving phosphodiester bond in one or both the strands. Types -> 1-IV

RNA Primer

  • 10 to 60 nucleotide long RNA chain synthesized by the enzyme primase at the ORI on the leading strand and at the replication fork on the lagging strand. 
  • Only one is synthesized in the leading strand at the ORI but several primers are formed in the lagging strand

Elongation

  • Elongation phase includes two distinct but related operations.
  • Leading strand synthesis.
  • Lagging strand synthesis
  • In the initiation phase the parent DNA is first unwound by enzyme helicase and topological stress is relieved by topoisomerases. Each separated strand is stabilized by SSB. 
  • From this point leading and lagging strand synthesis are sharply different. Because DNAP III can synthesize new strand only in 5’ to 3’ direction not in 3’ to 5’ direction.

Leading strand synthesis

  • Begins with synthesis by primase of a short (10-60 nucleotide) RNA primer at ORI. DNTPs are added to this primer by DNAP III. Leading strand synthesis proceeds continuously , keeping with the unwinding of DNA at the replication fork. Proof reading is also done.
  • DNA strand is read by DNAPIII in 3’to5’ direction and the new strand is formed in 5’to3’ direction , Towards the replication fork Continuously.

Lagging strand synthesis.

  • Accomplished by short Okazaki fragments. First an RNA primer is synthesized by primase at the replication fork and as in the leading strand DNTPs are added to the RNA primer by DNAP III. Proof reading is also done

Synthesis of Okazaki fragment

  • Helicase and primase constitute a functional unit within the replication complex, the primosome.
  • DNAPIII synthesizes the leading strand continuously but it cycles from one Okazaki fragment to the next on the lagging strand.

Okazaki Fragment

  • Helicase unwinds the DNA at the replication fork.
  • Primase component of primosome synthesizes short RNA primer from time to time. On each RNA primer DNAPIII adds DNTPs.
  • The entire complex responsible for DNA synthesis at the replication fork is a Replisome.

Removal of RNA primer & replacement by DNA

  • Once the Okazaki fragment has been completed , its RNA primer is removed & replaced with DNA by DNAPI & the nick is sealed by ligases.
  • As the new DNA strand encounters the previous RNA primer, DNAPI comes and removes the primer and replaces it by DNA. Also proof reads.

Termination 

  • Two replication forks meet at a terminus region containing multiple copies of a 20 base pair sequence called ’Ter” sequence. ‘Ter’ sequence binds with a protein called ‘Tus’ protein. 
  • When either replication fork encounters a ‘Ter-Tus’ complex it halts.
  • separation of 2 strands by topoisomerase IV.

Proteins at the replication fork

  • SSB.
  • Helicase.
  • Primase.
  • DNAPIII
  • DNAPI
  • DNA ligase.
  • Topoisomerase II (DNA gyrase)

Eukaryotic replication

  • DNA molecules in eukaryotic organisms are considerably larger & are organized into complex nucleoprotein structure, chromatin.
  • Essential features of replication are the same in both the organisms, but there are some variations.

Eukaryotic replication, Initiation

  • Rate is slower, 1/20th that in E. coli.
  • Origin of replication, called autonomously replicating sequences (ARS) or replicators have been identified.
  • Initiation requires a multi subunit protein the origin recognition complex (ORC), which binds to several sequences within the replicator. 

Elongation

  • As the rate is slower replication proceeds bidirectionally from many origins.
  • DNAP α,β,γ,δ & Є.
  • Two other protein complexes, Replication factor C (RFC) and replication protein A (RPA) function 

Termination

  • Involves synthesis of telomeres at the ends of each chromosome.
  • After replication there is reconstitution of chromatin structure.

DNA Polymerase (S/N)

  • The replication enzyme is DNAP. In prokaryotes 3 types -> I, II, &III.
  • DNAPI -> removes the RNA primer, replaces it with DNA , also proof reads.
  • DNAPII -> DNA repair.
  • DNAPIII -> main enzyme of replication. Adds DNTPs to both the leading and lagging strand. Also proof reads.

Eukaryotic DNAP.

  • DNAP α, β, γ, δ & Є.
  • α -> primase activity, synthesizes short primers for the Okazaki fragments on both the leading and lagging strand. The primers are extended by DNAP δ.
  • β -> repair.
  • γ -> mitochondrial DNA synthesis

Eukaryotic DNAP

  • DNAP δ -> multi subunit complex. 5’ -> 3’ polymerase activity in both leading & lagging strand., 3’ -> 5’ exonuclease activity (proof reading ). Associated with proliferating cell nuclear antigen (PCNA) which activates the enzyme.
  •   Є -> Removal of RNA primer from Okazaki fragments, also in DNA repair,

INHIBITORS OF REPLICATION

  • Antibiotics e.g. ciprofioxacin, naiidixic acid inhibit bacterial replication. Nalidixic acid inhibits bact. DNA gyrase.
  • Anti cancer drugs e.g. 6 mercapto purine, 5 fluoro uracil inhibit human DNA polymerases. Doxorubicin, adriamycin, etoposide inhibit topoismerase and telomerase.

SUMMARY

  • 1 Genetic information flows from DNA to RNA to PROTEIN.
  • 2  Replication is semi conservative & semi discontinuous.
  • 3  Helicase separates the two strands of DNA to form the replication fork where the synthesis of the new DNA strands takes place.
  • 4.Replication in the leading strand is continuous and but that in the lagging strand it is discontinuous.
  • 5. DNA Polymerases are the enzymes of replication.
  • 6. Drugs inhibiting bacterial replication are used as antibiotics.
  • 7.Drugs inhibiting human replication are used as anti cancer drugs.

Other Biochemistry Notes

Biochemistry of Proteins

Carbohydrate Metabolism

VITAMIN PYRIDOXINE AND BIOTIN

Vitamin A

Pyrimidine metabolism

Purine Metabolism

Cardiac Biomarkers

VITAMIN D

. Blood Metabolism (Heme synthesis and breakdown)

DNA Repair

Regulation of gene expression

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