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ENZYMES AND PROTIENS INVOLVED IN DNA REPLICATION

Presentation on

Submitted to:Dr. K. Jeevaratnam,Professor, DBMB.

Submitted By:B.Devadatha,Reg no. 1236829,DBMB.

Subject: Molecular Biology

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IndexContent Slide no

dnaA Protein 3

DNA Helicase 4

Single-strand- binding protein (SSBP) 5

Primase 6

DNA Polymerase III 7

DNA polymerase I 9

DNA ligase 10

Topoisomerase ІІ 11

Topoisomerase І 12

Differences between prokaryotic and eukaryotic enzymes 13

Reference 15

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dnaA Protein

Recognizes ori C sequence.

Binds to 9mers sequences forming an Initiation complex.

Melts DNA strands at oriC 13mers in presence of ATP to

form open complex.

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DNA Helicase

Opens up the duplex at the replication

fork to provide a single stranded template.

Helicase uses energy from the ATP to

break the hydrogen bonds holding the base

pairs together.

Shows processivity and polarity.

E. coli contains at least 6 different

helicases--some involved in DNA repair

and others in conjugation, the principal

helicase in DNA replication is DnaB.

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Single-strand- binding protein (SSBP)

Single-strand- binding protein (SSB) binds to the single-stranded

portion of each DNA strand, inhibiting the strands from reannealing and

protecting them from degradation by nucleases.

E.Coli SSBP is a tetramer and cooperatively in sequence independent

manner.

In eukaryotic cells, a heterotrimeric protein called replication factor A

serves the role of SSB in DNA replication.

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Primase

Primase synthesizes a short (about 10 nucleotides) RNA

primer in the 5’ 3’ direction to prime DNA chain elongation.

RNA primers are required because DNA polymerases are unable

to initiate synthesis of DNA, but can only extend a strand from

the 3' end of a preformed “primer”.

Korenberg coined the term primosome to refer to collection of

protiens needed to make primers.

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DNA Polymerase IIIDNA pol III Holoenzyme is

major replicative polymerase for

both leading and lagging strand

synthesis.

DNA polymerase III begins

synthesizing DNA in the 5’

3’direction, beginning at the 3’

end of each RNA primer.

This is a multiprotein complex

that consists of 10 distinct

polypeptides.

Hallmarks of pol III are its very

high catalytic potency and

processivity.

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Catalytic core of DNA pol III composed of α ,ε θ- subunits ,contains the

polymerase activity and a 3’ 5’ exonuclease for proofreading.

A dimer of β-subunit forms a ringshaped structure and act as sliding

clamp.

The ϒ-complex is a group of five proteins that comprise clamp loader;

the clamp loader places the clamp on DNA.

Two core proteins are bound together by τ-subunit.

In eukaryotic cells, a multi-subunit protein called replication factor C

(RF-C) is the clamp loader, and proliferating cell nuclear antigen

(PCNA) is the sliding clamp.

Clamp and clamp loader

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DNA polymerase I

DNA polymerase I and RNAse H are involved in removing RNA primers in the

processing of DNA after replication.

This enzyme removes the ribonucleotides one at a time from the 5' end of the

primer (5‘ 3' exonuclease).

DNA polymerase I also fills in the resulting gaps by synthesizing DNA,

beginning at the 3' end of the neighbouring Okazaki fragment.

Both DNA polymerase I and III have the ability to "proofread" their

work by means of a 3' 5' exonuclease activity.

Proteases such as subtilisin or trypsin cleave DNA pol І into two fragments

Klenowfragment and a smaller N-terminal fragment.

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DNA ligase seals the "nicks"

by forming phosphodiester

bonds between the 3‘OH and

5’phosphate of adjacent

Okazaki fragments,

converting them to a

continuous strand of DNA.

The ligase enzyme from Ecoli

requires NAD as a cofactor,

where as that of Viruses and

Eukaryotes requires ATP.

DNA ligase

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Topoisomerase ІІMake a transient double-

strand break in the helix and

forms a covalent linkage.

DNA gyrase can introduce

negative supercoils into DNA

using ATP.

GyrA subunit cuts and rejoins

the DNA.

GyrB subunit provides

energy by ATP hydrolysis.

Inhibited by Nalidixic acid

and Novobiocin.

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Topoisomerase І

Produces a transient single strand break forming 5’phospho tyrosine linkage. Reactions catalyzed:

Relaxation.Knotting/unknotting.Catenation/decatenation.

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DNA pol 111 is

involved in invivo

replication of DNA.

DNA pol 1 is the

repair enzyme.

Primase synthesizes

primer in Ecoli.

DNA pol α and δ are involved in

invivo replication of nuclear DNA

where as DNA pol gama for

mitochondrial DNA.

DNA pol beta and Є functions as

DNA repair enzyme.

DNA pol α synthesizes primers for

both leading and lagging strands.

Prokaryotes Eukaryotes

Differences between prokaryotic and eukaryotic enzymes

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In Ecoli SSB protiens are present.

β subunit of DNA pol 111 acts clamp

Gama subunit of DNA pol 111 acts as clamp loading factor.

DNA helicase is associated with DNA pol111.

Eukaryotic topoisomerase 1 relaxes both positive and negative supercoils.RF-A is the eukaryotic

SSB.PCNA acts as clamp.

RF-C acts as clamp loading factor.DNA helicase A is

associated with DNA α , and DNA helicase Є is associated with DNA pol Є

Prokaryotes Eukaryotes

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Text book of Molecular Biology By David Clark Genes VII

Text book of Lehninger Principles of Biochemistry, Fourth Edition - David

Reference

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Thank you

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