Presented by R.Parthasarathy

Enzymes and proteins in DNA replication

Introduction

• Multiple proteins are required for DNA replication at a replication fork.

• These include DNA polymerases, single-strand DNA binding proteins, helicases, primase,topoisomerases, and DNA ligase. Some of these are multisubunit protein complexes.

dnaA Protein

• The base sequence at the origin of replication is recognized and bound by the dna A protein.

Helicase

• Helicase uses energy from the ATP to break the hydrogen bonds holding the base pairs together.

• This allows the two parental strands of DNA to begin unwinding and forms two replication forks.

• Each strand of parental DNA has it own helicase.• In humans, two inherited diseases, Werner's

syndrome and Bloom's syndrome, result from helicase defects.

• 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

SSB Protein• Single-stranded DNA binding protein (SSB) binds to the

single-stranded portion of each DNA strand, preventing the strands from reassociating and protecting them from degradation by nucleases.

• gp32, the most studied SSB protein, binds in a strongly cooperative fashion to single-strand DNA.

• That is, binding adjacent to another gp32 is much more likely than the binding of a single gp32 in isolation.

• This property helps promote the denaturation of duplex DNA and helps keep the DNA template in an extended, single-strand conformation, with the purine and pyrimidine bases exposed so that they can base-pair readily with incoming nucleotides.

• In E. coli, the protein is called ssb. • In eukaryotic cells, a heterotrimeric protein called replication

factor A serves the role of SSB in DNA replication.

Primase • Primase is an enzyme that copies a DNA template strand by making an RNA strand complementary to it. • Primase synthesizes a short (about 10 nucleotides)

RNA primer in the 5’ 3’ direction.• The parental strand is used as a template for this

process. • 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”

• The enzyme is active only in the presence of other proteins (including a helicase), which create a complex called the primosome

DNA polymerase III

• It catalyzes the chemical reactions for polymerization of nucleotides.

• DNA polymerase III begins synthesizing DNA in the 5’ 3’direction, beginning at the 3’ end of each RNA primer.

• The newly synthesized strand is complementary and antiparallel to the parental strand used as a template.

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.

• If DNA polymerase makes a mistake during DNA synthesis, the resulting unpaired base at the 3' end of the growing strand is removed before synthesis continues.

Comparison of DNA and RNA polymerases

DNA Polymerase RNA Polymerase

Nucleic acid synthesized (5’ 3’)

DNA RNA

Required template DNA* DNA*

Required substrates dATP, dGTP, dCTP, dTTP ATP, GTP, CTP, UTP

Required primer RNA (or DNA) None

Proofreading activity(3’ 5’ exonuclease)

Yes No

Clamps and clamp loaders

• Protein from the DNA polymerase III holoenzyme

complex holds the polymerase to the DNA.• This helps the DNA polymerase complex to

stay on the DNA through an entire cycle of replication.

• A multi subunit entity called the complex functions as the "clamp loader". That is, it loads the clamp onto the DNA.

Clamps and clamp loaders

• a protein dimer that encircles the DNA strand and helps hold the DNA polymerase to the DNA strand.

• 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.

DNA ligase

• DNA ligase seals the "nicks" between Okazaki fragments, converting them to a continuous strand of DNA.

• Covalently closes nicks in double-stranded DNA.

DNA gyrase • DNA gyrase (DNA topoisomerase II) provides a

"swivel" in front of each replication fork.• As helicase unwinds the DNA at the replication

forks, the DNA ahead of it becomes overwound and positive supercoils form.

• DNA gyrase inserts negative supercoils by nicking both strands of DNA, passing the DNA strands through the nick, and then resealing both strands again.

• DNA topoisomerase I can relieve supercoiling in DNA molecules by the transient breaking and resealing of just one of the strands of DNA.

Action of a gyrase

Action of a type I topoisomerase

Step in Replication Prokaryotic cells Eukaryotic cells

Recognition of origin of replication

Dna A protein Unknown

Unwinding of DNA double helix Helicase(requires ATP)

Helicase(requires ATP)

Stabilization of unwoundtemplate strands

Single-stranded DNA-binding protein (SSB)

Single-stranded DNA-binding protein (SSB)

Synthesis of RNA primers Primase Primase

Synthesis of DNALeading strandLagging strand

DNA polymerase IIIDNA polymerase III

DNA polymerase δDNA polymerase α

Removal of RNA primers DNA polymerase I(5 3' exonuclease)

Unknown

Replacement of RNA with DNA DNA polymerase I Unknown

Joining of Okazaki fragments DNA ligase(requires NAD)

DNA ligase(requires ATP)

Removal of positive supercoils ahead of advancing replication forks

DNA topoisomerase II(DNA gyrase)

DNA topoisomerase II

References

MOLECULAR BIOLOGY by David ClarkGenes VII