•Replication of duplex DNA is a complex endeavor involving a conglomerate of enzyme activities.• Different activities are involved in the stages of initiation, elongation, and termination.•Initiation involves recognition of an origin by a complex of proteins. •Before DNA synthesis begins, the parental strands must be separated and (transiently) stabilized in the single-stranded state.• Then synthesis of daughter strands can be initiated at the replication fork. •Elongation is undertaken by another complex of proteins. •The replisome exists only as a protein complex associated with the particular structure that DNA takes at the replication fork. •As the replisome moves along DNA, the parental strands unwind and daughter strands are synthesized. •At the end of the replicon, joining and/or termination reactions are necessary. Following termination, the duplicate chromosomes must be separated from one another, which requires manipulation of higher-order DNA structure.

flowchart Introduction to replication

•The replisome complex assumes three principle functions, DNA polymerase, DNA primase and DNA helicase.• The helicase, a donut-shaped enzyme, initiates replication by unwinding the two parental DNA strands.• The structure of the helicase was elucidated in 1999 using the technique of x-ray crystallography. • It has been determined that the helicase moves along the DNA strand 300 paired nucleotides per second•.Once the DNA strands are unwound, ssb proteins attach to each unwound strand•DNA polymerase catalyzes the elongation of each strand. •The polymerase is able to operate in a continuous fashion on the leading parental strand that is unwinding. •On the other strand, the DNA primase molecule builds primers, which are eventually connected to the other replicated DNA strand.

• Inability to replicate DNA is fatal for a growing cell.• Mutants in replication must therefore be obtained as conditional lethals.• They accomplish replication under permissive conditions • and they are defective under nonpermissive conditions• . A comprehensive series of such temperature-sensitive mutants in E. coli

identifies a set of loci called the dna genes.• The dna mutants distinguish two stages of replication by their behavior

when the temperature is raised 1. The major class of quick-stop mutants 2. The smaller class of slow-stop mutants.

•An important assay used to identify the components of the replication apparatus is called in vitro complementation.• An in vitro system for replication is prepared from a dna mutant and operated under conditions in which the mutant gene product is inactive. •Extracts from wild-type cells are tested for their ability to restore activity. •The protein coded by the dna locus can be purified by identifying the active component in the extract.

in vitro complementation

The replisome is a complex molecular machine that carries out replication of DNA. It is made up of a number of subcomponents that each provide a specific function during the process of replication.Contents [hide]1 Major Components 1.1 Helicase1.2 Gyrase1.3 Primase1.4 DNA polymerase III holoenzyme1.5 DNA polymerase I1.6 Ligase2 Single-strand binding proteins3 Exonuclease Activity

•an enzyme that separates the strands of DNA, using the hydrolysis of ATP to provide the necessary energy.

•Helicases separate the strands of a duplex nucleic acid in a strand separation at the growing point of a replication fork.

•There are 12 different helicases in E. coli.

• A helicase is generally multimeric.

• A common form of helicase is a hexamer .

•This typically translocates along DNA by using its multimeric structure to provide multiple DNA-binding sites.

•In eukaryotes, the Mcm2-7 complex ,This helicase translocates in the same direction as the DNA polymerase (3' to 5' with respect to the template strand).

•In prokaryotic systems, the helicases are better identified and include dnaB, which moves 5' to 3' on the strand opposite the DNA polymerase.

helicase

Helicase figure

•A primer is a short sequence of RNA that is paired with one strand of DNA and provides a free 3 -OH end at which a DNA polymerase starts synthesis of a deoxyribonucleotide chain.

•The primase is a type of RNA polymerase that synthesizes short segments of RNA that will be used as primers for DNA replication. (priming reaction)

• This is provided by a special RNA polymerase activity,

• the product of the dnaG gene.

• The enzyme is a single polypeptide of 60 kD .

•DnaG primase associates transiently with the replication complex, and typically synthesizes an 11-12 base primer.

• Primers start with the sequence pppAG, opposite the sequence 3 –GTC-5 in the template.

primase

Primase figure

•DNA polymerase III holoenzyme is the multisubunit replicase of the Escherichia coli chromosome. • distinguished from other DNA polymerases in the cell by its high processivity (>50 kbp) and rapid rate of synthesis (750 nucleotides/s) •. The high processivity and speed is rooted in a ring-shaped subunit, The ring-shaped clamp assembled around DNA by . multisubunit clamp loader coupled with the energy of ATP hydrolysis to the assembly of the clamp onto DNA.

•There are two copies of the catalytic core. Each catalytic core contains the α subunit (the DNA polymerase activity), ε subunit (3 –5 proofreading exonuclease), and θ subunit (stimulates exonuclease).

•There are two copies of the dimerizing subunit, τ, which link the two catalytic cores together .•There are two copies of the clamp, which is responsible for holding catalytic cores on to their template strands. •Each clamp consists of a homodimer of β subunits that binds around the DNA and ensures processivity. •Beta clamp is made of 3 subunits.•Helps pol III to remain on the strand for 1000-2000 nucleotides.•The γ complex is a group of 5 proteins, the Clamp loader, that places the clamp on DNA•The replisome contains a mechanical-like structure called the beta clamp, which contains three subunits that come together to enclose the strand, which helps DNA pol III stay on the strand for 1000-2000 nucleotides, as opposed to 100-200.

•First the clamp loader uses hydrolysis of ATP to bind β subunits to a template-primer complex.

•Binding to DNA changes the conformation of the site on β that binds to the clamp loader, and as a result it now has a high affinity for the core polymerase. This enables core polymerase to bind, and this is the means by which the core polymerase is brought to DNA.

•A τ dimer binds to the core polymerase, and provides a dimerization function that binds a second core polymerase (associated with another β clamp). The holoenzyme is asymmetric, because it has only 1 clamp loader. The clamp loader is responsible for adding a pair of β dimers to each parental strand of DNA.

Pol I and ligase

•DNA ligase makes a bond between an adjacent 3 -OH and 5 -phosphate end where there is a nick in one strand of duplex DNA.

•synthesis of an Okazaki fragment terminates just before the start of the RNA primer of the preceding fragment.

•When the primer is removed, there will be a gap.

•The gap is filled by DNA polymerase I.

• The 5 –3 exonuclease activity removes the RNA primer while simultaneously replacing it with a DNA sequence extended from the 3 –OH end of the next Okazaki fragment. •In mammalian systems ,Okazaki fragments are removed by a two-step process. 1) RNAase HI (an enzyme that is specific for a DNA-RNA hybrid substrate) makes an endonucleolytic cleavage; •2) a 5 –3 exonuclease called FEN1 removes the RNA.•Once the RNA has been removed and replaced, the adjacent Okazaki fragments must be linked together.• The 3 –OH end of one fragment is adjacent to the 5 –phosphate end of the previous fragment.• The responsibility for sealing this nick lies with the enzyme DNA ligase. •Ligases are present in both prokaryotes and eukaryotes.

Requirements of topoisomerases

1)In replication, as the replication fork moves ahead ,thus separating the strands and also creating negative supercoils in the unseparated DNA region.

2) In the case of transcription, the movement of RNA polymerase creates a region of positive supercoiling in front and a region of negative supercoiling behind the enzyme.

3) When a circular DNA molecule is replicated, the circular products may be catenated, with one passed through the other.

They must be separated in order for the daughter molecules to segregate to separate daughter cells.

•DNA topoisomerases are enzymes that catalyze changes in the topology of DNA by transiently breaking one or both strands of DNA, passing the unbroken strand(s) through the gap, and then resealing the gap

•Topoisomerases are divided into two classes, •Type I topoisomerases act by making a transient break in one strand of DNA. •Type II topoisomerases act by introducing a transient double-strand break.• Some topoisomerases can relax (remove) only negative supercoils from DNA; others can relax both negative and positive supercoils.• Enzymes that can introduce negative supercoils are called gyrases; those that can introduce positive supercoils are called reverse gyrases •There are four topoisomerase enzymes in E. coli, called topoisomerase I, III, IV and DNA gyrase. DNA topoisomerase I and III are type I enzymes. Gyrase and DNA topoisomerase IV are type II enzymes. •Most eukaryotes contain a single topoisomerase I enzyme that is required both for replication fork movement and for relaxing supercoils generated by transcription. •A topoisomerase II enzyme(s) is required to unlink chromosomes following replication.

Single-strand binding proteinsExposed, single-strand DNA is highly unstable (particularly in the aqueous environment of the cell). The single-strand DNA can hydrogen bond to itself and form dangerous hairpin structures. To counteract this instability, single-strand binding proteins (SSB) (e.g. Replication protein A) bind to the exposed bases.

Exonuclease Activity:-Incorrect base pairing occurs rarely during replication. Some DNA polymerases contain "proofreading" mechanism that removes nucleotides that have been mistakenly added. This is termed as exonuclease activity.