DNA Replication Enzymes and Their Functions

In this DNA replication enzymes and their functions post we have briefly explained about enzyme of DNA replication, key features of DNA polymerases, DNA polymerase structure and types, DNA ligase, and replication fork.

Enzyme of DNA Replication

DNA replication is a highly enzyme-dependent process. There are many enzyme of DNA replication involved in replication which includes the enzymes DNA-dependent DNA polymerase, helicase, ligase, etc.  Among them, DNA-dependent DNA polymerase is the main enzyme of DNA replication.

DNA Replication Enzymes

Enzyme of DNA Replication

DNA Replication Enzymes and Their Functions

Helicase 

Helicases are nucleic acid or nucleic acid protein complex binding enzymes that can also remodel them. There are helicases for both DNA and RNA. DNA helicases are required during DNA replication because they split double-stranded DNA into single strands, allowing each strand to be copied independently.

Primase

Primase is an enzyme of DNA replication that produces short RNA sequences known as primers. These primers act as a jumping off point for DNA synthesis. Primase is an RNA polymerase because it generates RNA molecules.

DNA polymerase

A DNA polymerase is an enzyme of DNA replication that catalyses the synthesis of DNA molecules from nucleoside triphosphates, DNA’s molecular precursors. These enzymes are required for DNA replication and typically work in groups to produce two identical DNA duplexes from a single original DNA duplex.

DNA Ligase

DNA ligases are enzyme of DNA replication that catalyse the joining together of two DNA ends, in a manner that requires either adenosine triphosphate (ATP) or NAD+

Nucleases

Nucleases an enzyme of DNA replication are broad and diverse class of enzymes that hydrolyse the phosphodiester bonds of DNA and RNA.

Primosome

Primosome is a protein complex responsible for creating RNA primers on single stranded DNA during DNA replication. The primosome consists of seven proteins: DnaG primase, DnaB helicase, DnaC helicase assistant, DnaT, PriA, Pri B, and PriC. The primosome is utilized once on the leading strand of DNA and repeatedly, initiating each Okazaki fragment, on the lagging DNA strand

Replisome

Replisome is composed of the following: 2 DNA Pol III enzymes, made up of α, ε and θ subunits. The α subunit has polymerization activity, the ε subunit has proofreading activity, the θ subunit stimulates the ε subunit’s proofreading.

2 β units which act as sliding DNA clamps, they keep the polymerase bound to the DNA. 2 τ units which connect the 2 DNA Pol III enzymes.  1 γ unit which acts as a clamp loader for the lagging strand Okazaki fragments, helping the two β subunits to form a unit and bind to DNA. The γ unit is made up of 5 γ subunits.

Features of DNA polymerases

In 1957, Arthur Kornberg and his colleagues discovered the first DNA polymerase. A primer is essential because DNA polymerases can elongate only pre-existing chains; this primer must possess a free 3′-OH end to which an incoming deoxynucleoside monophosphate is added.

All four dNTPs are substrates, pyrophosphate (PPi) is released, and the dNMP is linked to the 3′-OH of the primer chain through formation of a phosphoester bond. The deoxynucleoside monophosphate to be incorporated is chosen through its geometric fit with the template base to form a Watson-Crick base pair.

As DNA polymerase I catalyses the successive addition of deoxynucleotide units to the 3′-end of the primer, the chain is elongated in the 5′ – 3′ direction, forming a polynucleotide sequence that runs antiparallel to the template but complementary to it.

DNA polymerase I can proceed along the template strand, synthesizing a complementary strand of about 20 bases before it falls off (dissociates from) the template. All DNA polymerases, whether from prokaryotic or eukaryotic sources, share the following properties:

(a) The incoming base is selected within the DNA polymerase active site, as determined by Watson-Crick geometric interactions with its corresponding base in the template strand, (b) chain growth is in the 5′ → 3′ direction and is antiparallel to the template strand, and (c) DNA polymerases cannot initiate DNA synthesis de novo—all require a primer oligonucleotide with a free 3′-OH to build upon.

E.coli DNA Polymerases enzymes are numbered I, II, and III, in order of their discovery. DNA polymerases I and II function principally in DNA repair; DNA polymerase III is the chief enzyme of DNA replication of E. coli.

Catalytic function

DNA polymerase I joins deoxynucleoside monophosphate units to the 3′-OH carries out a nucleophilic attack on the alpha-phosphoryl group of the incoming dNTP to form a phosphoester bond, and PPi is released. The subsequent hydrolysis of PPi by inorganic pyrophosphatase renders the reaction effectively irreversible. The reaction is

dNTP+pNpNpNpN-3’OH ——- PPi+pNpNpNpNpN*-3’OH

The structure of most of the DNA polymerases resembles a hand, which is holding active sites. The active site of the enzyme has two parts. At the insertion site, nucleotides are added. After adding, the newly formed base-pair migrates to the post-insertion site.

Prokaryotic DNA Polymerase

There are five DNA polymerases identified in E.coli. All the DNA polymerases differ in structure, functions and rate of polymerization and processivity.

DNA Polymerase I is coded by polA gene. It is a single polypeptide and has a role in recombination and repair. It has both 5’→3’ and 3’→5’ exonuclease activity. It removes the RNA primer from lagging strand by 5’→3’ exonuclease activity and also fills the gap.

DNA Polymerase II is coded by polB gene. It is made up of 7 subunits. Its main role is in repair and also a backup of DNA polymerase III. It has 3’→5’ exonuclease activity.

DNA Polymerase III is the main enzyme of DNA replication in E.coli. It is coded by polC gene. The polymerization and processivity rate is maximum in DNA polymerase III. It also has proofreading 3’→5’ exonuclease activity.

DNA Polymerase IV is coded by dinB gene. Its main role is in DNA repair during SOS response, when DNA replication is stalled at the replication fork. DNA polymerase II, IV and V are translesion polymerases.

DNA Polymerase V is also involved in translesion synthesis during SOS response and DNA repair. It is made up of UmuC monomer and UmuD dimer.

Eukaryotic DNA Polymerase

Like prokaryotic cells, eukaryotic cells also have many DNA polymerases, which perform different functions, e.g. mitochondrial DNA replication, nuclear DNA replication, etc. The nuclear DNA replication is mainly done by DNA polymerase 𝝳 and 𝜶. There are at least 15 DNA polymerases identified in human beings.

DNA polymerase 𝝳, It is the main enzyme of DNA replication in eukaryotes. It also has 3’→5’ exonuclease activity for proofreading.

DNA polymerase 𝜶, The main function of DNA polymerase 𝜶 is to synthesize primers.  The smaller subunit has a primase activity. The largest subunit has polymerization activity. It forms a primer for Okazaki fragments, which is then extended by DNA polymerase 𝝳.

DNA polymerase 𝟄, The main function is DNA repair. It removes primers for Okazaki fragments from the lagging strand.

DNA polymerase 𝝲, DNA polymerase γ (Pol γ) is the only replicative DNA polymerase found in the mitochondria and is essential for copying and repair of mitochondrial DNA.

DNA ligase

DNA ligase seals nicks in double-stranded DNA where a 3′-OH and a 5′-phosphate are juxtaposed. This enzyme of DNA replication is responsible for joining Okazaki fragments together to make the lagging strand a covalently contiguous polynucleotide chain.

DNA ligase from eukaryotes and bacteriophage T4 is ATP-dependent; the E. coli enzyme requires NAD+. Both types of DNA ligase act via an adenylylated ε-amino group of a Lys residue.

Adenylylation of the 5′- phosphoryl group activates it for formation of a phosphoester bond with the 3′-OH, covalently sealing the sugar-phosphate backbone of DNA.

Replication Fork

DNA gyrase (topoisomerase) and helicase unwind the DNA double helix, and the unwound, single-stranded regions of DNA are maintained through interaction with SSB. Primase synthesizes an RNA primer on the lagging strand; the leading strand, which needs priming only once, was primed when replication was initiated.

The lagging strand template is looped around, and each replicative DNA polymerase moves 5′ -3′ relative to its strand, copying template and synthesizing a new DNA strand. Each replicative polymerase is tethered to the DNA by its b – subunit sliding clamp. DNA pol III complex periodically unclamps and then reclamps. Downstream on the lagging strand, DNA polymerase I excises the RNA primer and replaces it with DNA, and DNA ligase seals the remaining nick.

DNA Replication Enzymes

Replication Fork

Further Readings

Reference