DNA polymerase enzyme (DNAP of eukaryotes)
Eukaryotes consist of large number of DNA polymerases as compared to prokaryotes. Out of these polymerases three polymerases α, δ and ε are needed for replication of chromosomal DNA, while others needed for repair of damaged DNA.
1) DNA polymerase α
- DNA polymerase α holoenzyme has both primase and polymerase activity and is involved in the primer synthesis during DNA replication.
- Primase activity synthesizes ~12-nt RNA primers. The polymerase activity extends the primer to some 30-40 nucleotide lengths (The polymerase subunit extends the primer synthesized by primase subunit).
- After that polymerase switching takes place. The polymerase switching is required as polymerase α has low processivity.
- In polymerase switching polymerase α is displaced from the template and the synthesis is taken over by polymerase δ and polymerase ε.
2) DNA polymerase δ
- During polymerase switching Replication factor C displaces polymerase α and load PCNA on the template DNA near the primer strand, following which polymerase δ binds to the PCNA and extends the DNA strand.
- Pol δ in complex with PCNA is required for lagging strand synthesis.
3) DNA polymerase ε
- PCNA interact with DNA polymerase ε to facilitate leading strand synthesis.
- Pol ε is highly processive in the absence of PCNA and has a 3’ to 5’ exonuclease activity that degrades single-stranded DNA to 6- or 7-residue oligonucleotides rather than to mononucleotides.
Family of DNA polymerases
The DNA polymerases have been classified into 7 families. These classes have been denoted by letter A, B, C, D, X, Y and RT (reverse transcriptase).
Family A: The prototype enzyme of this family is pol I and T7 DNA polymerase (DNA replication of T7 phage). The members of this family have 3′-5′ exonuclease activity (proofreading activity) which is conserved.
Family B: This family consists of main replicative enzymes of eukaryotes which are involved in replicating large DNAs of eukaryotes. Family B polymerases are mainly multi subunit enzymes. The members of this family also have 3′-5′ exonuclease activity (proofreading activity).
Family C: This family consist of main replicative enzyme of prokaryotes i.e. polymerase III.
Family D: The family D polymerases are exclusive to the archaea. The members of this family have 3′-to-5′ exonuclease activity
Family X: Polymerases belonging to this family are small and monomeric like DNA pol λ, DNA pol μ etc. These polymerases are mainly involved in DNA repair.
Family Y: The members belonging to this family do not have any exonuclease activity. These polymerases are mainly involved in Translesion synthesis.
How the processivity of polymerases increased?
- The processivity of DNA polymerase is increased by their association with the sliding clamps.
- In eukaryotes these sliding clamps are known as PCNA (Proliferating cell nuclear antigen).
PCNA: Named so because it occurs only in the nuclei of proliferating cells
Function of PCNA:
- Multi-subunit proteins that prevent the dissociation of the polymerase from template.
- Act as a sliding platform to recruit other replication proteins such as DNA helicase, nuclease, ligase, and histone chaperones.
How they act:
- These proteins encircle the double helix of DNA and slide along the DNA with the polymerase without getting dissociated from it.
- The sliding clamps prevent the diffusion of the polymerase once it gets dissociated from the template.
- Thus, the sliding clamps ensure the close proximity between DNA and the polymerase increasing the processivity of the enzyme.
Replication factor C
- Clamp loaders are known as RF-C (Replication Factor C) in eukaryotes.
- PCNA is loaded onto DNA by replication factor C.
- RFC is a pentameric complex consisting of Rfc1, Rfc2, Rfc3, Rfc4, and Rfc5 subunits (Rfc1–5 subunits).
How they load PCNA?
It binds to the 3′ end of the DNA and uses ATP to open the ring of PCNA so that it can encircle the DNA. ATP hydrolysis causes release of RFC, with concomitant clamp loading onto DNA.
Figure 2: Loading of PCNA on to template by Relication factor C
- Unloading of PCNA must take place repeatedly during replication—on completion of every Okazaki fragment and also at replication fork termination.
- Elg1 replication factor C-like complex (Elg1-RLC) functions in PCNA unloading.