Question

What other DNA polymerases are involved in DNA replication and when are they active?

Although Dpb3 and Dpb4 are considered non-essential, DNA polymerase ? dissociates more frequently from the template when Dpb3 and Dpb4 are deleted. What are the explanations for this result?

What do you think would happen if, in addition to Dpb3 and Dpb4 deletion, the exonuclease activity of Pol2 was also mutated in such a way that it did not function? How might replication fork progression and fidelity be effected?

Answer 1

E. coli DNA Replication

Overview of DNA replication

DNA polymerases can only add deoxynucleotides to 3' ends (i.e., strands are synthesized only in a 5' to 3' direction), but both strands at replication forks are synthesized at the same time. Thus, at the replication fork (Figure H2), the leading strand is synthesized continuously, but the lagging strand is made discontinuously as Okazaki fragments.

DNA polymerase III holoenzyme

There are five DNA polymerases in E. coli. We have already discussed DNA polymerase I. DNA polymerase III is the replicative enzyme, also called the replicase (we will not discuss the other three DNA polymerases; they are primarily involved in various pathways to repair damaged DNA). The Table compares DNA polymerases I and III.

Quick Comparison of DNA polymerases I and III
DNA polymerase IIIDNA polymerase I
StructureDNA Pol III holoenzyme is an asymmetric dimer; i. e., two cores with other accessory subunits. It can thus move with the fork and make both leading and lagging strands.DNA Pol I is a monomeric protein with three active sites. It is distributive, so having 5'-to-3' exonuclease and polymerase on the same molecule for removing RNA primers is effective and efficient.
ActivitiesPolymerization and 3'-to-5' exonuclease, but on different subunits. This is the replicative polymerase in the cell. Can only isolate conditional-lethal dnaE mutants. Synthesizes both leading and lagging strands. No 5' to 3' exonuclease activity.Polymerization, 3'-to-5' exonuclease, and 5'-to-3' exonuclease (mutants lacking this essential activity are not viable). Primary function is to remove RNA primers on the lagging strand, and fill-in the resulting gaps.
Vmax (nuc./sec)250-1,000 nucleotides/second. This is as fast as the rate of replication measured in Cairns' experiments. Only this polymerase is fast enough to be the main replicative enzyme.20 nucleotides/second. This is NOT fast enough to be the main replicative enzyme, but is capable of "filling in" DNA to replace the short (about 10 nucleotides) RNA primers on Okazaki fragments.
ProcessivityHighly processive. The beta subunit is a sliding clamp. The holoenzyme remains associated with the fork until replication terminates.Distributive. Pol I does NOT remain associated with the lagging strand, but disassociates after each RNA primer is removed.
Molecules/cell10-20 molecules/cell. In rapidly growing cells, there are 6 forks. If one processive holoenzyme (two cores) is at each fork, then only 12 core polymerases are needed for replication.About 400 molecules/cell. It is distributive, so the higher concentration means that it can reassociate with the lagging strand easily.
Note: Processivity is generally examined by diluting active replication forks into reaction mixtures that contain no additional template but in which the concentrations of all the enzyme components except the one under investigation have been kept constant. After dilution, the reaction proceeds unchanged if the enzyme acts processively, whereas it stops or slows if the enzyme acts distributively.

DNA Polymerase III (pol III) from E. coli is a single protein of molecular weight 130 kDa (130,000 grams per mole). It is also referred to as polC, dnaE, or the alpha subunit. Though the molecule has DNA polymerase activity by itself, polIII works to replicate DNA in the bacterial cell in conjunction with other proteins. This multi-protein complex is referred to as the pol III holoenzyme (Figure P). The proteins (called subunits) that associate with pol III in the holoenzyme perform several functions. The most interesting subunit is called beta, which forms a donut shaped ring around the DNA and helps to anchor the holoenzyme to the DNA during replication (Figure Q). By acting as a sliding "clamp", beta helps the holoenzyme to replicate long stretches of DNA without "falling off" the strand (this is called processivity). Pol III holoenzyme directs both leading and lagging strand synthesis simultaneously by virtue of having two polymerase subunits. The Table summarizes the pol III subunits, subassemblies, and their functions:

DNA polymerase III subunits and subassemblies
Subunit
Function
Subassembly (complex)
alphaDNA polymerasecore (there are two cores per DNA polymerase III holoenzyme)
epsilon3'-to-5' exonuclease (editing exonuclease)
thetastimulates 3'-to-5' exonuclease
taudimerizes cores, activates DnaB helicase activity  
gammabinds ATPgamma complex (clamp loader), uses ATP energy when loading beta onto primed DNA.
deltaunknown
delta primestimulates clamp loading
chiinteracts with SSB to allow removal of DnaG primase from primer
psiunknown
betasliding clamp. The beta subunit can be loaded onto DNA by the clamp loader (gamma complex) in an ATP-dependent reaction (Figure G1). (The clamp loader also unloads clamps!) Beta cannot be loaded ont

Answer 2

1)

The DNA polymerases are enzymes that create DNA molecules by assembling nucleotides, the building blocks of DNA. These enzymes are essential toDNA replication and usually work in pairs to create two identical DNA strands from a single original DNA molecule. During this process, DNA polymerase