Question

You are curious how the three eukaryotic DNA polymerases assemble at the replication origin. To study this, your lab has purified DNA polymerases, a DNA template with a replication origin, and a procedure for measuring DNA-bound polymerases. You carry out experiments to see the effect of leaving out a DNA polymerase and obtain the results in the table.

POLYMERASES INCLUDED	DNA-BOUND POLYMERASE
α, ε	ε
α, δ	δ
δ, ε	δ, ε
α, δ, ε	α, δ, ε

4. What can you conclude about the order of polymerase assembly at the origin? Explain your answer.

The same experiment yields different results when you add NaCl to the reaction as shown below.

POLYMERASES INCLUDED	DNA-BOUND POLYMERASE	DNA-BOUND POLYMERASE
	Low salt	High salt
α, ε	ε	none
α, δ	δ	none
δ, ε	δ, ε	none
α, δ, ε	α, δ, ε	α, δ, ε

5a. What are some possible explanations for this observation?

5b. What does this finding suggest about the mechanism of polymerase assembly?

Answer 1

Ans-4)

A gel filtration assay can be used to measure the assembly of DNA polymerases at the origin. This assay would require an origin containing plasmid DNA and 32P labeled DNA polymerases, which are incubated with the DNA to allow loading. To measure assembled polymerases, one must separate bound (to DNA) polymerases from unbound (free) by using gel filtration (bound polymerases will elute first with DNA, whereas free will elute later in the fractions). In order to determine whether protein is DNA associated or free, the amount of radioactivity present in the fractions is determined by using a scintillation counter, which will indicate the presence of the labeled polymerase. By performing this analysis with all combinations of polymerases alone and with other polymerases, each time labeling the polymerase one is assaying with 32P, one can determine which polymerase(s) can load by themselves, which can load in the presence of other polymerases, as well as the relative order of loading. Using your assay, you determine what effect leaving out one polymerase has on the assembly of the other two DNA polymerases at the origin.and obtain the results.

Ans-5)a)-

Only the most stable complex can load on DNA in presence of high salt.the combination of DNA can occur when pol α /Primase, Pol δ and Pol ε are all present makes the complex more stable than the complexes formed with a subset of the polymerases present. By interacting with proteins and the DNA, NaCl can compete for charge interactions between the proteins and DNA and between the different proteins (that is why salt gradients are used in ion exchange columns, etc). Therefore, only a very stable complex stays associated with the DNA in high salt. You want to determine whether DNA synthesis activity is required to assemble the DNA polymerases onto the DNA. To address this question, you take advantage of the fact that your assay monitors association with the DNA template and not DNA synthesis. You perform your assays again using mutant DNA polymerases that have a single point mutation that prevents DNA synthesis by altering binding to a catalytic Mg+2 but does not alter the binding to substrate. At the same time you also produce a Pol α/Primase that has a similar point mutation in its Primase subunit.

Ans-5)b)-

The mechanism behind the polymerase assembly is In order for the Polα/Primase to load, both Pol ε and Pol δ must be already loaded.

Pol ε and Pol δ can load independently of one another and Pol α/Primase. Pol α/Primase only loads in the last experiment when both Pol ε and Pol δ are present, and cannot load in the first two lanes when only one or the other is present. The simplest hypothesis is that Pol ε and Pol δ both can load by themselves as seen in the first three lanes. The relative order of Pol ε and Pol δ loading cannot be determined from the data present. The data suggests that Pol ε and Pol δ can load by themselves, although it is possible that the loading of Pol ε and Pol δ requires either Pol α/Primase or the other Pol.