DNA Replication 2

Howdy folks! Let's continue.

Which enzyme is responsible for the elongation of the new DNA chain?

DNA Polymerase!

Which gene in E.coli codes for DNA Polymerase I?

The PolA – gene.

What is the fundamental reaction that all DNA Polymerases (in all spieces, that is) catalyzes?

The negatively charged oxygen attached on the 3' carbon of the deoxyribose acts as a nucleophile; attacking the positive dipole that is the phosphate of the incoming deoxyribose. The phosphate is a positive dipole in this case because it is surrounded by electronegative oxygens. A diphosphate is thus released and the two nucleotides are linked to each other by a phosphodiester bond! See picture below!

How can the reaction described above proceed without being too costly energywise?

Non covalent base-pairing offers extra stability, as well as the fact that 19kJ/mol is given through the hydrolysis of pyrophosphate product formed in the reaction. The latter reaction is catalyzed by the enzyme pyrophosphate!

Two fundamental conditions have to be met in order for the DNA polymerase to function – describe them!

First and foremost : the DNA Polymerase needs a template. It needs it in order to “know” which nucleotide to attach to the growing chain – if there is an guanine on the opposite side (on the original DNA strand, that is) the DNA polymerase will fetch a cytosine.

This feature is important to be familiar with also because it is used in guided biosynthesis.

Secondly; the DNA Polymerase is unable to work without a primer!

What is a primer?

A primer is a small fragment of nucleotides – usually RNA oligotides – that is complementary to the DNA template. The function of a primer is to provide the first free 3' end for the DNA Polymerase to start from.

What is the free 3'end of a primer called?

Primer terminus!

What is the next step for the DNA Polymerase after it has added a nucleotide to the free 3´end of the primer or the previous DNA-base?

The enzyme may either continue along a the DNA template, adding nucleotides as it goes, or it can dissociate. Dissociation will of course limit the rate of the process, whereas moving along the template makes it go faster. The number of nucleotides a DNA Polymerase adds to the growing chain before it fucks off defines its processivity. The processivity varies between different DNA Polymerases, from a handful to many thousands bp!

How can replication of a whole genome be so accurate? Describe the possible responsible

processes and enzymes!

First of all : DNA Polymerase will only add C-G and A-T bases to eachother. The hydrogen bonding between two complementary bases are favourble whereas hydrogen bonding between two incorrect bases (i.e. A-C) is not favourble.

This is partly because there are three hydrogen bonds between C and G but only two hydrogen bonds between A and T. Thus, if an Adenine and a Cytosine would bind to eachother, one of oxygens or nitrogens on cytosine would be “left hanging” there with no hydrogen on the other side to connect to. That causes instability.

There is also another reason as to why two non complementary bases wont bind to eachother : geometry. The active site of the DNA Polymerase only accepts the correct geometry, which is formed by two complementary bases! Two non complementary bases might actually hydrogen bond to eachother, but since the resulting geometry wont fit in the active site of the enzyme, the incorrect base will be dismissed before a phosphodiester bond is formed!

Secondly, DNA Polymerases all have two active sites; one that is responsible for the actual elongation of the new DNA strand and another that is responsible for the process known as proofreading!

Describe proofreading!

This mechanism is conserved in most DNA Polymerases (even though some exceptions occur).

It is a exonuclease activity that proceeds in 3' – 5' direction!

It double-checks each nucleotide after it has been added to the growing chain. If the DNA Polymerase has indeed added an incorrect nucleotide the proofreading mechanism will inhibit the DNA Polymerase from moving forward. The pause that results from this provides an oppurtunity to correct the mistake. First of all the 3'-5´exonuclease activity will remove the incorrect base. Secondly the the DNA Polymerase slides back one step and adds a correct base instead.

In short : the DNA Polymerase has two active sites; one for elongation and one for proofreading.

Is proofreading simply the reverse reaction of polymerization?

No, not quite, sine pyrophosphate is not involved in proofreading.