Finishing replication

Completion of DNA replication requires a set of specific events. These events are different for circular versus linear chromosomes. For a circular chromosome, the conventional replication machinery can replicate the entire molecule, but the resulting daughters are topologically linked to one another. Type II DNA topoisomerass are required to separate (or decatenate) daughter DNA molecules.

Topoisomerase II catalyzes the decatenation of replication products

 Replication of the very ends of linear chromosomes cannot be completed by the replication machinery we have discussed so far.
The requirement for an RNA primer to initiate all new DNA synthesis creates a dilemma of the ends of linear chromosomes, called the end replication problem. Prokaryotic cells use a protein priming protein instead of RNA primers, the protein provides the priming 3OH to initiate DNA synthesis. Eukaryotic cells use the enzyme, telomerase to replicate their chromosome ends (telomeres). Telomerase is a polymerase acts to extend the 3 end of its substrate.

The end replication problem

DNA ligase
As each newly formed segment of the lagging strand approaches the 5end of the adjacent Okazaki fragment (the one just completed), DNA polymerase I takes over. The DNA polymerase I has two functions: 1- The 5 3 exonuclease activity of this enzyme removes the RNA primer of the adjacent fragment. 2- Fills the gap between the DNA fragments. Finally DNA ligase joins the DNA fragments by the formation of Enz-AMP Complex which binds to the 5 phosphate of one fragment activating it, so It is susceptible to 3 OH attack to form a phosphodiester linkage. Thus, the two DNA fragments are ligated into one fragment.

Mode of action of DNA ligase

Definition of mutation
Mutation: a change in the genetic material (ie. DNA).
Let's further define mutation as a heritable change in the genetic material.

This point becomes important in multicellular organisms where we must distinguish between changes in gametes (germline mutations) and changes in body cells (somatic mutations).

Types of Mutations
A. Base pair (nucleotide pair) substitutions
These are of two types: 1- Transitions (purine to purine or pyrimidine to pyrimidine) 2- Transversions (purine to pyrimidine or pyrimidine to purine). The two categories because they can occur in different ways.

B. Frameshift mutations
These result from the insertion or deletion of one or more nucleotides in the coding region of a gene.

This causes an alteration of the reading frame: since codons are groups of three nucleotides, there are three possible reading frames for each gene although only one is used.
eg. mRNA with sequence AUG CAG AUA AAC GCU GCA UAA amino acid sequence from the first reading frame: met gln ile asn ala ala stop The second reading frame gives: cys arg stop

Origins of spontaneous mutation

A spontaneous mutation is one that occurs as a result of natural processes in cells. Induced mutations; those that occur as a result of interaction of DNA with an outside agent or mutagen. . Some so-called "spontaneous mutations" probably are the result of naturally occurring mutagens in the environment; nevertheless there are others that definitely arise spontaneously, for example, DNA replication errors.

DNA replication errors and polymerase accuracy

Mistakes in DNA replication where an incorrect nucleotide is added

will lead to a mutation in the next round of DNA replication of the strand with the incorrect nucleotide. The frequency at which a DNA polymerase makes mistakes (inserts an incorrect base) will influence the spontaneous mutation frequency and it has been observed that different polymerases vary in their accuracy. One major factor affecting polymerase accuracy is the presence of a "proofreading" 3'-5' exonuclease which will remove incorrectly paired bases inserted by the polymerase and to prevent mutations .

A mutation can be permanently incorporated by replication

Base alterations and base damage

The bases of DNA are subject to spontaneous structural alterations called tautomerization. If during DNA replication, G is in the enol form, the polymerase will add a T across from it instead of the normal C because the base pairing rules are changed (not a polymerase error). The result is a G :C to A :T transition; tautomerization causes transition mutations only.

 Another mutatgenic process occurring in cells is spontaneous base degradation. The deamination of cytosine to uracil happens at a significant rate in cells.
Deamination can be repaired by a specific repair process which detects uracil, not normally present in DNA; otherwise the U will cause A to be inserted opposite it and cause a C:G to T:A transition when the DNA is replicated.

Deamination of cytosine creates uracil

 Deamination of methylcytosine to thymine can also occur. Methylcytosine occurs in the human genome at a sequence which is normally avoided in the coding regions of genes. If the meC is deaminated to T, there is no repair system which can recognize and remove it (because T is a normal base in DNA). This means that wherever the sequence containing meC occurs in genes it is a "hot spot" for mutation.

Deamination of 5-methyl cytosine creates thymine

DNA is damaged by alkylation, oxidation, and radiation

In alkylation, methyl or ethyl groups are transferred to reactive sites On the bases and to phosphates in the DNA backbone.

Alkylating chemicals include nitrosamines and N-methyl-N-nitro-NNitrosoguanidine.

Example: is the formation of O6-methylguanine, often mispairs with thymine, resulting in the change of a G:C to A:T when damaged DNA is replicated

Specific sites on guanine that can be damaged by alkylation, oxidation or radiation

Ultraviolet induces the formation of a cyclobutane ring between adjacent thymines