Lecture 4 Dna Repair&mutation

DNA MUTATION/DAMAGE The most common lesion that occurs in DNA is depurination.

Molecules in the gene do undergo major changes due to thermal fluctuations. About 5,000 purine bases (adenine and guanine) are lost per day from the DNA of each human cell because of the thermal disruption of their N-glycosyl linkages to deoxyribose (depurination)

DNA MUTATION/DAMAGE Similarly spontaneous deaminations of cytosine to uracil in DNA are estimated to occur at a rate of 100 per genome per day.

DNA MUTATION/DAMAGE DNA bases are also subject to change by reactive metabolites that alter their base-pairing abilities and by ultraviolet light from the sun, which can promote a covalent linkage of two adjacent thymine bases in DNA (forming thymine dimers). These changes lead to either deletion of one or more base pairs in the daughter DNA chain after DNA replication or to a base-pair substitution (e.g., each C → U deamination would eventually change a C-G base pair to a T-A base pair, since U closely resembles T and forms a complementary base pair with A).

DNA REPAIR The altered position of a damaged or mutated strand is recognised and removed by one set of enzymes and then replaced in its original by another enzyme, DNA polymerase (which copies the information stored in the good strand by means of complementary base-pairing). DNA ligase then seals the nick that remains in the DNA helix to complete restoration of an intact DNA strand.

DNA REPAIR Depurination is very efficiently repaired. First, the presence of the missing base is recognised by a repair nuclease that cuts the phosphodiester backbone at the altered site. The neighbouring nucleotides (including the damage one) have been removed by further cuts around the initial site of incision, an undamaged DNA sequence is restored.

DNA REPAIR Another important repair pathway involves a battery of different enzymes called DNA glycoylases, each of which recognises a single type of altered base in DNA and catalyses its hydrolytic removal from the deoxyribose sugar. At least 20 different such enzymes are thought to exist, including those for removing deaminated C’s, deaminated A’s, different types of alkylated bases, bases with opened rings, and bases in which a carbon-carbon double bond has been accidentally converted to a carbon-carbon single bond.

The reaction catalysed by all DNA glycosylase enzymes. There are many different DNA glycosylases, each recognising a different altered base.

Excision and Restoration-fundamental to DNA repair.

DNA REPAIR In cases where the C bases are methylated to produce 5-methylcytosine, at specific points in the DNA sequence it yields a T residue which is a normal component of DNA, rather than a U residue. As a result, these particular C deaminations are not recognised by the cell’s uracil DNA glycosylases, and they are consequently not repaired. The deamination of a methylated cytosine residue in DNA produces thymine instead of uracil, which cannot be recognised and removed by uracil DNA glycosylase

DNA REPAIR Multiple-step excision pathway capable of removing almost any type of DNA damage that creates a very large lesion. Such “bulky lesions” include those created by the covalent reaction of DNA bases with large hydrocarbons, such as benzpyrene, that have carcinogenic potential and the thymine dimers caused by sunlight. In such cases, a large multi-enzyme complex recognises the large distortion in the DNA double helix that results, rather than a specific base change.

Pyrimidine dimer- Two adjacent pyrimidine residues in the same strand of DNA, which have become covalently cross-linked (e.g by UV radiation) The first step is cleavage of the phosphodiester backbone next to the distortion. The second step is excision of the lesion and resynthesis of DNA in its place

Excision and Restoration

Restoration reaction: two steps A filling by DNA polymerase of the gap created by excision events. The sealing by DNA ligase of a nick left in the repaired strand

DNA REPAIR The importance of these repair processes to life is reflected in the large investment that cells make in DNA repair enzymes. For example, a genetic analysis of human patients with xeroderma pigmentosum suggest that at least five different enzymes are required for the excision of “bulky lesions” alone. In yeast there are more than 50 different types of DNA repair enzymes, each monitoring the DNA of each cell at all times.

DNA REPAIR Genetic information can be stably stored in DNA sequences only because a large variety of different DNA repair enzymes are continuously scanning the DNA and removing damaged nucleotides. The process of DNA repair depends on the fact that a separate copy of the genetic information is stored in each strand of the DNA double helix. An accidental lesion on one strand can therefore be cut out by a repair enzyme and a good strand resynthesised from the information in the undamaged strand.