PTT 207 Biomolecular and Genetic Engineering Semester 1 2012/2013 BY: PUAN NURUL AIN HARMIZA ABDULLAH
We totally missed the possible role of enzymes in DNA repair…. I later came to realize that DNA is so precious that probably many distinct repair mechanisms would exist. Nowadays one could hardly discuss mutation without considering repair at the same time. Francis Crick, Nature (1974), 248:766
7.1 Introduction
• Damage can occur to all cellular molecules. • DNA repair refer to a collection of process by which a cell identifies and corrects damage to the DNA molecules that encoded its genome. • Because DNA is genetic material, changes in its structure can result in mutations .
7.2 Mutations and DNA damage
Mutation and DNA Damage • Mutations result from changes in the nucleotide sequence of DNA or from deletions, insertions, or rearrangements of DNA sequences in the genome. • These changes can be spontaneous or induced.
Spontaneous mutations • Occur as a result of natural processes in cells. e.g. DNA replication errors Induced mutations • Occur as a result of interaction of DNA with an outside agent that causes DNA damage.
Mutation and DNA Damage • The simplest type of mutation is a nucleotide substitution or point mutation . • A nucleotide pair in DNA duplex is replaced with a different nucleotide pair.
Transitions and transversions can lead to silent, missense, or nonsense mutations • Transition mutations replace one pyrimidine base with another, or one purine base with another. • Transversion mutations replace a pyrimidine with a purine or vice versa. • In humans, the ratio of transitions to transversions is approximately 2:1
• A transition or transversion mutation can be permanently incorporated by DNA replication.
SILENT MUTATIONS • Mutations that change the nucleotide sequence without changing the amino acid sequence are called synonymous mutations or silent mutations.
MISSENSE MUTATIONS • Nucleotide substitutions in protein-coding regions that do result in changed amino acids are called nonsynonymous mutations or missense mutations. • May alter the biological properties of the protein. • Sickle cell anemia is an AT→TA transversion: • Glutamic acid codon in the -globin gene replaced by a valine codon
NONSENSE MUTATIONS • A nucleotide substitution that creates a new stop codon is called a nonsense mutation. • Causes premature chain termination during protein synthesis. • Nearly always a nonfunctional product .
Frameshift Mutations • If the length of an insertion or deletion is not an exact multiple of three nucleotides, this results in a shift in the reading frame of the resulting mRNA. • Usually leads to production of a nonfunctional protein .
Table : Codons (displayed as mRNA triplets)
EXPANSION OF TRINUCLEOTIDE REPEATS LEADS TO GENETIC INSTABILITY • Trinucleotide repeats can adopt triple helix conformations and unusual DNA secondary structures that interfere with transcription and DNA replication. Refer chapter 2.4 pg 31. • Expansion of trinucleotide repeats leads to certain genetic neurological disorders.
REPEAT EXPANSION CAN OCCUR BY TWO DIFFERENT MECHANISMS: • Unequal crossing over. • Slippage during DNA replication.
Unequal crossing over • A trinucleotide repeat in one chromosome misaligns for recombination during meiosis with a different copy of the repeat in the homologous chromosome, instead of with the corresponding copy. • Recombination increases the number of repeats on one chromosome, resulting in a duplication. • On the other chromosome, there is a deletion .
Slippage during DNA replication • During DNA replication the DNA melts and then reanneals incorrectly in the repeated region, resulting in re- replication of an additional repeat .
http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/mutations/deletions.html
DNA DAMAGE • Damages are physical abnormalities in the DNA , such as single- and double-strand breaks, 8- hydroxydeoxyguanosine residues, and polycyclic aromatic hydrocarbon adducts. • DNA damages can be recognized by enzymes, and, thus, they can be correctly repaired if redundant information, such as the undamaged sequence in the complementary DNA strand or in a homologous chromosome, is available for copying. • If a cell retains DNA damage, transcription of a gene can be prevented, and, thus, translation into a protein will also be blocked. Replication may also be blocked and/or the cell may die.
DIFFERENCE BETWEEN MUTATION AND DNA DAMAGE • In contrast to DNA damage, a mutation is a change in the base sequence of the DNA . • A mutation cannot be recognized by enzymes once the base change is present in both DNA strands, and, thus, a mutation cannot be repaired. • At the cellular level, mutations can cause alterations in protein function and regulation. • Mutations are replicated when the cell replicates. In a population of cells, mutant cells will increase or decrease in frequency according to the effects of the mutation on the ability of the cell to survive and reproduce. • Although distinctly different from each other, DNA damages and mutations are related because DNA damages often cause errors of DNA synthesis during replication or repair; these errors are a major source of mutation.
Three General Classes of DNA Damage • Single base changes • Structural distortion • DNA backbone damage
Single base changes • Deamination , alkylation , and oxidation are all capable of causing a modification in one or more bases in a DNA sequence. • Deamination is the loss of an amino group (-NH 2 ) of the DNA bases. • Alkylation occurs when a reactive mutagen transfers an alkyl group (typically a small hydrocarbon side chain such as a methyl or ethyl group, denoted as-CH 3 and-C 2 H 5 , respectively) to a DNA base. • Oxidative damage to DNA bases occurs when an oxygen atom binds to a carbon atom in the DNA base
Structural distortion • UV radiation induces that formation of a cyclobutane ring between adjacent thymines, forming a T-T dimer. • The T-T dimer distorts the double helix and can block transcription and replication. • UV radiation can also induce dimers between cytosine and thymine.
thymine-thymine dimer thymine-cytosine dimer
GENERAL CLASSES OF DNA DAMAGE • Structural distortion can be caused by intercalating agents and base analogs : • Ethidium bromide has several flat polycyclic rings that insert between the DNA bases. • 5-bromouracil , an analog of thymine, can mispair with guanine.
DNA backbone damage Formation of abasic sites • Loss of the nitrogenous base from a nucleotide. • Generated spontaneously by the formation of unstable base adducts. Double-stranded DNA breaks • Induced by ionizing radiation and a wide range of chemical compounds. • The most severe type of DNA damage.
DNA REPAIR • Cells cannot function if DNA damage corrupts the integrity and accessibility of essential information in the genome (but cells remain superficially functional when so-called "non- essential" genes are missing or damaged).
TYPES OF DNA REPAIR • Damage bypass • not truly repair but a way of coping with damage so that life can go on • Damage reversal • simplest; enzymatic action restores normal structure without breaking backbone • Damage removal • involves cutting out and replacing a damaged or inappropriate base or section of nucleotides
1. LESION BYPASS Translesion synthesis (TLS) • Specialized low-fidelity, “error - prone” DNA polymerases transiently replace the replicative polymerases and copy past damaged DNA .
1. LESION BYPASS • The genome of a cell is continuously exposed to different compounds and types of radiation that can alter the chemical composition of the DNA. In response, the cell has developed different types of DNA repair mechanisms that can remove the lesion. When the lesion is not removed before replication is initiated, it can result in a block of the replication machinery that can ultimately lead to cell death. To bypass these blocks, specialized translesion synthesis (TLS) DNA polymerases are recruited to the site of the lesion. The TLS polymerases are capable of DNA synthesis over the damaged DNA, after which the replicative DNA polymerase can continue normal DNA synthesis. These TLS polymerases are generally error prone and have been implicated in drug resistance in bacteria and in different forms of cancer in humans.
1. LESION BYPASS Error-prone DNA polymerases • May insert incorrect nucleotides opposite the lesion: nucleotide substitution • May skip past and insert correct nucleotides opposite bases downstream: frameshift
1. LESION BYPASS DNA polymerase eta ( ) • Performs translesion synthesis past TT dimers by inserting 2 Adenine residues. • Has an extra wide active site that can accommodate two dNTPs instead of one. • Van der Waals forces and hydrogen-bonding interactions hold the TT dimer so that the two thymines can be paired with two adenines.
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