PTT 207 Biomolecular and Genetic Engineering Semester 1 2012/2013 - - PowerPoint PPT Presentation

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.

• Expansion of trinucleotide repeats leads to

certain genetic neurological disorders.

Refer chapter 2.4 pg 31.

REPEAT EXPANSION CAN OCCUR BY TWO DIFFERENT MECHANISMS:

• Unequal crossing over.
• Slippage during DNA replication.

• 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
• n one chromosome, resulting in a duplication.
• On the other chromosome, there is a deletion.

Unequal crossing over

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 (-NH2) 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-CH3 and-C2H5, 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
• r 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
• pposite 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.

• 2. DIRECT REVERSAL OF DNA

DAMAGE

Reversal of thymine-thymine dimers by DNA photolyase

• In most organisms, UV radiation damage to DNA

can be directly repaired.

• DNA photolyase uses energy from near UV to blue

light to break the covalent bonds holding two adjacent pyrimidines together.

• 2. DIRECT REVERSAL OF DNA

DAMAGE

DNA photolyase has two cofactors:

• A pigment that absorbs UV/blue light
• Fully reduced flavin dinucleotide (FADH-)

• 2. DIRECT REVERSAL OF DNA

DAMAGE

Damage reversal by DNA methyltransferase

• O6-alkyl-guanine is the major carcinogenic lesion

in DNA induced by alkylating mutagens. This DNA adduct is removed by the repair protein, O6-methylguanine-DNA methyltransferase.

• Methyltransferase catalyzes the transfer of the methyl

group on O6-methylguanine to the sulfhydryl group of a cysteine residue on the enzyme.

• 3. REPAIR OF SINGLE BASE CHANGES AND

STRUCTURAL DISTORTIONS BY REMOVAL OF DNA DAMAGE

Base excision repair

• The correction of single base changes that are

due to conversion of one base to another.

• Specific DNA glycosylases recognize and excise

the damaged base.

• How do DNA repair proteins find the rare sites of

damage in a vast expanse of undamaged DNA?

• 3. REPAIR OF SINGLE BASE CHANGES AND

STRUCTURAL DISTORTIONS BY REMOVAL OF DNA DAMAGE

• DNA's bases may be modified by deamination or

alkylation.

• The position of the modified (damaged) base is

called the "abasic site" or "AP site".

• In E.coli, the DNA glycosylase can recognize the AP

site and remove its base.

• Then, the AP endonuclease removes the AP site

and neighboring nucleotides.

• The gap is filled by DNA polymerase I and DNA

ligase.

• 3. REPAIR OF SINGLE BASE CHANGES AND

STRUCTURAL DISTORTIONS BY REMOVAL OF DNA DAMAGE

Mismatch repair

• The correction of mismatched base pairs which

result from DNA polymerase errors during replication.

• A large region of DNA including the mismatch is

excised.

• The method of strand discrimination in

mammalian cells is currently unknown.

• 3. REPAIR OF SINGLE BASE CHANGES AND

STRUCTURAL DISTORTIONS BY REMOVAL OF DNA DAMAGE

Nucleotide excision

• In E. coli, proteins UvrA, UvrB, and UvrC are involved in removing

the damaged nucleotides (e.g., the dimer induced by UV light). The gap is then filled by DNA polymerase I and DNA

• ligase. In yeast, the proteins similar to Uvr's are named RADxx

("RAD" stands for "radiation"), such as RAD3, RAD10. etc.

• Repair of structural distortion
• e.g. bulges from thymine-thymine dimers induced by UV irradiation.
• Global genome repair (GGR) pathway: repair of lesions in the

whole genome.

• Transcription coupled repair (TCR) pathway: repair of lesions in

the transcribed strand of active genes.

• 3. DOUBLE-STRAND BREAK REPAIR BY

REMOVAL OF DNA DAMAGE

• Double-strand breaks in DNA are induced by

reactive oxygen species, ionizing radiation, and chemicals the generate reactive oxygen species (free radicals).

• Repaired by homologous recombination or

nonhomologous end-joining.

• 4. DOUBLE-STRAND BREAK REPAIR BY

REMOVAL OF DNA DAMAGE

Homologous recombination

• Repairs double-strand breaks by retrieving genetic

information from an undamaged homologous chromosome. Nonhomologous end-joining (NHEJ)

• Rejoins double-strand breaks via direct ligation of the DNA

ends without any requirement for sequence homology.

The End

Thank You