台大農藝系 遺傳學 601 20000Chapter 2

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DNA As The Genetic Material,DNA Damage & Repair Mechanisms.

Preety Sweta Hembrom. M.Sc. Genomic science.

Central University Of Kerala

GENETICSTOPIC:

The genetic material must perform three essential function:

1.The Genotypic function,2.The Phenotypic function,3.The Evolutionary function.

This means that genetic material must replicate in order to transmit copies from parents to offspring.

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In the early 1900s, chromosomes were shown to be the carriers of hereditary information.

In eukaryotes they are composed of both DNA and protein, and most scientists initially believed that protein must be the genetic material.

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Chromosome consists of protein and nucleic acid

Protein v.s. nucleic acid Protein: 20 kinds of amino acid Nucleic acid: 2 types – DNA & RNA

During the 1940s and early 1940s through the result of elegant experiments it was clearly established that genetic information is stored in nucleic acids & not in proteins.

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Frederick Griffith in 1928 performed experiment with Streptococcus pneumoniae bacteria in mice.

This showed that something passed from dead bacteria into nearby living ones, allowing them to change their cell surface.

He called this agent the transforming principle, but did not know what it was or how it worked.

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In 1944, Avery, MacLeod and McCarty published results of a study that identified the transforming principle from S. pneumoniae.

Chemically separate the components (e.g., protein, nucleic acids) and determine which was capable of transforming live S. pneumoniae cells.

Only the nucleic acid fraction was capable of transforming the bacteria.

nucleic acid fraction was contaminated with proteins.

The researchers treated this fraction with either RNase or protease and still found transforming activity, but when it was treated with DNase, no transformation occurred, indicating that the transforming principle was DNA.

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1. More evidence for DNA as the genetic material came in 1953 with Alfred Hershey and Martha Chase’s work on E. coli infected with bacteriophage T2.

2. In one part of the experiment, T2 proteins were labeled with 35S, and in the other part, T2 DNA was labeled with 32P.

3. The 35S-labeled protein was found outside the infected cells, while the 32P-labeled DNA was inside the E. coli, indicating that DNA carried the information needed for viral infection. This provided additional support for the idea that genetic inheritance occurs via DNA.

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A BRIEF INTRODUCTION:

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Nucleic acids are composed of nucleotides. Each nucleotide consists of:

1. A Phosphate group,2. 5 carbon sugar &3. A cyclic nitrogen containing base.

In DNA: Sugar is 2-deoxyribose

Four bases are adenine, guanine, thymine & cytosine.

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The faithful transmission of genetic material from one cell to another is based on the ability to replicate.

The process of DNA replication is based on the complementary nature of the strands that makeup DNA duplex.

These strands are held together by hydrogen bonds between specific base pairs- A paired with T & G paired with C.

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Thus the expression of genetic information is that the flow of information is from DNA to RNA to polypeptide which is known as:-

Central Dogma Of Biology

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DNA is easily damaged under normal physiological conditions.

The return of damaged DNA to its normal sequence and structure is called Repair.

Many different kinds of physical & chemical agents damage DNA. Some of these are:-

1. Endogenous agents2. Exogenous agents Cells usually can survive DNA damage

provided that the replication and transcription machinery can still perform their functions.

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Hence, agents that damage DNA can be mutagenic, cytotoxic or both.

DNA damaging agents that cause mutations are called Mutagens.

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Mutations are inherited changes in the genetic material which provide new genetic variation that allows organisms to evolve.

Mutations occur in 2 ways:

1.Spontaneous mutations

2. Induced mutation

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Damages are physical abnormalities in the DNA, such as single and double strand breaks etc.

In DNA damage transcription of a gene can be prevented & thus translation as well as replication of a gene will also be blocked & the cell may die.

Mutation is a change in the base sequence of the DNA. It is replicated when the cell replicate.

Cause alteration in protein function & regulation.

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Although distinctly different from each other, DNA damages & mutations are related to each other because DNA damage often cause errors of DNA synthesis during replication or repair, these errors are a major source of mutation.

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The 4 major types of DNA damages are:

1.Radiation damage2.DNA instability in water3.Oxidative damage4.Alkylation damage

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The high energy electromagnetic radiation to the exposure of which cell experience considerable damage to their DNA are:

1.Ultraviolet light: The major type of damage caused by UV

light is divided into three bands:I. UV-A (321-400 nm)II. UV-B (296-320 nm)III.UV-C (100-295 nm)

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Two major photoproducts account for nearly all of the UV induced DNA damage are:

a)Cyclobutane pyrimidine dimer- 75% of the UV induced damage. Formed by introducing 2 new bonds between adjacent pyrimidines (C & T) on the same DNA strand.

b)(6-4) photoproducts- Remaining of the UV induced damage. Formed by introducing a bond between the c-6 atom of one pyrimidine & the c-4 atom of atoms of adjacent pyrimidine on the same DNA strand.

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2. X- Rays3. Gamma Rays Through these direct damage takes

place when DNA or water tightly bound to it absorbs the radiation.

Indirect damage takes place when water or other molecules surrounding the DNA absorbs the radiation & form reactive species that then damage DNA.

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DNA is damaged by hydrolytic cleavage reactions.

DNA has 2 kinds of bonds with the potential for hydrolytic cleavage:

1.Phosphodiester bonds- Introduces a nick into a DNA strand.

2.N- glycosyl bond- leads to the formation of an abasic site also known as AP site. AP site sensitizes the neighbouring 3’-phosphodiester bond to cleavage.

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Two of the oxidized base products that cause damage are:

1. 8- oxoguanine (oxoG)- Base pair with A or C. If uncorrected 8- oxoG- A base pair will be replicated to form A-T base pair causing transversion mutation.

Guanine oxidizes to 8-oxoguanine, causes single & double strand breaks.

2. Thymine glycol- Inhibits DNA replication & is therefore cytotoxic.

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Alkylating agents damage DNA by transferring alkyl groups to centers of negative charge.

Formation of monoadduct.

Some of the DNA methylating agents are:

1.Dimethylguanosine2.Dimethylsulfate.

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There are five types of DNA repair mechanisms:

1.Light- Dependent repair.2.Excision repair.3.Mismatch repair.4.Post replication repair.5.SOS response.

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Carried out by light activated enzyme called photolyase.

Production of thymine dimers. DNA photolyase recognises & binds to

thymine dimers in DNA & uses light energy to cleave the covalent cross- links.

Photolyase also splits cytosine dimers & cytosine- thymine dimers.

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Two major types of excision repair:I. Base-Excision repair- Remove abnormal or modified

bases from DNA.II. Nucleotide-Excision Repair- Remove larger defects like

thymine dimers.

Base- Excision Repair:- Initiated by a group of enzymes- DNA

glycosylases( recognize abnormal bases in DNA). The glycosylases cleave glycosidic bond b/w the

abnormal base & 2-deoxyribose creating AP sites.

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AP sites are recognized by enzymes- AP endonucleases

DNA polymerase then replaces the missing nucleotide according to the specifications of the complementary strand.

Dna ligase seals the nick.

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Base Excision Repair (BER)

Variety of DNA glycosylases, for different types of

damaged bases.

AP endonuclease recognizes sites with a missing base; cleaves sugar-phosphate

backbone.

Deoxyribose phosphodiesterase removes the sugar-phosphate lacking

the base.

Nucleotide Excision Repair:- Removes bulky DNA lesions that distort the

double helix. An enzyme complex recognizes the

distortion resulting from damage. Additional enzymes separate the two

nucleotide strands at the damaged region, & single strand binding proteins stabilize the separated strands.

The sugar phosphate backbone is cleaved on both sides of the damage.

Part of the damaged is peeled away & the gap is filled by DNA polymerase & sealed by DNA ligase.

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Chapter 2 slide 36

Nucleotide Excision Repair

Excinuclease cuts on either side of damage (~20 nt

altogether).

Strands unwound by helicase.

Many incorrectly inserted nucleotides detected by proofreading are corrected by mismatch repair.

Enzymes cut out the distorted section of the newly synthesized strand of DNA & replace it with new nucleotides.

The proteins that carry out this in E.coli differentiate b/w old & new strands of DNA by the presence of methyl group.

Adenine nucleotides in GATC sequence is methylated.

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The mismatch repair complex brings the mismatch bases close to the methylated GATC sequence & the new strand is identified.

Exonucleases remove nucleotides on the new strand b/w the GATC sequence & the mismatch.

DNA polymerase then replaces the nucleotides correcting the mismatch & DNA ligase seals the nick in the sugar phosphate backbone.

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Takes place after replication. When DNA polymerase III encounters a

thymine dimer in a template strand, its progress is blocked.

The damaged DNA is repaired by a recombination- dependent repair process mediated by E.coli recA gene product.

The recA protein binds to the single strand of DNA at the gap & mediates pairing with the homologous segment of the sister double helix.

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The gap opposite the dimer is filled with the homologous DNA strand from sister DNA molecule.

The resulting gap in the sister double helix is filled by DNA polymerase

And the nick is sealed by DNA ligase.

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Discovered by Miroslav Radman in 1975. Two regulatory protein that controls SOS

response- LexA & RecA protein. Synthesized at low background levels. LexA binds to the DNA region that regulate

the transcription of the genes that are induced during the SOS response.

When the cells are exposed to UV lights or other agents that cause DNA damage, the RecA protein binds to the ss region of DNA.

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The interaction of RecA with DNA activates RecA, which then stimulates LexA to inactivate itself by self-cleavage.

With LexA inactivate, the level of expression of the SOS genes increases & the error prone repair system is activated.

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