Genetic materials and chromosomes.

transcript

1 Prakash Pokhrel

Genetic materials and its organization into chromosome (DNA)

In this session...

Identification of genetic material

Components of DNA

Structure of DNA

Replication

Damage and repair

IntroductionThe progeny of organism develops characters similar to that

organism

The resemblance of offspring to their parents depends on the

precise transmission of principle component from one generation

to the next

That component is- The Genetic Material

The genetic material of a cell or an organism refers to those materials found in the nucleus, mitochondria and cytoplasm, which play a fundamental role in determining the structure and nature of cell substances, and capable of self-propagating and variation.

What is genetic material?

Four requirements for a genetic material

• Must carry information– Cracking the genetic code

• Must self replicate– DNA replication

• Must allow for information to change

– Mutation

• Must govern the expression of the phenotype

– Gene function

Identification of genetic material:

PROTEINDNA

The process of identification of genetic material began in

1928 with experiments of Griffith and concluded in 1952 with

the studies of Hershey and Chase.

Between these two experiments other three scientists, Avery,

Macloed and McCarty were did an experiment to identify the

genetic material.

Discovery of Transformation in Bacteria:

In 1928, Frederick Griffith discovered bacterial transformation. He worked on Streptococcus pneumonieae (Pneumococcus) Pneumococci have various strains which can be classified by- 1. The presence or absence of a polysaccharide capsule2. The molecular composition of the capsuleWhen grown on blood agar medium, pneumococci with capsules are virulent and form large, smooth colonies and designated as typeIII S

S pneumococci mutate to an avirulent form that has no capsules.

When grown on blood agar medium, these noncapsulated pnuemococci form small, rough-surfaced colonies and designated as typeII R

Based on the molecular composition of the capsule, these pneumococci cells are type I, II, III, and so forth.

(Griffith,1928)

Based on these observations he concluded that some of the cells of typeIIR had changed into typeIIIS due to influence of dead typeIIIS cells

He called this phenomenon as transformation

Principle Component of typeIIIS cells which induced the conversion of type IIR cells into type IIIS was named transforming principle

Griffith’s Conclusions:

Griffith’s transforming principle was the genetic material

Transformation assay to identify actual biomolecule

Major constituents - DNA, RNA, proteins, carbohydrates & lipids

Made cell extracts from type IIIS cells containing each of these macromolecules

1944 - Avery, MacLeod & McCarty Identify the Genetic Material

Avery, MacLeod, McCarty Experiment: The transforming principle is DNA

13(Avery, et al., 1944)

(Avery, et al., 1944) 14

The evidence presented supports the belief that a nucleic

acid of the deoxyribose type is the fundamental unit of the

transforming principle of Pneumococcus TypeIIIS.

(Avery, et al., 1944)

What is DNA?

• nitrogen base and sugar make a nucleoside. • Phosphate group and a nucleoside make a

nucleotide.

•DNA is deoxyribo nucleic acid. A German chemist,Friedrich Miescher, discovered DNA in 1869.

•DNA contains three main components (1) Phosphate (PO4) groups;

(2) Five-carbon sugars; and(3) Nitrogen-containing bases called purines and pyrimidines.

Components of DNA:

Assembly into nucleotides

Nucleotides linked in a chain

The phosphate group of one nucleotide is attached to the sugar of the next nucleotide in line.

• The result is a “backbone” of alternating phosphates and sugars, from which the bases project

5’ PO4

PO4 5’

3’ OH

Structure of DNA:

• Two polynucleotide

chains are held

together by

hydrogen bonding

between bases in

opposing strands.

Watson and Crick’s structure :

They proposed that DNA as

a right handed double helix

with two poly nucleotide

chains are coiled about one

another in a spiral.

(Watson and Crick,1953)21

The strands of DNA are antiparallel

The strands are complimentary

There are Hydrogen bond forces

There are base stacking interactions

There are 10 base pairs per turn

Properties of a DNA double helix

(Watson and Crick,1953)22

23 Watson and Crick with their model of DNA structure

Basis for double helix:

Rosalind Franklin’s DNA X-ray diffraction photograph.

Central cross mark indicates –helical structure of DNA.

Top and bottom dark bands indicates bases perpendicular to axis of molecule.

Chargaff’s base pairing rule:

Percent of adenine = percent of thymine (A=T)

Percent of cytosine = percent of guanine (C=G)

A+G = T+C (or purines = pyrimidines)

(Chargaff et al.,1950)25

DNA Replication:

Replication is one of the most important requirement for a genetic material.

The parent molecule unwinds, and two new daughter strands are built based on base-pairing rules.

It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material’. (Watson and Crick,1953) 26

extreme accuracy of DNA replication is necessary in order

to preserve the integrity of the genome in successive

generations.

DNA has to be copied before a cell divides

DNA is copied during the S or synthesis phase of

interphase

New cells will need identical DNA strands

Biological significance:

Models of DNA replication:

Steps in DNA replication:

Initiation

Proteins bind to DNA and open up double helix

Prepare DNA for complementary base pairing

Elongation

Proteins connect the correct sequences of nucleotides

into a continuous new strand of DNA

Termination

Proteins release the replication complex

Binding proteins prevent single strands from rewinding.

Helicase protein binds to DNA sequences called origins and unwinds DNA strands.

5’ 3’

Primase protein makes a short segment of RNA complementary to the DNA, a primer.

3’ 5’

5’ 3’

Proteins in replication:

Overall directionof replication 5’ 3’

5’ 3’

3’ 5’

DNA polymerase III enzyme adds DNA nucleotides to the RNA primer. DNA polymerase proofreads bases added and replaces

incorrect nucleotides.

3’ 5’

5’ 3’

5’ 5’ 3’

Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

3’ 3’ 5’

5’ 3’

Exonuclease activity of DNA polymerase I removes RNA primers.

Polymerase activity of DNA polymerase I fills the gaps.

Ligase forms bonds between sugar-phosphate backbone.

3’ 5’

5’ 3’

Origin of replication:

Initiator proteins identify specific base sequences on DNA called sites of origin.

Prokaryotes – single origin site E.g E.coli Eukaryotes – multiple sites of origin (replicator)

Prokaryotes Eukaryotes

Most eukaryotes except for budding yeast have ill-defined

origins of replication that rely on epigenetic mechanisms for

molecular recognition by initiator proteins.

Replication is initiated at multiple origins along the DNA

using a conserved mechanism that consists of four steps:

origin recognition, assembly of a prereplicative initiation

complex, followed by activation of the helicase and loading of

the replisome.

(Sclafani and Holzen,2007)36

Uni or bidirectionalReplication forks move in one or opposite directions

Replication Fork

View of bidirectional movement of the DNA replication machinery

Semi-discontinuous replication

Anti parallel strands replicated simultaneouslyLeading strand synthesis continuously in 5’– 3’Lagging strand synthesis in fragments in 5’-3’

DNA synthesis only in 5’ 3’:

Eukaryotic enzymes:

Five common DNA polymerases from mammals.

1. Polymerase (alpha): nuclear, DNA replication, no proofreading

2. Polymerase (beta): nuclear, DNA repair, no proofreading

3. Polymerase (gamma): mitochondria, DNA replication, proofreading

4. Polymerase (delta): nuclear, DNA replication, proofreading

5. Polymerase (epsilon): nuclear, DNA repair, proofreading

Polymerases vary by species.

Model of DNA Replication:

End-replication problem:

Every time a linear chromosome replicates, the laggaing strand at each end gets shorter by about 150 nucleotides. Because there is a minimum length of DNA needed for initiation of an Okazaki fragment.

DNA polymerase/ligase cannot fill gap at end of chromosome after RNA primer is removed. If this gap is not filled, chromosomes would become shorter each round of replication.

Eukaryotes have tandemly repeated sequences at the ends of their chromosomes. Telomerase binds to the terminal telomere repeat and catalyzes the addition of of new repeats.Compensates by lengthening the chromosome.

DNA Damage and Repair:

DNA polymerase do great job during DNA replication by proof reading the new DNA strand.

But its not enough to maintain the 100% fidelity in replication.

Several kinds of damage occurs by endogenous and exogenous agents.

DNA has its own mechanisms to repair this damages and maintain the accuracy of copying mechanism.

Natural polymerase errorEndogenous DNA damage

oxidative damage depurination

Exogenous DNA damageradiation

chemical adducts“Error-prone” DNA repair

Sources of damage

DNA Damage Response(DDR):

To respond to these threats, eukaryotes have evolved the

DNA Damage Response (DDR).

The DDR is a complex signal transduction pathway that has

the ability to sense DNA damage and transduce this

information to the cell to influence cellular responses to

DNA damage.

(Ciccia and Elledge, 2010)

“Mutation is rare because of repair”

Over 200 human genes known to be involved in DNA repairMajor DNA repair pathways:1. Base excision repair (BER)2. DNA Mismatch repair (MMR)3. Nucleotide excision repair (NER)4. DNA strand break repair pathways:

Single strand break repair (SSBR)Double-strand break repair pathways (DSBR)Homologous Recombination (HR)Nonhomologous end joining (NHEJ)

Direct reversal of damage - Photoreactivation (bacteria, yeast, some vertebrates - not humans) Two thymines connected together by UV light.

Excision Repair - removal of defective DNA. There are three distinct types1) base-excision2) nucleotide-excision3) mismatch repair

Base-excision repair(BER):Presence of the Uracil in DNA is a great example of this typeSpecial enzymes replace just the defective base

snip out the defective basecut the DNA strandAdd fresh nucleotideLigate gap

deoxycytosine deoxyuracil

2’3’

thymine

glycosidic bond

Nucleotide-excision repair(NER):

Same as previous except that-

It removes entire damaged nucleotide

Remove larger segments of DNA

Example:Xeroderma pigmentosum

• Extreme sensitivity to sunlight

• Predisposition to skin cancer

Mismatch repair (MMR):Despite extraordinary fidelity of DNA synthesis, errors do

persistSuch errors can be detected and repaired by the post-

replication mismatch repair systemSpecial enzymes scan the DNA for bulky alterations in the

DNA double helixThese are normally caused by mismatched bases

A GA CC T

These are excised and the DNA repaired

MMR also processes mispairs that result from heteroduplex DNA

formed during genetic recombination: act to exclude “homeologous”

recombination.

Repair involving two or more close sites in same heteroduplex occur

much more often on the same strand than the opposite strands.

Analysis of the pattern of repair suggest that repair tracks initiates at

mismatches and propagate preferentially in 5’ to 3’ direction.

(Wagner and Meselson, 1976)52

The problem of strand discrimination:

MMR can only aid replication fidelity if repair is targeted to newly synthesized strand

The cell has a mechanism of identifying new strand synthesis by leaving nicks that DNA. There are enzymes which scan these new regions looking for errors.

Other forms of DNA damage:Depurination - the base is simply ripped out of the DNA molecule

leaving a gap.

Deamination - An amino group of Cytosine is removed and the base becomes Uracil.

Basic mechanism is the same for all three types

1) Remove damaged region

2) Resynthesis DNA3) Ligate

Genetic materials and chromosomes.

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