Introduction to Molecular Biology and DNA Replication

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Presentation transcript:

Introduction to Molecular Biology and DNA Replication Dr. Shumaila Asim Lecture No # 1

Genetic Material in the Living Cells Cells contain a nucleus surrounded by a nuclear membrane in eukaryotic cells, and a nuclear region in the prokaryotic cells. In a non-dividing cell the nucleus is filled with a thread- like material known as "chromatin". Chromatin is made up of DNA and proteins (mainly histones and some non-histone acidic proteins).

The Normal Human Chromosomes Normal human cells contain 23 pairs of homologous chromosomes: 22 pairs of autosomes. 1 pair of sex chromosomes. Autosomes are the same in males and females Sex chromosomes are: XX in females XY in males. Both X are homologous. Y is much smaller than X and has only a few genes.

Composition of Chromosome DNA Histones (Major proteins) q Non-histone (Small amounts) The chromosomes themselves are macromolecular entities that must be synthesized, packaged, protected, and properly distributed to daughter cells at cell division. Significant segments of every chromosome are dedicated to these functions.

Nucleosomes: The Basic Units of DNA Condensation The material of chromosomes, both protein and DNA, is often referred to as chromatin. The protein component is about equal in mass to the DNA component. Histones constitute the largest protein component of chromatin, are highly conserved, basic proteins that assemble into octameric complexes containing two each of four different histone subunits. DNA wraps around the histones to form condensed nucleosomes.

Nucleosome (10 nm diameter): 8 histones in bead & 1 outside. Each bead: is surrounded by 140 bpDNA and there are 60 bp in the linker region. Space between beads is about 14 nm.

The Central Dogma of Life. replication

Replication of DNA It is the process in which each strand of DNA molecule is copied to give two daughter DNA molecules identical to parent DNA The genetic information found in DNA is copied and transmitted to daughter cells Entire chromosome is copied

Three possible models were proposed for DNA replication: a. Conservative model proposed both strands of one copy would be entirely old DNA, while the other copy would have both strands of new DNA. b. Dispersive model was that dsDNA might fragment, replicate dsDNA, and then reassemble, creating a mosaic of old and new dsDNA regions in each new chromosome. c. Semiconservative model is that DNA strands separate, and a complementary strand is synthesized for each, so that sibling chromatids have one old and one new strand. This model was the winner in the Meselson and Stahl experiment.

Fig. 3.1 Three models for the replication of DNA

DNA Replication is Semi-Conservative DNA replication of one helix of DNA results in two identical helices. If the original DNA helix is called the "parental" DNA, the two resulting helices can be called "daughter" helices. Each of these two daughter helices is a nearly exact copy of the parental helix. Each newly synthesized strand of DNA (daughter strand) is made by the addition of a nucleotide that is complementary to the parent strand of DNA. In this way, DNA replication is semi-conservative, meaning that one parent strand is always passed on to the daughter helix of DNA.

Salient features of replication Semi-conservative Ordered , sequential & complete Proceeds bi-directionally (5  3) from many origins Uses activated substrates (dATP, dGTP, dCTP, dTTP) Continuous on one strand & discontinuous on other strand Far more accurate than any other enzyme-catalyzed process

The mechanism of 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

Requirements Substrates Primer Mg++ Four deoxy-nucleoside triphosphates (dATP, dGTP, dCTP, dTTP) (liberation of pyrophosphate provides energy for the addition of the nucleotide at 3-end of primer) Primer RNA primer (10 – 200 nucleotide long) Sequence complementary & anti-parallel to DNA template Synthesized by “primase” Mg++ Optimizes DNA polymerase activity

Requirements Template Each strand of DNA serves as template for the synthesis of complementary strand Double stranded DNA (ds DNA) unwinds to form single stranded DNA (ss DNA) DNA synthesis is continuous on one strand (leading strand) and discontinuous on other strand (lagging strand) (Okazaki fragments), every fragment needs primer

Enzymes & other proteins Requirements Enzymes & other proteins Protein Function DNA polymerases Polymerization of deoxynucleotides Helicases Unwinding of DNA Topo-isomerases (gyrase) Relieve torsional strain that results from helicases-induced unwinding DNA primase Initiates synthesis of RNA primers Single-strand binding proteins Prevent premature re-annealing of dsDNA DNA ligase Seals the single strand nick between the nascent chain and Okazaki fragments on lagging strand

Steps involved in DNA replication in eukaryotes Identification of the origins of replication (ori) Unwinding (denaturation) of dsDNA to provide a ssDNA template Formation of the replication fork Elongation and Termination Reconstitution of chromatin structure

Steps 1. Identification of origin of replication (ori) Sequence-specific dsDNA-binding proteins interact with special sequence sites (consensus sequences)(replicators) (A+T rich regions) of DNA to cause local denaturation and un-winding of DNA These sites of interactions are known as “ori” “Ori” are essential for initiation of DNA synthesis

Steps Identification of origin of replication (ori)

Steps 2. Unwinding of DNA Interaction of proteins with “Ori” defines the start site DNA helicase unwinds the DNA molecule (separates the strands) by breaking the H-bonds between bases ssDNA-binding-proteins help in this separation and certain other proteins stabilize the ssDNA

The first major step for the DNA Replication to take place is the breaking of hydrogen bonds between bases of the two antiparallel strands. The unwinding of the two strands is the starting point. The splitting happens in places of the chains which are rich in A-T. That is because there are only two bonds between Adenine and Thymine (there are three hydrogen bonds between Cytosine and Guanine).  The initiation point where the splitting starts is called "origin of replication".

3. Formation of Replication Fork Helicase is the enzyme that splits the two strands. The structure that is created is known as "Replication Fork".

Steps RNA primer is required for initiation, synthesized by primase (10 – 200 nucleotides) One RNA primer for leading strand Separate RNA primers for each Okazaki fragment DNA polymerase simultaneously catalyzes the leading and lagging strand synthesis (both in 5 -3 directions i.e. opposite to one-another) First deoxy -ribonucleotide triphosphate attacks 3-free end of RNA primer

Diagrammatic representation of DNA replication Diagrammatic representation of DNA replication. direction of replication with both the leading strand and lagging strands of replication shown separated from each other.

Elongation The elongation process is different for the 5'-3' and 3'-5' template. One of the strands is oriented in the 3’ to 5’ direction (towards the replication fork), this is the leading strand. The other strand is oriented in the 5’ to 3’ direction (away from the replication fork), this is the lagging strand. As a result of their different orientations, the two strands are replicated differently

Leading Strand: A short piece of RNA called a primer (produced by an enzyme called primase) comes along and binds to the end of the leading strand. The primer acts as the starting point for DNA synthesis. DNA polymerase binds to the leading strand and then ‘walks’ along it, adding new complementary Nucleotide bases (A, C, G and T) to the strand of DNA in the 5’ to 3’ direction. This sort of replication is called continuous.

Lagging strand: Numerous RNA primers are made by the primase enzyme and bind at various points along the lagging strand. Chunks of DNA, called Okazaki fragments, are then added to the lagging strand also in the 5’ to 3’ direction. This type of replication is called discontinuous as the Okazaki fragments will need to be joined up later

DNA elongation

In prokaryotes, there are three main types of DNA polymerase Polymerase Polymerization (5’-3’) Exonuclease (3’-5’) Exonuclease (5’-3’) I Yes Yes Yes II Yes Yes No III Yes Yes No 3’ to 5’ exonuclease activity = ability to remove nucleotides from the 3’ end of the chain Important proofreading ability Without proofreading error rate (mutation rate) is 1 x 10-6 With proofreading error rate is 1 x 10-9 (1000-fold decrease)

DNA polymerase I also has 3' to 5' and 5' to 3' exonuclease activity, which is used in editing and proofreading DNA for errors. The 3' to 5' can only remove one mononucleotide at a time, and the 5' to 3' activity can remove mononucleotides or up to 10 nucleotides at a time.

Eukaryotic enzymes: Five common DNA polymerases from mammals. Polymerase  (alpha): nuclear, DNA replication, no proofreading Polymerase  (beta): nuclear, DNA repair, no proofreading Polymerase  (gamma): mitochondria, DNA repl., proofreading Polymerase  (delta): nuclear, DNA replication, proofreading Polymerase  (epsilon): nuclear, DNA repair , proofreading

Termination Once all of the bases are matched up (A with T, C with G), an enzyme called exonuclease (DNA Pol I) strips away the primer(s). The gaps where the primer(s) were are then filled by more complementary nucleotides. The new strand is proofread to make sure there are no mistakes in the new DNA sequence. Finally, an enzyme called DNA ligase seals up the sequence of DNA into two continuous double strands.

The last step of DNA Replication is the Termination The last step of DNA Replication is the Termination. This process happens when the DNA Polymerase reaches to an end of the strands. Now in the last section of the lagging strand, when the RNA primer is removed, it is not possible for the DNA Polymerase to seal the gap (because there is no primer). So, the end of the parental strand where the last primer binds isn't replicated. These ends of linear (chromosomal) DNA consists of noncoding DNA that contains repeat sequences and are called telomeres. Telomerase is a ribonucleoprotein recruited to replicate ends of linear chromosomes because normal DNA polymerase cannot replicate the ends, or telomer. In the end, a part of the telomere is removed in every cycle of DNA Replication.

Synthesis of telomeric DNA by telomerase

Editing & proofreading DNA The DNA Replication is not completed before a mechanism of repair fixes possible errors caused during the replication. Enzymes like nucleases remove the wrong nucleotides and the DNA Polymerase fills the gaps. 1000 bases/second DNA polymerase I proofreads & corrects repairs mismatched bases removes abnormal bases repairs damage throughout life reduces error rate from 1 in 10,000 to 1 in 100 million bases

Final Step - Assembly into Nucleosomes: As DNA unwinds, nucleosomes must disassemble. Histones and the associated chromatin proteins must be duplicated by new protein synthesis. Newly replicated DNA is assembled into nucleosomes almost immediately.