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  • MCD Nucleic Acids Alexandra Burke-Smith

    1

    1. Nucleic Acids and Chromosomes Dr Birgit Leitinger ([email protected])

    DNA and RNA bases - purines and pyrimidines, the four bases of DNA (A, T, G, C) and of RNA (A, U, G, C)

    Sugars in DNA and RNA - deoxyribose and ribose

    Nucleotides and nucleosides

    DNA: polymer of deoxyribonucleotide units

    Primary sequence of DNA specifies genetic information

    DNA double helix - antiparallel complementary strands

    Melting and annealing of complementary strands

    Watson-Crick base pairing: G-C and A-T

    Genomes of E.coli (a single circular DNA molecule) and Homo Sapiens (46 chromosomes contain linear DNA)

    The human karyotype

    Packaging of eukaryotic DNA - chromatin and chromosomes

    1. Outline the Chemical composition of DNA

    Nucleic Acids

    DNA and RNA are nucleic acids; linear polymer chains consisting of nucleotides joined by phosphodiester

    bonds

    DNA is a linear polymer of deoxyribonucleotide units

    A nucleotide consists of a nitrogenous base, a sugar and one or more phosphate groups

    A nucleoside is composed of a nitrogenous base and a sugar (NO PHOSPHATE)

    DNA and RNA each contain 4 different types of nucleotides that are arranged in different sequences.

    Sugars- deoxyribose or ribose

    The sugar in DNA is deoxyribose, lacking an oxygen atom that is present in ribose, the parent compound. Ribose is the sugar in RNA.

    Primes () are used in numbering the carbon atoms in the ribose: 1 to 5. The 1 C is linked to the base; the 5 C is linked to the phosphate. The 3 C has an OH group which is important in the structure of DNA.

    DNA and RNA nitrogenous bases

    There are 5 different bases; but each type of nucleic acid (DNA or RNA) contains only 4 bases:

    Purines (big): Adenine (A) and Guanine (G)

    Consists of TWO aromatic rings including carbon and nitrogen

    Pyrimidines (Small): Cytosine (C) and Thymine (T)/ Uracil (U)- in RNA

    Consists of ONE aromatic ring including carbon and nitrogen

    The nitrogenous bases are then linked to the pentose sugar (ribose or doxyribose) by a beta-Glycosidic

    linkage

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  • MCD Nucleic Acids Alexandra Burke-Smith

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    Nucleosides vs Nucleotides

    Base + (deoxy)ribose = nucleoside

    Base Nucleoside

    Adenine (deoxy)adenosine

    Guanine (deoxy)guanosine

    Cytosine (deoxy)cytidine

    Uracil (deoxy)uridine

    Thymine (deoxy)thymidine

    Note: a nucleoside can also involve a ribose sugar instead of a deoxyribose, e.g. adenosine

    Base + ribose + phosphate = nucleotide

    E.g. adenosine monophosphate (AMP), deoxycytidine triphosphate (dCTP)

    2. Describe the Structure of DNA

    A single DNA strand is a long chain of deoxyribonucleotide units linked by phosphodiester links.

    The 3 C-OH group on a sugar of one nucleotide is linked to phosphate group, which in turn is joined to 5-OH of an adjacent sugar.

    On each deoxyribose there is a base.

    The chain has two ends, the 5 end and the 3 end. It is not symmetrical.

    The primary sequence is the linear sequence of the bases. By convention, the nucleotide sequence is specified in the 5 to 3 direction.

    The bases carry genetic information, the sugar and phosphates perform a structural role.

    The secondary structure of DNA is a right-handed double helix. The two chains in the helix run in opposite directions; antiparallel

    There are 10 base pairs per helical turn

    The deoxyribose and phosphate groups run along the outside of the helix, with the negative charges outside.

    The bases point inwards and the flat planes are perpendicular to the helix.

    The two chains are held together by hydrogen bonds between the bases.

    The two strands are complementary in their sequence due to the specificity of base-pairing. Adenine always pairs with Thymine (or Uracil in RNA); Guanine always pairs with Cytosine.

    A and T form 2 Hydrogen bonds, which is less stable than C and G, which forms 3 hydrogen bonds

    The DNA strands can be separated by Melting (Heat or low salt) and re-annealing (Cool or high salt)

    RNA can assume a variety of shapes.

    3. Explain How cells package DNA

    Genome The entire DNA coding for an organism constitutes its genome.

    E.coli has 4.6 x 106 base pairs in a single circular double-stranded molecule. The length of E.coli DNA is 1.4

    mm.

    The human genome is approxiamately 3 x 109 base pairs of DNA divided into chromosomes that each

    contain a single, linear double-helical DNA molecule of ~ 200 x 106 base pairs. Chromosomes are visible only just before cells divide but not in non-dividing cells. Mitotic chromosomes: highly condensed form of

    chromosomes. Interphase chromosomes: extended form

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    The karyotype A karyotype is an organised profile of someones chromosomes.

    A diploid human cell has 46 chromosomes o 22 pairs of normal chromosomes (autosomes) and o 2 sex chromosomes (X and Y)

    Abnormal Karyotypes in breast tumour: 48 chromosomes instead of 46, multiple translocations, eg two pieces of

    chromosome 8 (green) plus piece of chromosome 17 (purple).

    Packaging of eukaryotic DNA

    tightly packaged, forming a complex with proteins. This complex is called chromatin. During interphase,

    when cells are not dividing, chromatin is more extended. Further condensation during mitosis produces

    chromosomes.

    The lowest level of packaging is the nucleosome, which consists of DNA wrapped around histone proteins.

    Nucleosome includes ~ 200 bp DNA (linker DNA plus core DNA)

    Structure of the nucleosome

    The nucleosome causes an approximately 7-fold condensation of DNA. (200 bp ~68nm; wrapping around

    histone octamer ~10 nm).

    The chain of nucleosomes is further packed to generate a more compact structure: the 30 nm fiber, giving

    ~40-fold condensation.

    In the interphase nucleus the DNA is further condensed about 100-1000 fold.

    Chromosomes

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    2. DNA Replication, the cell cycle and mitosis Dr Birgit Leitinger ([email protected])

    DNA replication is semi-conservative

    DNA synthesis is mediated by DNA polymerases

    DNA polymerases require a template and a primer

    DNA synthesis occurs in the 5 to 3 direction

    Nucleoside analogs lacking a 3-OH terminate the chain

    The replication fork, leading and lagging strands

    Proof reading mechanism

    Replication of E.coli and mammalian chromosomes

    Phases of the life cycle of a eukaryotic cell

    Stages of mitosis

    1. Explain how cells duplicate DNA

    DNA replication

    Before each cell division, a faithful copy of the DNA needs to be made to ensure

    genetic continuity in the daughter cell

    DNA replication is semi-conservative. Each daughter cell inherits one old and

    one new strand.

    The two strands are complementary to each other so each strand serves as a

    template for the synthesis of the other strand. This generates two identical copies.

    The DNA helix is very stable and has to be unwound before replication can occur. This is done by a DNA

    helicase, an enzyme that uses ATP as source of energy to break hydrogen bonds between base pairs.

    New DNA is synthesised by enzymes called DNA polymerases. DNA polymerases add nucleotides to the 3

    end of a growing chain. DNA polymerases require a template strand, an olinucleotide primer (sequence of

    polymerized nucleotides made by a different enzyme) , and a supply of of deoxynucleotide triphosphates

    (dNTPs)

    The Enzyme reaction

    DNA polymerases add dNTPs to the 3 end of a DNA molecule.

    DNA (and RNA) synthesis occurs in 5 to 3 direction.

    Energy is released by hydrolysis of the triphosphate. This drives the reaction.

    A free 3 hydroxyl group is required.

    Some nucleoside analogs are used as drugs- they act as chain terminators e.g. Acyclovir- herpes, and

    dideoxycytosine (ddC)- Drug for HIV (zalcitabine). Chain terminators do not have a free 3 hydroxyl group,

    therefore polymerase cannot add anymore free dNTPs

    The Replication Fork

    Replication begins at discrete points on the DNA molecule called origin of replication.

    The site of DNA synthesis is called a replication fork: the fork moves along during the process.

    The templates for the two new daughter strands have opposite orientations: 3 to 5 and 5 to 3

    The replication fork is asymmetric. Both strands are synthesised in a 5-3 direction. The leading strand is

    synthesised continuously (i.e. the top one in the diagram above), whereas the lagging strand is synthesised

    in short pieces termed Okazaki fragments

    (bottom strand)

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  • MCD Nucleic Acids Alexandra Burke-Smith

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    RNA Primers

    RNA primes the synthesis of new DNA

    A specialised RNA polymerase called DNA primase synthesises a short RNA fragment (~ 5 nucleotides). The RNA primer is only transient and removed at a later stage of replication.

    For the synthesis of the leading strand, an RNA primer is needed only to start replication at a replication origin.

    Synthesis of the Lagging Strand

    DNA primase synthesises multiple short RNA fragment primers

    DNA polymerase adds to RNA primer, starting the Okazaki fragment,