DNA, Chromosomes and DNA Replication

Dr.Aida Fadhel Biawi

DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance.

The process starts when one double-stranded DNA molecule produces two identical copies of the molecule.

DNA REPLICATION

How does DNA replicate?

DNA Replication is a semiconservative process that results in a double-stranded molecule that synthesizes to produce two new double stranded molecules such that each original single strand is paired with one newly made single strand.

Semiconservative replication would produce two copies that each contained one of the original strands and one new strand.

replication begins at specific sites on DNA molecule called "origins of replication , "

origins are specific sequence of bases

mammalian DNA have many origins

The replication fork is a structure that forms within the nucleus during DNA replication. It is created by helicases, which break the hydrogen bonds holding the two DNA strands together.

Replication fork is where the parental DNA strands hasn't untwist. Replication bubbles allow DNA replication to speed up therefore the untwisted DNA would not be attacked by enzymes while replicating. . ( Which enzymes can attack DNA?? )

Specific enzymes & proteins recognize origins & bind DNA :

1- primase and DNA polymerase will find these specific portions and will bind to the template DNA at the correct location .

( DNA replication requires a RNA primer , primer synthesized by the enzyme primase , primer is a short strand RNA about 5 bases and RNA primer is complementary to DNA )

- new DNA synthesized by DNA polymerase , DNA polymerase binds to parent DNA strand with primer.

- DNA polymerase sequentially adds deoxyribonucleotides to RNA primer , deoxyribonucleotides added have bases complementary to parent strand DNA .

- The rate nucleotide additions in bacteria add about 500 bases/second while in mammels add about 50 bases/second ??!!

2-replication requires strand separation a. strand separation begins at origin of

replication (Helicase)

b. specific proteins prevent the two separated DNA strands from coming back together (single strand binding protein)

At origin of replication, one strand of DNA is made in a continuous manner (the leading strand) and the other in a discontinuous manner (the lagging strand(.

DNA is made in only the 5-prime to 3-prime direction and the replication bubble opens the original double stranded DNA to expose both a 3-prime to 5-prime template (Leading strand template) and it complement.

The lagging strand must be synthesized as a series of discontinuous segments of DNA. ??

These small fragments are called Okazaki fragments and they are joined together by an enzyme known as DNA ligase.

EnzymeFunction in DNA replication

DNA HelicaseAlso known as helix destabilizing enzyme. Unwinds the DNA double helix at the Replication Fork.

DNA Polymerase

Builds a new duplex DNA strand by adding nucleotides in the 5' to 3' direction. Also performs proof-reading and error correction.

Single-Strand Binding (SSB) Proteins

Bind to ssDNA and prevent the DNA double helix from re-annealing after DNA helicase unwinds it thus maintaining the strand separation.

TopoisomeraseRelaxes the DNA from its super-coiled nature.

DNA LigaseRe-anneals the semi-conservative strands and joins Okazaki Fragments of the lagging strand.

PrimaseProvides a starting point of RNA (or DNA) for DNA polymerase to begin synthesis of the new DNA strand.

DNA CONDENSATION

A cell's genetic information, in the form of DNA, is stored in the nucleus. The space inside the nucleus is limited and has to contain billions of nucleotides that compose the cell's DNA. Therefore, the DNA has to be highly organized or condensed. There are several levels to the DNA packaging.

At the finest level, the nucleotides are organized in the form of linear strands of double helices. The DNA strand is wrapped around histones, a form of DNA binding proteins. Each unit of DNA wrapped around a histone molecule is called a nucleosome . The nucleosomes are linked together by the long strand of DNA.

Characteristics of Histone ProteinsCharacteristics of Histone Proteins

- -Octamere structureOctamere structure - -Responsible for packaging DNA into nucleosomesResponsible for packaging DNA into nucleosomes

- -44 different types: H2A, H2B, H3, H4different types: H2A, H2B, H3, H4

Nucleosome StructuresHistone octamer

2 H2A2 H2B2 H32 H4

Beads on a String—10 nm Fiber

histones (= 1g per g DNA) DNA

proteinpurification

H1

H3H2A

H2BH4

•Basic (arg, lys);•+ charges bind to - phosphates on DNA

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To further condense the DNA material, nucleosomes are compacted together to form chromatin fibers. The chromatin fibers then fold together into large looped domain. During the mitotic cycle, the looped domains are organized into distinct structures called the chromosomes.

Packing of DNA into ChromatinPacking of DNA into Chromatin

- histone-DNA complexes, referred to as nucleosomes, which are further folded into higher-order chromatin structures .

Nucleofilament or solenoid

30 nm fiber

Orders of chromatin structure from naked DNA to chromatin to fully condensed chromosomes...

Fig. 9

- Chromosomes are also used as a way of referring to the genetic basis of an organism as either diploid or haploid. Many eukaryotic cells have two sets of the chromosomes and are called diploid. Other cells that only contain one set of the chromosomes are called haploid.

• A diploid cell has two sets of each of its chromosomes• A human has 46 chromosomes (2n = 46)• In a cell in which DNA synthesis has occurred all the chromosomes are

duplicated and thus each consists of two identical sister chromatids

Maternal set ofchromosomes (n = 3)

Paternal set ofchromosomes (n = 3)

2n = 6

Two sister chromatidsof one replicatedchromosome

Two nonsisterchromatids ina homologous pair

Pair of homologouschromosomes(one from each set)

Centromere

Chromosome Duplication

0.5 µm

Chromosomeduplication(including DNA synthesis)

Centromere

Separation of sister

chromatids

Sisterchromatids

Centrometers Sister chromatids

An eukaryotic cell has multiplechromosomes, one of which is

represented here. Before duplication, each chromosome

has a single DNA molecule.

Once duplicated, a chromosomeconsists of two sister chromatids

connected at the centromere. Eachchromatid contains a copy of the

DNA molecule.

Mechanical processes separate the sister chromatids into two chromosomes and distribute

them to two daughter cells.

• In preparation for cell division, DNA is replicated and the chromosomes condense

• Each duplicated chromosome has two sister chromatids, which separate during cell division

• Because of duplication, each condensed chromosome consists of 2 identical chromatids joined by a centromere.

• Each duplicated chromosome contains 2 identical DNA molecules (unless a mutation occurred), one in each chromatid:

Chromosome Duplication

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Two unduplicatedchromosomes

Centromere

Sisterchromatids

Sisterchromatids

Duplication

Non-sisterchromatids

Two duplicated chromosomes

Structure of Chromosomes• The centromere is a constricted region of the chromosome containing a

specific DNA sequence, to which is bound 2 discs of protein called kinetochores.

• Kinetochores serve as points of attachment for microtubules that move the chromosomes during cell division.???!!!

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Metaphase chromosome

Kinetochore

Kinetochoremicrotubules

Centromereregion ofchromosome

Sister Chromatids

Chromosome structure

p: short arm

q: long arm

C: constriction point or centromere , the location of centromere give the chromosome its shape and can be used to help describe the location of the genes.

A typical mitotic chromosome at metaphase

– Diploid - A cell possessing two copies of each chromosome (human body cells).

• Homologous chromosomes are made up of sister chromatids joined at the centromere.

– Haploid - A cell possessing a single copy of each chromosome (human sex cells).

Phases of the Cell Cycle• Interphase

– G1 - primary growth

– S - genome replicated

– G2 - secondary growth

• M - mitosis• C - cytokinesis

Cell cycle begins with the formation of two cells from the division of a parent cell and ends when the daughter cell does so as well.

Observable under the microscope, M phase consists of two events, mitosis (division of the nucleus) and cytokinesis (division of the cytoplasm).

As replication of the DNA occurs during S-phase, when condensation of the chromatin occurs two copies of each chromosome remain attached at the centromere to form sister chromatids.

After the nuclear envelope fragments, the microtubules of the mitotic spindle separate the sister chromatids and move them to opposite ends of the cell.

Cytokinesis and reformation of the nuclear membranes occur to complete the cell division.

-Most of the time, cells are in interphase, where growth occurs and cellular components are made. DNA is manufactured during S phase.

-To prepare the cell for S phase (DNA synthesis), G1 phase occurs (the preparation of DNA synthesis machinery, production of histones).

-In an analogous manner, the cell prepares for mitosis in the G2 phase by producing the machinery required for cell division. -The length of time spent in G1 is variable. In growing mammalian cells often spend ??? hours in G1 phase. G2 is usually shorter than G1 and is usually ??? hours. And S phase ???

Interphase

• G1 - Cells undergo majority of growth

• S - Each chromosome replicates (Synthesizes) to produce sister chromatids– Attached at centromere– Contains attachment site (kinetochore)

• G2 - Chromosomes condense - Assemble

machinery for division such as centrioles

Mitosis Some haploid & diploid cells divide by mitosis. Each new cell receives one copy of every

chromosome that was present in the original cell. Produces 2 new cells that are both genetically

identical to the original cell.

DNA duplication

during interphase Mitosis

Diploid Cell