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.living organismsDNAbiological 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 processDNA 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

Genetic studies (in prokaryotic) suggested that initiation of replication at oriC most likely depended on the protein encoded by a gene designated dnaA., dnaB, dnaC.proteingene

Eukaryotic DNA have many origins of replication DNA have many origins

We have seen how the activities of helicase and primase solve two of the problems inherent to DNA replication — unwinding of the duplex template and the requirement of DNA polymerases for a primer Remember, though, that both strands of the DNA template are copied as the replication bubble enlarges. Each end of the bubble represents a growing fork where both new strands are synthesized. helicaseprimaseDNA templateprimer growing fork Growing fork Site in double-stranded DNA at which the template strands are separated and addition of deoxyribonucleotides to each newly formed chain occurs; also called ( replication fork. (template

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 separationstrand 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 oAt 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.

sites As synthesis of the leading strand progresses, sites uncovered on the single-stranded template of the lagging strand are copied into short RNA primers (<15 nucleotides) by primase.leading strandtemplatelagging strandRNA. Each of these primers is then elongated by addition of deoxyribonucleotides to its 3′ end. In E. coli, this reaction is catalyzed by DNA polymerase III (Pol III), one of three DNA polymerases produced by E. coli.DNA polymerase

Thus each lagging strand grows in a direction opposite to that in which the growing fork is moving. The resulting short fragments, containing RNA covalently linked to DNA, are called Okazaki fragments After their discoverer Reiji Okazaki. In bacteria and bacteriophages, Okazaki fragments contain 1000 – 2000 nucleotides, and a cycle of Okazaki-strand synthesis takes about 2 seconds to complete. In eukaryotic cells, Okazaki fragments are much shorter (100 – 200 nucleotides.growing forkOkazaki fragments

As each newly formed segment of the lagging strand approaches the 5′ end of the adjacent Okazaki fragment (the one just completed), E. coli DNA polymerase I takes over.lagging strandDNA polymerase Unlike polymerase III, polymerase I has 5′ → 3′ exonuclease activity, which removes the RNA primer of the adjacent fragment; the polymerization activity of polymerase I simultaneously fills in the gap between the fragments by addition of deoxyribonucleotides. Finally, another critical enzyme, DNA ligase, joins adjacent completed fragments.RNAprimerenzymeligase

DNA Polymerase in Pro and Eukaryotic See attached word file

Function in DNA replication Enzyme Also known as helix destabilizing enzyme. Unwinds the DNA double helix at the Replication Fork.Replication Fork DNA Helicase Builds a new duplex DNA strand by adding nucleotides in the 5' to 3' direction. Also performs proof-reading and error correction. DNA Polymeras e Bind to ssDNA and prevent the DNA double helix from re- annealing after DNA helicase unwinds it thus maintaining the strand separation. Single- Strand Binding (SSB) Proteins Relaxes the DNA from its super-coiled nature. Topoisome rase Re-anneals the semi-conservative strands and joins Okazaki Fragments of the lagging strand.Okazaki Fragments DNA Ligase Provides a starting point of RNA (or DNA) for DNA polymerase to begin synthesis of the new DNA strand. Primase

Telomerase Prevents Progressive Shortening of Lagging Strands during Eukaryotic DNA Replication

Unlike bacterial chromosomes, which are circular, eukaryotic chromosomes are linear and carry specialized ends called telomeres. Telomeres consist of repetitive oligomeric sequences; for example, the yeast telomeric repeat sequence is 5′-G1 – 3 T-3′. telomeres

The need for a specialized region at the ends of eukaryotic chromosomes is apparent when we consider that all known DNA polymerases elongate DNA chains from the 3′ end, and all require an RNA or DNA primer.DNARNAprimer As the growing fork approaches the end of a linear chromosome, synthesis of the leading strand continues to the end of the DNA template strand; the resulting completely replicated daughter DNA double helix then is released.growing forkchromosomeleading strandtemplate

However, because the lagging-strand template is copied in a discontinuous fashion, it cannot be replicated in its entirety. When the final RNA primer is removed, there is no upstream strand onto which DNA polymerase can build to fill the resulting gap. Without some special mechanism, the daughter DNA strand resulting from lagging-strand synthesis would be shortened at each cell division.upstreamDNA polymerasecell division

The enzyme that prevents this progressive shortening of the lagging strand is a modified reverse transcriptase called telomerase, which can elongate the lagging-strand template from its 3′-hydroxyl end.enzymelagging strand reverse transcriptasetemplate This unusual enzyme contains a catalytic site that polymerizes deoxyribonucleotides directed by an RNA template, and the RNA template itself, which is brought to the site of catalysis as part of the enzyme (Figure 12-13).RNAFigure 12-13

The repetitive sequence added by telomerase is determined by the RNA associated with the enzyme, which varies among telomerases from different sources. Once the 3′ end of the lagging-strand template is sufficiently elongated, synthesis of the lagging strand can take place, presumably from additional primers.

Cell Cycle in Prokaryotic and Eukaryotic

The Prokaryotic Cell Cycle 1- The prokaryotic cell cycle is a relatively straightforward process. Essentially, unicellular prokaryotic organisms grow until reaching a critical size, and synthesize more cytoplasm, cell membrane, ribosomes, cell wall, and other cell constituents. They then replicate their DNA, segregate copies of the chromosome, and divide by a process called binary fission to produce two new genetically identical daughter cells.

Binary fission in prokaryotic

Binary fission in a prokaryotic 1- The bacterium before binary fission is when the DNA tightly coiled. 2- The DNA of the bacterium has replicated. 3- The DNA is pulled to the separate poles of the bacterium as it increases size to prepare for splitting. 4- The growth of a new cell wall begins to separate the bacterium. 5- The new cell wall fully develops, resulting in the complete split of the bacterium. 6- The new daughter cells have tightly coiled DNA, ribosomes, and plasmids

2- Most research suggests that the rate of fission in prokaryotic organisms is largely controlled by environmental conditions. For example, most prokaryotic organisms have an optimum temperature range for cell growth. When environmental temperatures are above or below the optimum, cell division tends to decrease.

3-Under ideal environmental conditions, many prokaryotic species undergo binary fission at a fairly rapid rate with generation times of one to several hours. This can lead to an astonishing growth in population size over a relatively short period of time. In some instances, populations of prokaryotes may increase by a million or even a billion fold in a matter of days.

Phases of the Cell Cycle in Eukaryotic Interphase –G 1 - primary growth –S - genome replicated –G 2 - secondary growth M - mitosis C - cytokinesis

Cell cycleCell 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 G 1 - Cells undergo majority of growth S - Each chromosome replicates (Synthesizes) to produce sister chromatids –Attached at centromere –Contains attachment site (kinetochore) G 2 - 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