1. DNA REPLICATION

2. When the two strands of the DNA double helix are separated, each can serve as a template for the replication of a new complementary strand. This produces two daughter molecules, each of which contains two DNA strands with an antiparallel orientation.

3. DNA synthesis(DNA Replication) was first described in E. coli. DNA synthesis in higher organisms is less well understood, but involves the same types of mechanisms. In either case, initiation of DNA replication commits the cell to continue the process until the entire genome has been replicated.

4. In eukaryotic cells, replication begins at multiple sites along the DNA double helix, thus providing a mechanism of rapidly replicating the great length of the eukaryotic DNA molecule.

5. As the two strands unwind and separate, they form a “V” where active synthesis occurs. This region is called the “replication fork” and it moves along the DNA molecule as synthesis occurs. Replication of dsDNA is bidirectional—that is, the replication forks move in both directions away from the origin

7. The initiation of DNA replication requires certain proteins which are responsible for maintaining the separation of the parental strands including: 1- Dna A protein It binds to specific nucleotide sequences at the origin of replication, causing short, tandemly arranged (one after the other) AT-rich regions in the origin to melt. Melting is ATP-dependent, and results in strand separation with the formation of localized regions of ssDNA.

8. 2. DNA helicases These enzymes bind to ssDNA near the replication fork, and then move into the neighboring double-stranded region, forcing the strands apart—in effect, unwinding the double helix. Helicases require energy provided by ATP

9. 3. Single-stranded DNA-binding (SSB) proteins These proteins (not enzymes) that bind to the ssDNA generated by helicases. These proteins exert two functions; 1 st they keep the two strands of DNA separated in the area of the replication origin, thus providing the single- stranded template required by polymerases, second they protect the DNA from “nucleases” that cleave ssDNA.

10. Action of DNA Polymerase Once the two strands of DNA are separated a group of enzymes called “DNA polymerases” act to synthesize two anti parallel stretches of nucleotide chains. They “read” the parental nucleotide sequences in the 3′→5′ direction, and they synthesize the new complementary strands in the 5′→3′ (antiparallel) direction. Therefore, beginning with one parental double helix, the two newly synthesized stretches of nucleotide chains must grow in opposite directions—one in the 5′→3′ direction toward the replication fork and one in the 5′→3′ direction away from the replication fork.

11. “leading strand” The strand that is being copied in the direction of the advancing replication fork is called the “leading strand” and is it is synthesized continuously. However, the other strand that is being copied in the direction away from the replication fork represents a problem!!. Okazaki fragments. lagging strand”. The strand is synthesized discontinuously and slowly, with small fragments of DNA being copied near the replication fork. These short stretches of discontinuous DNA, termed Okazaki fragments. Later on, they will be eventually joined to become a single, continuous strand. The new strand of DNA produced by this mechanism is termed the “lagging strand”.

12. RNA primers T In fact DNA polymerases cannot initiate synthesis of a complementary strand of DNA on a totally single-stranded template. For this reason, there should be areas on each DNA strand where double stranded regions are formed. These regions are called RNA primers. They are short (about 10 nucleotides length), double-stranded regions consisting of RNA base-paired to the DNA template formed in complementary anti parallel way.

13. These primers are formed by RNA Primase. They are constantly being synthesized at the replication fork on the lagging strand, while there is only one RNA primer at the origin of replication on the leading strand. Now, the DNA polymerase will consider these RNA short stretches as templates to copy them. The mechanism for that is that free “OH” at the 3′ end of the RNA primer serves as an acceptor for the new complementary deoxyribonucleotides when DNA polymerases join them forming phosphodiester bonds & remove PPi group. Later on, the RNA primers will be removed leaving only DNA nucleotides.

15. Chain elongation The newly formed 5′→3′ DNA strands are being elongated using DNA- polymerase III. This enzyme will begin to add new nucleotides along the single-stranded template that specifies the sequence of bases in the newly synthesized chain. DNA polymerase III is a highly “processive” enzyme—that is, it remains bound to the template strand as it moves along, and does not have to diffuse away and rebind before adding each new nucleotides.

16. The nucleotide substrates are 5′-deoxyribonucleoside triphosphates. Pyrophosphate (PPi) is released when each new nucleoside monophosphate is added to the growing chain. All four deoxyribonucleoside triphosphates (dATP, dTTP, dCTP, and dGTP) must be present for DNA elongation to occur. If one of the four is in short supply, DNA synthesis stops when that nucleotide is depleted.

17. On the lagging strand, this enzyme act to add the first deoxyribonucleotide to the 3′- end of RNA primer, later on, it will add other deoxyribonucleotides consequently according to base- pairing role with the DNA template.

19. Excision of RNA primers and their replacement by DNA DNA polymerase I. This enzyme has got a 5′→3′ exonuclease activity that act to remove the RNA primer in 5′→3′ direction removing one nucleotide at each time and replacing the gap by DNA nucleotides until RNA is totally removed and replaced by DNA. Once DNA polymerase III add the first nucleotide to the 3′-OH end of the RNA primers, it continuously add nucleotide monophosphates in 5′→3′ direction over the DNA template until it is blocked by another neighboring RNA primer. At this time, RNA primer will be excised and replaced by DNA polymerase I. This enzyme has got a 5′→3′ exonuclease activity that act to remove the RNA primer in 5′→3′ direction removing one nucleotide at each time and replacing the gap by DNA nucleotides until RNA is totally removed and replaced by DNA.

20. DNA ligase The final phosphodiester linkage between the 5′- phosphate group on the DNA chain synthesized by DNA polymerase III and the 3′-hydroxyl group on the chain made by DNA polymerase I is catalyzed by DNA ligase. The joining of these two stretches of DNA requires energy, which in eukaryotes is provided by the cleavage of ATP to AMP + PPi.

22. Reverse transcriptase It is involved in the replication of retroviruses, such as (HIV). These viruses carry their genetic information form of ssRNA molecules. Following infection of a host cell, the viral enzyme, reverse transcriptase, uses the viral RNA as a template for the 5′→3′ synthesis of viral DNA, which then becomes integrated into host chromosomes.

23. Inhibition of DNA synthesis by nucleoside analogs DNA chain growth can be blocked by the insertion of certain nucleoside analogs that have been modified in the sugar moeity of the nucleoside. By blocking DNA replication, these compounds slow the division of rapidly growing cells and viruses. For example, cytosine arabinoside (cytarabine, or araC) has been used in anticancer chemotherapy, whereas adenine arabinoside (vidarabine, or araA) is an antiviral agent. The chemical modification of the sugar moiety, as seen in zidovudine (AZT), accomplishes the same goal of termination of DNA chain elongation.

24. [Note: These drugs are generally supplied as nucleosides, which are then converted to the active nucleotides by cellular “salvage” enzymes]

26. Organization of Eukaryotic DNA histones A typical human cell contains 46 chromosomes, whose total DNA length is about 1m long! DNA interacts with a large number of proteins, each of which performs a specific function in the ordered packaging of these long molecules of DNA. Eukaryotic DNA is associated with tightly bound basic proteins, called histones. These serve to order the DNA into basic structural units, called nucleosomes, that resemble beads on a string. Nucleosomes are further arranged into increasingly more complex structures that organize and condense the long DNA molecules into chromosomes that can be segregated during cell division. The complex of DNA and protein found inside the nuclei of eukaryotic cells is called chromatin.

27. There are five classes of histones, H1, H2A, H2B, H3, and H4 which are positively charged and thus they form ionic bonds with negatively charged DNA. Histones, along with positively charged ions such as Mg+2, help neutralize the negatively charged DNA phosphate groups.

28. Two molecules each of H2A, H2B, H3, and H4 form the structural core of the individual nucleosome “nucleosome core” around which a segment of the DNA double helix is wound nearly twice. Histone H1, is not found in the nucleosome core, but instead binds to the “linker DNA” chain between the nucleosome beads. H1 is the most tissue-specific and species-specific of the histones. It facilitates the packing of nucleosomes into the more compact structures.

30. nucleofilament Nucleosomes can be packed more tightly to form a polynucleosome (also called a nucleofilament). This structure assumes the shape of a coil, often referred to as a 30-nm fiber. The fiber is organized into loops that are anchored by a nuclear scaffold containing several proteins. Additional levels of organization lead to the final chromosomal structure.