Replication PDF

Replication  Replication Definition:- Replication is the synthesis of DNA. It occurs during the S phase of cell cycle. Steps:- 1. Identification of the origins of replication (ori) 2. Unwinding (denaturation) of dsDNA to provide a ssDNA template 3. Formation of the...

Replication  Replication Definition:- Replication is the synthesis of DNA. It occurs during the S phase of cell cycle. Steps:- 1. Identification of the origins of replication (ori) 2. Unwinding (denaturation) of dsDNA to provide a ssDNA template 3. Formation of the replication fork 4. Initiation of the DNA synthesis and elongation 5. Excision and replacement of the RNA primers 6. Reconstitution of chromatin structure  1- Identification of the origins of replication (ori) The ori is a specific sequence of bases of DNA at which replication starts. In bacteria: It is called ori C. A set of proteins help in identification of the origin of replication and bind it.  2- Unwinding (denaturation) of dsDNA to provide a ssDNA template 2 types of proteins are responsible for unwinding:- 1. DNA helicase:- Allows progressive unwinding of the DNA. 2. Single stranded DNA-binding proteins (SSBs):- That stabilizes the complex. This lead to DNA unwinding and active replication.  3- Formation of the replication fork As the 2 strands unwind and separate, they form a "V" shape where active synthesis occurs called the replication fork.  The replication fork is formed of 4 components:- 1. The DNA helicase: - That unwinds a short segment of the parental duplex DNA. 2. A primase: - That initiates synthesis of an RNA molecule, needed for priming DNA synthesis. 3. The DNA polymerase: - That initiates nascent, daughter strand synthesis. 4. SSBs: - That binds to ssDNA and prevents premature reannealing of ssDNA to ds DNA. The rep. fork moves along the DNA molecule as synthesis occurs. Replication is bidirectional, moves in both directions away from the origin of replication.  4- Initiation of the DNA synthesis and elongation Primosome: - is a mobile complex formed of the helicase and primase enzymes, and responsible for unwinding and formation of RNA primers. Formation of the RNA primer: - – DNA polymerase is unable to synthesize DNA except, by the presence of RNA primer. – RNA primer: - is a short fragment of RNA, about 10 nucleotides in length, synthesized by primase. – It has a free-OH group at 3' end, this -OH group serves as the acceptor of the first nucleotide from DNA polymerase.  Initiation of DNA synthesis:- – It requires priming by a short length of RNA. – It involves nucleophilic attack by the 3' hydroxyl group of the RNA primer on the α phosphate of the first entering deoxynucleoside, with the release of pyrophosphate. Elongation of the newly synthesized DNA strand:- – The 3' hydroxyl group of the first deoxynucleoside monophosphate is then free to carry out a nucleophilic attack to the next deoxynucleoside triphosphate. – Then, a stepwise addition of deoxynucleotides occurs according to the base pairing rule  Replication exhibits polarity The replication of DNA occurs in both strands simultaneously. DNA polymerase III synthesizes DNA in the 5' to 3' direction only. The DNA strands are anti parallel, therefore the newly replicated strands grow in opposite directions. 2 types of strands are present: – Leading strand:- Is the strand that is being copied in the direction towards the replication fork, it is synthesized continuously. It needs one RNA primer. – Lagging strand: - Is the strand being copied in the direction away from the replication fork. It is synthesized discontinuously. It is synthesized in short segments of DNA attached to an RNA primer called Okazaki fragments. Many RNA primers are needed for the lagging strand. This process is called semi discontinuous DNA synthesis.  5- Excision and replacement of the RNA primers – DNA polymerase III synthesizes the DNA fragment of the lagging strand, until it becomes very close to the next RNA primer. – Then, DNA polymerase I excise the primer and replace it with DNA nucleotides. – The reaming nick is then filled by DNA ligase. 6- Reconstitution of chromatin structure:- – Newly replicated DNA is rapidly assembled into nucleosomes and reforms the chromatin again.  Classes of proteins involved in replication Protein Function DNA polymerases Deoxynucleotide polymerization. Helicases Processive unwinding of DNA. Topoisomerases Relieve torsional strain that results from helicase-induced unwinding. DNA primase Initiates synthesis of RNA primer Single-strand binding proteins (SSBPs) Prevent premature reannealing of dsDNA. DNA ligase Seals the single strand nick between the nascent chain and Okazaki fragments on lagging strand.  DNA polymerase A number of different DNA polymerase molecules engage in DNA replication, they share 2 common characters; 1) Chain elongation:- That accounts for the rate (No of nucleotides per second) at which polymerization occur. 2) Proofreading function: - In which it identifies the copying errors and corrects them.  Swivels and nick resealing During unwinding and separation of the 2 Strands, there are multiple swivels interspersed in the DNA molecule. The swivel function is made by enzymes that introduce nicks in one strand of the double helix, thus allowing the unwinding to proceed. The nicks are quickly resealed by two ways: - 1. Resealing without requiring energy :- – By using the DNA topoisomerase. – Topoisomerases are also capable of unwinding supercoiled DNA. 2. ATP-dependent resealing:- – It is carried out using DNA ligase enzyme  DNA synthesis occurs during the S phase of cell cycle The replication of the DNA genome occurs at a specified time during the life span of the cell called the synthetic or S phase. The life span of the cell undergoes 4 phases:- 1. Gap 1 (G1):- The cell prepares for DNA synthesis. Damage DNA detected. 2. Synthetic (S):- Replication of DNA genome. Incomplete replication detected. 3. Gap 2 (G2):- The cell prepares for mitosis. Damage DNA detected. 4. Mitotic (M):- The cell undergoes mitosis. Improper spindle detected.  All eukaryotic cells have gene products that govern the transition from one phase of the cell cycle to another e.g cyclin proteins.