replication-and-recombination.pdf

DNA REPLICATION AND RECOMBINATION HOPE RINETTE P. WONG, R.N., L.P.T. Lecturer Objective At the end of the topic lesson, the learners should have: 1. Illustrated the Replication Fork and the associated enzymes OVERVIEW DNA REPLICATION THEORIES REVIEW OF THE DNA ST...

DNA REPLICATION AND RECOMBINATION HOPE RINETTE P. WONG, R.N., L.P.T. Lecturer Objective At the end of the topic lesson, the learners should have: 1. Illustrated the Replication Fork and the associated enzymes OVERVIEW DNA REPLICATION THEORIES REVIEW OF THE DNA STRUCTURE ENZYMES PROCESS/STEPS DNA RECOMBINATION CROSSING OVER PROCESS SUMMARY DNA REPLICATION DNA replication is the process by which DNA makes a copy of itself during cell division.  DNA replication occurs in all living organisms acting as the most essential part for biological inheritance This is essential for cell division during growth and repair of damaged tissues, while it also ensures that each of the new cells receives its own copy of the DNA. THEORIES CONSERVATIVE MODEL According to the conservative replication model, the entire original DNA double helix serves as a template for a new double helix, such that each round of cell division produces one daughter cell with a completely new DNA double helix and another daughter cell with a completely intact old (or original) DNA double helix. DISPERSIVE MODEL The original DNA double helix breaks apart into fragments, and each fragment then serves as a template for a new DNA fragment. As a result, every cell division produces two cells with varying amounts of old and new DNA. SEMICONSERVATIVE MODEL The two original DNA strands (i.e., the two complementary halves of the double helix) separate during replication; each strand then serves as a template for a new DNA strand, which means that each newly synthesized double helix is a combination of one old (or original) and one new DNA strand. NEW OLD REVIEW: DNA STRUCTURE ANTIPARALLEL- two molecules that are side by side but run in opposite directions 5’ 2’ 1’ 3’ 4’ 4’ 3’ 1’ 5’ 2’  KEY ENZYMES IN DNA REPLICATION HELICASE- (“the unzipping enzyme”) unwind DNA at positions called origins where synthesis will be initiated DNA POLYMERASE- (“the builder”) build new strands of DNA DNA POLYMERASE I- main function is excision repair of DNA strands from the 3′-5′ direction to the 5′-3 direction, as an exonuclease also helps with the maturation of Okazaki fragments, which are short DNA strands that make up the lagging strand during DNA replication DNA POLYMERASE III- the primary enzyme that is used in DNA replication It is responsible for the synthesis of new strands by adding nucleotides to the 3′- OH group of the primer It has a 3′-5′ exonuclease activity hence it can also proofread the errors that may arise during DNA strand replication  PRIMASE- (“the initializer”) an enzyme that synthesizes short RNA sequences called primers. These primers serve as a starting point for DNA synthesis LIGASE- (“the gluer”) facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond TOPOISOMERASE- (“the nicker”) During DNA replication and transcription, DNA becomes overwound ahead of a replication fork In order to prevent and correct these types of topological problems caused by the double helix, topoisomerases bind to DNA and cut the phosphate backbone of either one or both the DNA strands. This intermediate break allows the DNA to be untangled or unwound, and, at the end of these processes, the DNA backbone is resealed again PROCESS of DNA REPLICATION 1.The first step in DNA replication is to ‘unzip’ the double helix structure of the DNA molecule. 2.This is carried out by an enzyme called helicase which breaks the hydrogen bonds holding the complementary bases of DNA together (A with T, C with G). 3.The separation of the two single strands of DNA creates a ‘Y’ shape called a replication ‘fork’. The two separated strands will act as templates for making the new strands of DNA. 4. One of the strands is oriented in the 3’ to 5’ direction (building is towards the replication fork), this is the leading strand. The other strand is oriented in the 5’ to 3’ direction (building is away from the replication fork), this is the lagging strand. As a result of their different orientations, the two strands are replicated differently: Leading Strand: 5. A short piece of RNA called a primer (produced by an enzyme called primase) comes along and binds to the end of the leading strand. The primer acts as the starting point for DNA synthesis. 6. DNA polymerase binds to the leading strand and then ‘walks’ along it, adding new complementary Nucleotide bases (A, C, G and T) to the strand of DNA in the 5’ to 3’ direction. 7. This sort of replication is called continuous. Lagging strand: 5.Numerous RNA primers are made by the primase enzyme and bind at various. points along the lagging strand. 6. Chunks of DNA, called Okazaki fragments, are then added to the lagging strand also in the 5’ to 3’ direction. 7. This type of replication is called discontinuous as the Okazaki fragments will need to be joined up later.. 8.Once all of the bases are matched up (A with T, C with G), an enzyme called exonuclease strips away the primer(s). The gaps where the primer(s) were, are then filled by yet more complementary. nucleotides. 9.The new strand is proofread to make sure there are no mistakes in the new DNA sequence. 10. Finally, an enzyme called DNA ligase seals up the sequence of DNA into two continuous double strands. 11. The result of DNA replication is two DNA molecules consisting of one new and one old chain of nucleotides. This is why DNA replication is described as semi- conservative, half of the chain is part of the original DNA molecule, half is brand new. 12. Following replication the new DNA automatically winds up into a double helix. DNA RECOMBINATION Recombination is a process by which pieces of DNA are broken and recombined to produce new combinations of alleles.  This recombination process creates genetic diversity at the level of genes that reflects differences in the DNA sequences of different organisms. In eukaryotic cells, which are cells with a nucleus and organelles, recombination typically occurs during meiosis. During the first phase of meiosis (prophase I), the homologous pairs of maternal and paternal chromosomes align.  During the alignment, the arms of the chromosomes can overlap and temporarily fuse, causing a crossover. Crossovers result in recombination and the exchange of genetic material between the maternal and paternal chromosomes. As a result, offspring can have different combinations of genes than their parents. Genes that are located farther apart on the same chromosome have a greater likelihood of undergoing recombination, which means they have a greater recombination frequency. paternal.  Holliday Model for General Recombination - Single Strand Invasion In 1964, Robin Holliday proposed a model that accounted for heteroduplex formation and gene conversion during recombination. Although it has been supplanted by the double-strand break model (at least for recombination in yeast and higher organisms), it is a useful place to start and understand recombination. It illustrates the critical steps of pairing of homologous duplexes, formation of a heteroduplex, formation of the recombination joint, branch migration and resolution.  It begins with two paired DNA duplexes, or homologs. (a) In each of which an endonuclease introduces a single-stranded nick at an identical position. (b)  The internal strand endings produced by these cuts are then displaced and subsequently pair with their complements on the opposite duplex. © A ligase seals the loose ends (d)  Creating hybrid duplexes called heteroduplex DNA molecules, held together by a cross-bridge structure and the position of this cross can move down the chromosome by a process called branch migration. (e.) Hydrogen bonds are broken and then reformed between complementary bases.  (If )the duplexes bend and the bottom portion is rotated by 180 degrees (f) The intermediate planar structure called x(chi) form- or Holliday structure- is created. (g)  If the two strands on opposite homologs previously uninvolved in the exchange are now nicked by an endonuclease. (h) Ligation occurs, two recombinant duplexes are created. (i) SUMMARY DNA REPLICATION HELICASE – unwinds the helix and separates the strands PRIMASE- produce RNA primers POLYMERASE III- copies each strand (once continuously on leading strand and Okazaki fragments on lagging strand) POLYMERASE I- replaces the primers with DNA nucleotides LIGASE- seals everything up DNA RECOMBINATION THANK YOU. ☺