7. 3. replication. Unit 7: Molecular biology and genetics

Transcription

1 7. 3 DN replication he fact that DN is a self-replicating molecule and can make copies of itself is the basis of all life forms. It is the essence of what life is. Indeed, according to Richard Dawkins in his book he Selfish ene, DN is the life form on planet Earth, perpetuating itself by using its code to direct the building of living organisms. hose best adapted organisms will survive and their DN will proliferate in their descendants. On successful completion of this topic you will: be able to carry out experimental techniques involving manipulating DN, RN and protein (LO3). o achieve a Pass in this unit you will need to show that you can: explain the process of DN replication (3.1) safely perform techniques to isolate DN and mrn (3.2) describe the polymerase chain reaction (3.3). 1

2 1 DN Every time a cell divides to produce new cells its DN is copied. Each molecule of DN undergoes semi-conservative replication. Put very simply, the DN unwinds and unzips to expose nucleotide bases. DN polymerases catalyse the addition of activated DN nucleotides, according to complementary base-pairing rules, to make two new identical molecules of DN, each one containing one old strand and one new strand. Hence each new molecule contains half of the original molecule. Before DN synthesis begins the original strands are separated and the synthesis of the daughter strands begins at the replication fork at a site called an origin of replication where a replisome is assembled from many proteins. he initiation complex that is formed attracts DN polymerases. Synthesis of the new strands is called elongation and is aided by the proteins in the replisome. Lastly the termination site replicates (see Figures and 7.3.2). Figure 7.3.1: he DN replication fork. Because both daughter strands are synthesised in the to direction, the DN complementary to the lagging strand is synthesised in small fragments called Okazaki fragments. hese fragments are then joined together. Leading strand Lagging strand with Okzaki fragments Most recently synthesized DN Figure 7.3.2: Enzymes involved in DN replication. Enzyme Primase synthesises RN DN polymerase III extends RN primer into Okazaki fragments Next Okazaki fragment is synthesised DN polymerase I uses nick translation to replace RN primer with DN Ligase seals the nick 7.3: DN replication 2

3 Key terms Lagging strand: he DN strand that is synthesised (during replication) in a to direction away from the replication fork in short, Okazaki fragments that are then joined. Leading strand: he DN strand that is synthesised (during replication) with no or few interruptions, in a to direction towards the replication fork. Primers: Short, single-stranded sequences of DN or RN, usually of around bases long. he replisome he replisome consists of many proteins, including helicase, gyrase/ topoisomerase, primase, DN polymerases, RNse H and ligase. One DN polymerase complex synthesises the lagging strand and another synthesises the leading strand. here are also factors, called replication proteins, that protect both the unstable single-stranded unwound leading and lagging strands from making hydrogen bonds with themselves and forming hairpins. Helicase Helicase causes the hydrogen bonds between complementary base pairs to break and so catalyses the separation of the two parental strands that will act as templates for synthesis of the daughter molecules. Helicase moves along the DN in a to direction. yrase yrase (a form of topoisomerase) unwinds the resulting supercoil that forms upstream of the section of unwound DN. DN polymerases DN polymerases catalyse the elongation phase of replication. lamp proteins lamp proteins help keep the DN polymerases attached to the leading and lagging strands and make sure the process proceeds at a suitably fast rate. Priming In eukaryotic cells a DN-dependent RN polymerase creates an RN primer, of about 10 bases long, on both the newly separated leading and lagging strands, once for the leading strand and once per Okazaki fragment (about 1000 base pairs long) on the lagging strand. he RN primer attached to its DN template is called -form DN. (Normal DN is called B-form DN.) In prokaryotes primase creates an RN primer at the beginning of the newly separated leading and lagging strands. DN polymerase enzymes cannot bind directly to single-stranded DN and these primers provide a short chain of nucleotides that give the correct configuration to allow the active site of DN polymerase to fit on and begin elongation. Elongation he leading and lagging strands are anti-parallel. In the leading strand nucleotide synthesis (catalysed by DN polymerase epsilon in eukaryotes and by DN polymerase III in prokaryotes) proceeds in the to direction ( to direction on the template strand) and makes a continuous complementary strand. Synthesis of the other strand in the opposite direction cannot occur at the same time so replication of the lagging strand is discontinuous. It involves making short discrete nucleotide chains, called Okazaki fragments, that are then joined by DN repair enzymes, such as DN polymerase I and ligase, so it is not made in one continuous strand. 7.3: DN replication 3

4 ctivity: Semidiscontinuous and semi-conservative 1 Explain why the replication of DN is described as semidiscontinuous. 2 Explain why the replication of DN is described as semiconservative. 3 Research and write an illustrated account to show how Meselson and Stahl s experiment confirmed that DN replication is semiconservative. 4 Make a 3D poster showing how a piece of DN replicates. his can only happen once a sufficient length of DN has been unwound so replication of this strand lags behind that of the leading strand. RNse H enzymes remove the unstable RN primers from the newly synthesised fragments and replace them with DN fragments. DN ligase (aided by polymerase I in prokaryotes) enzyme connects the Okazaki fragments, closing the gaps between their sugar-phosphate backbones by catalysing the formation of phosphodiester bonds. Proofreading enzymes correct any mistakes due to insertion of incorrect bases. ase study: Investigation to find if the replisome moves along the DN molecule You probably envisage the replisome moving along the DN molecule. In 2000 Katherine Lemon and lan rossman, at the Massachusetts Institute of echnology (MI), carried out an experiment using the bacteria Bacillus subtilis. hey tagged the replisomes with a green fluorescent protein and used microscopy to observe its position in the cell during DN replication. he replisome was always in the same position. What can you conclude from this investigation does the replisome move along the DN molecule or is the DN fed through the replisome? he polymerase chain reaction (PR) First developed in 1983 by Kary Mullis, the polymerase chain reaction (PR) is a way of amplifying small amounts of DN in a laboratory for analysis. It is similar to DN replication that happens in cells but it can only be used to amplify short lengths of DN up to 40-kilo base pairs not whole chromosomes. Figure summarises the process. aq DN polymerase is obtained from a thermophilic bacterium, hermus aquaticus, so that the temperature does not have to be lowered to 37 at any stage, and this speeds up the process. Heat (95 ), rather than helicase, causes the DN strands to separate. he temperature is reduced to 55 and DN primers, complementary to the ends of each strand of the DN, are added to anneal at the ends of the separated chains and initiate DN polymerase activity. Now DN polymerase and a supply of activated DN nucleotides (P, P, P and P) are added, the temperature is raised to 72 and the DN is replicated. his one cycle has doubled the DN. It can be repeated, increasing the DN exponentially. his process used to be lengthy, as it involved using water baths and timers, but it is now carried out in a PR thermocycler that adjusts temperatures as necessary. Portfolio activity: PR reaction arry out PR amplification of a DN sample. ctivity: How many cycles? 1 How many PR cycles does it take to amplify one length of DN into (a) 1 million lengths (b) 2 million lengths? 2 If it takes 8.5 seconds for one PR cycle, how long does it take to amplify one length of DN to 1 million copies of it? 7.3: DN replication 4

5 5 Unit 7: Molecular biology and genetics 7.3: DN replication ctivity: omparing DN replication and PR ompare the process of DN replication with the polymerase chain reaction. You may want to use annotated diagrams or a table of information. 1 Double-stranded DN sample 3 dd primers and reduce temperature to 55 to allow primers to anneal 4 Raise temperature to 72 DN polymerase binds and extends primers using free nucleotides 2 Heat to 95 strands separated PR Figure 7.3.3: he polymerase chain reaction (PR). Kacper works in a hospital laboratory, using the PR reaction for pre-implantation genetic testing. If both parents carry a recessive allele, for example, for cystic fibrosis, they may choose to have IVF. Eggs are fertilised and grown in vitro until they reach the eight-cell stage. Without damaging the embryos, one cell can be taken and its DN extracted and amplified using PR. It can then be tested to see if it has normal alleles for the FR gene. One or two healthy embryos will then be implanted into the mother s uterus. Pre-implantation genetic testing technician Patience is a molecular biologist who carries out genetic testing for several conditions including cystic fibrosis, coeliac disease, Down s syndrome and HIV at a private pathology company. his involves extracting DN from biological samples and amplifying it using the PR. She begins each day by checking the worklist on the lab computer to see which tests have to be carried out that day. She then plans the day, according to how many tests and how long each one will take, so that she can get them all done. It takes a long time to extract DN for HPV (human papilloma virus) assay so she extracts these first. She has levels in biology, chemistry and maths and a degree in biomedical science. She hopes to become a registered clinical scientist, which will take about six years and involves passing an exam so she can register with the Health Professions ouncil. his will open up many more career opportunities for her. Molecular biologist

6 hecklist t the end of this topic guide you should be familiar with the following ideas: DN is a self-replicating molecule and is duplicated during the S phase of the cell cycle, before the cell divides replisomes, complexes of many proteins, most acting as enzymes, direct the DN replication, which is semi-conservative and semi-discontinuous as a molecule of DN begins to unwind it forms the replication fork where unpaired nucleotides are exposed and can bind to complementary activated nucleotides this process starts at the end of the template strand and is continuous on one strand (the leading strand), starting where an RN primer has been added on the other strand (the lagging strand), antiparallel to the leading strand, new DN is synthesised in short Okazaki fragments, each started by an RN primer that is then displaced, and ligase enzyme then joins the fragments together proofreading enzymes check for errors the polymerase chain reaction is a useful laboratory technique for augmenting small amounts of smaller lengths of DN for forensic or clinical analysis. cknowledgements he publisher would like to thank the following for their kind permission to reproduce their photographs: orbis: MedicalRF.com ll other images Pearson Education We are grateful to the following for permission to reproduce copyright material: Figure 7.3.1: he DN replication fork, from Molecular biology of the cell, 5th ed. lberts et al. opyright 2008 from Molecular Biology of the ell, Fifth Edition by lberts et al. Reproduced by permission of arland Science/aylor & Francis LL; Figure 7.3.2: Enzymes involved in DN replication, from enes by Benjamin Lewin, published by OUP, Used by permission; Figure 7.3.3: he polymerase chain reaction (PR), from OR 2 Biology, Pearson. Used with permission of Pearson Education Ltd. Every effort has been made to trace the copyright holders and we apologise in advance for any unintentional omissions. We would be pleased to insert the appropriate acknowledgement in any subsequent edition of this publication. 7.3: DN replication 6