W3_L9_DNA replication and repair.pptx

Lecture 9: DNA Replication and Repair Dr RAJ Radhakrishnan Lecturer in Biomedical Sciences St George’s, University of London In this lecture... • DNA replication • DNA repair DNA Replication • Since the DNA holds the genetic information required for heredity, it must be copied when a cell divid...

Lecture 9: DNA Replication and Repair Dr RAJ Radhakrishnan Lecturer in Biomedical Sciences St George’s, University of London In this lecture... • DNA replication • DNA repair DNA Replication • Since the DNA holds the genetic information required for heredity, it must be copied when a cell divides • This has to be done in an accurate and coordinated fashion DNA Replication • There are several characteristics of the DNA and its helical structure that facilitates and hinders this process – the antiparallel strands are templates for each other, as postulated as a copying mechanism by Watson and Crick. – specific base pair hydrogen bonding allows for accurate copying – tightly wound helical structure is stable and needs to have the hydrogen bonds broken for strands to unwind – torsional stress in winding the extremely long molecule need to be overcome DNA replication A simple schematic showing how DNA could be copied. The helix opens out, and the 2 new strands of DNA are produced. The reality of the mechanism is much more complex. Meselson Stahl experiment Bacteria grown with a source of nitrogen (15N), rather than normal 14N. Heavier isotope is incorporated into nitrogenous bases. Cells collected after division and DNA extracted, centrifuged and the density measured. Only semiconservative replication made sense with their results. Phases of DNA replication • Initiation • Elongation • Termination Initiation in bacteria In bacteria DNA replication starts from one point of origin. Specific proteins (initiation complex) recognise DNA sequences at the origin and bind to the DNA and begin to open out the helix and unwind it. This forms a replication bubble, which allows access to proteins that synthesise the new strands. They move away from the origin as they make new DNA. A replication fork is produced. Bidirectional replication. Directionality of DNA replication Origin 3’ 5’ 5’ 3’ unidirectional Origin 5’ 3’ 3’ 5’ Bidirectional* This is by far the most common Eukaryotes have multiple origins The replication bubbles merge and coalesce Molecular Mechanism of DNA Replication Incorporation of new nucleotides Nucleotide triphosphate is attacked by the OH group of the ribose. It forms a phosphodiester bond with the release of pyrophosphate. DNA elongates in a 5’ to 3’ direction • DNA polymerase is the key enzyme responsible for elongation of DNA strands • It can only add new nucleotides at the 3’ end of the deoxyribose. • DNA therefore elongates in a 5’ to 3’ direction. • BUT – DNA has antiparallel strands. How is DNA elongated on the opposite strand? 3’ Movement of replication fork 5’ 3’ ? Discontinuous Replication • The strands must be copied in different ways • One strand can be copied continuously by adding to the 3’ end (leading strand) • The other strand needs to be replicated in small sections, as a discontinuous process (lagging strand) • This requires a whole set of enzymes and proteins to cope with this. Semi discontinuous replication DNA Polymerase • DNA polymerase is the enzyme responsible for elongation of the DNA • There are several types of DNA polymerase, each with specific roles in the process. • DNA polymerases cannot de novo synthesise DNA strands, they must have a template and they can only extend on the 3’ end. • They require a primer DNA Primase Primase is an enzyme that synthesizes short RNA sequences called primers. These primers serve as a starting point for DNA synthesis. Primase functions by synthesizing short RNA sequences that are complementary to a singlestranded piece of DNA, which serves as its template. DNA polymerases need a primer synthesised by DNA primase 5’ 3’ DNA polymerase 5’ 3’ 5’ RNA primer 5’ newly synthesized DNA 3’ Lagging Strand • DNA synthesis on the lagging strand has to be continuously re-initiated, resulting in fragments. • Okazaki fragments are short DNA nucleotide sequences, formed discontinuously on the lagging strand at the time of DNA replication. • They are initiated by the creation of a new RNA primer by the primase. • These fragments then need to be processed. 5’ 3’ 3’ Movement of replication fork DNA Ligase The ligase enzyme joins the Okazaki fragments together, making one strand. Joining of Okazaki fragments 1 RNA primer 5’ (Okazaki fragment) pol 5’ A second enzyme called RNase H may also be involved in removing the primer Joining of Okasaki fragments 2 DNA ligase 3’ 5’ 3’ 5’ Other Enzymes & Protein Required • DNA helicase • Single stranded DNA binding protein • Topoisomerase Enzymes needed for elongation Replication proteins Prokaryotes Polymerase III (10 subunits) Polymerase I DNAg DNAa DNAb SSB DNA ligase RNAseH Gyrase n n’ i i’ i’’ Eukaryotes Polymerase alpha (4subunits) Polymerase delta (several subunits) Polymerase epsilon (several subunits) MCM complex ( 6 proteins) ORCcomplex ( 6 proteins) CDC6 CDT1 Geminin Cdc45 Sld2 Sld3 Gins complex ( 3 proteins) RPA ( 3 subunits) Pcna RFC ( 5 subunits) MCM8 MCM10/cdc23 Fen DNA ligase RNAse H DNA2 Topoisomerase I Topoisomerase II DNA Polymerase Fidelity The fidelity of a DNA polymerase refers to its ability to accurately replicate a template. It is estimated that polymerases make errors approximately once every 104–105 nucleotide polymerized The human genome is about 3 billion bases, so this would represent a big problem in terms of fidelity (10,000 mistakes every generation!!) Obviously – this does not happen. The actual error rate is closer to 1 in 109 or 1 in 1010 nucleotides Base Pairing Rules • Correct base pairing at the active site of the polymerase enzyme is more energetically favourable in the rapid processivity of the enzyme • Correct base pairing is preferred in the geometry of the active site of the enzyme 3’ to 5’ exonuclease activities of polymerases Secondly – the DNA polymerase itself has a proofreading activity The enzyme has a 3’ to 5’ exonuclease activity This means it checks the base pairing as it goes. An incorrect base pair activates a second catalytic site, which removes the incorrect base pair Self correcting DNA polymerase DNA Damage and Repair How does DNA get damaged? endogenous sources • Replicative errors – Incorrect base inserted (point mutation) – Insertion – Deletions • Oxidative damage by free radicals (oxygen metabolism) • Spontaneous alteration in DNA • Alkylating agents (malondialdehyde - byproduct of polyunsaturated fatty acid peroxidation and arachidonic acid metabolism) How does DNA get damaged? exogenous sources • • • • UV Pollution (hydrocarbons) Carcinogens - smoking and chemicals in food / water Radiotherapy -Ionising radiation - X-rays • Chemotherapy - alkylating agents - cisplatin - mitomycin C - cyclophosphamide - melphalan What kind of damage can occur? Bases can become oxidised, damaged, alkylated, deaminated, lost Bases can become dimerised (covalently bonded) DNA backbone can break – single or double strand breaks Strands can become crosslinked What sort of changes occur? How are the errors dealt with? • Direct reversals • Nucleotide excision repair (NER) • Base excision repair (BER) • Mismatch repair • Recombinational repair • Non homologous end joining Direct reversals T T T T Examples: UV light causes 2 adjacent T dimers to become covalently bonded together – cell use a lyase enzyme to directly repair the bases Methylated bases can be repaired by enzymes that remove methyl groups **Does not require template information for repair Nucleotide Excision Repair NER • The error is removed as a stretch of nucleotides Damage recognition leads to removal of a short singlestranded DNA segment that contains the lesion. The undamaged single-stranded DNA remains is used as a template to synthesize a short complementary sequence. DNA ligase fills in to repair the phosphate backbone. Base Excision Repair - BER Only the affected base is removed. The damaged base is removed, leaving a apurinic/apyrimidinic site (AP). The DNA strand is then broken and the base removed, which is then filled in by DNA pol and ligase. Mismatch Repair Mechanism very similar to nucleotide excision repair although this term applies only to situations where the 2 different DNA strands contain a mismatch. ATCGGGACCTAGGA TAGCCGTGGATCCT May be more error prone, as a parental strand needs to be identified. Recombinational repair A very complicated process by which the DNA is repaired using sequences from a homologous piece of DNA. Non Homologous end joining NHEJ Binding together of 2 broken DNA ends. May or may not be the same DNA strands as original. Very toxic form DNA damage. Very prone to error. of Human Syndromes Related to DNA Repair Defects Thank you for your attention. Any questions: [email protected]