DNA Replication PDF

Molecular Biology I Lecture 2 - 3: DNA Replication & Repair Figure 16.7 G 5 end C Hydrogen bond...

Molecular Biology I Lecture 2 - 3: DNA Replication & Repair Figure 16.7 G 5 end C Hydrogen bond 3 end C G G C G C T A 3.4 nm T A G C G C C G A T 1 nm C G T A C G G C C G A T A T 3 end A T 0.34 nm T A 5 end (a) Key features of (b) Partial chemical structure (c) Space-filling DNA structure model Figure 16.8 Sugar Sugar Adenine (A) Thymine (T) Sugar Sugar Guanine (G) Cytosine (C) Figure 16.9-3 A T A T A T A T C G C G C G C G T A T A T A T A A T A T A T A T G C G C G C G C (a) Parent molecule (b) Separation of (c) “Daughter” DNA molecules, strands each consisting of one parental strand and one new strand  Watson and Crick’s semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) and one newly made strand Competing models were the conservative model (the two parent strands rejoin) and the dispersive model (each strand is a mix of old and new) © 2011 Pearson Education, Inc. Figure 16.10 Parent First Second cell replication replication (a) Conservative model (b) Semiconservative model (c) Dispersive model Getting Started Replication begins at particular sites called origins of replication, where the two DNA strands are separated, opening up a replication “ bubble” A eukaryotic chromosome may have hundreds or even thousands of origins of replication Replication proceeds in both directions from each origin, until the entire molecule is copied Animation: Origins of Replication © 2011 Pearson Education, Inc. Figure 16.12 (a) Origin of replication in an E. coli cell (b) Origins of replication in a eukaryotic cell Origin of Double-stranded Parental (template) strand Origin of replication DNA molecule replication Daughter (new) strand Parental (template) Daughter (new) strand strand Replication Double- fork stranded DNA molecule Replication bubble Bubble Replication fork Two daughter DNA molecules Two daughter DNA molecules 0.25 m 0.5 m Figure 16.13 Primase 3 Topoisomerase 5 RNA 3 primer 5 3 Helicase 5 Single-strand binding proteins  DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the 3 end The initial nucleotide strand is a short RNA primer © 2011 Pearson Education, Inc.  An enzyme called primase can start an RNA chain from scratch and adds RNA nucleotides one at a time using the parental DNA as a template The primer is short (5–10 nucleotides long), and the 3 end serves as the starting point for the new DNA strand © 2011 Pearson Education, Inc. Synthesizing a New DNA Strand Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork Most DNA polymerases require a primer and a DNA template strand The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells © 2011 Pearson Education, Inc.  Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate dATP supplies adenine to DNA and is similar to the ATP of energy metabolism The difference is in their sugars: dATP has deoxyribose while ATP has ribose As each monomer of dATP joins the DNA strand, it loses two phosphate groups as a molecule of pyrophosphate © 2011 Pearson Education, Inc. Roles of DNA Polymerases 1. DNA polymerase catalyzes the formation of a phosphodiester bond between the 3’-OH group of the deoxyribose on the last nucleotide and the 5’-phosphate of the dNTP precursor. The energy for the formation of the phosphodiester bond comes from the release of two of three phosphates from the dNTP 2. finds the correct precursor dNTP that can form a complementary base pair with the nucleotide on the template strand of DNA 3. The direction of synthesis of the new DNA chain is only from 5’-to-3’. 14  Other DNA polymerases Scientists have now identified a total of five DNA polymerases, DNA Pol I–V. Functionally, DNA Pol I and DNA Pol III are polymerases necessary for replication, and DNA Pol I, II, IV, and V are polymerases involved in DNA repair. Both DNA Pol I and DNA Pol III replicate DNA in the 5’-to-3’ direction. Both enzymes also have 3’ to 5’ exonuclease activity, meaning that they can remove nucleotides from the 3’ end of a DNA chain (error correction in a proofreading mechanism). DNA Pol I also has 5’-to-3’ exonuclease activity and can remove either DNA or RNA nucleotides from the end of a nucleic acid strand. 15 Figure 16.14 New strand Template strand 5 3 5 3 Sugar A T A T Phosphate Base C G C G G C G C DNA OH polymerase 3 A T A T P P Pi OH P P C Pyrophosphate 3 C OH Nucleoside 2Pi triphosphate 5 5 Antiparallel Elongation The antiparallel structure of the double helix affects replication DNA polymerases add nucleotides only to the free 3end of a growing strand; therefore, a new DNA strand can elongate only in the 5to3direction © 2011 Pearson Education, Inc.  Along one template strand of DNA, the DNA polymerase synthesizes a leading strand continuously, moving toward the replication fork Animation: Leading Strand © 2011 Pearson Education, Inc. Figure 16.15a Overview Leading strand Origin of replication Lagging strand Primer Lagging Leading strand strand Overall directions of replication  Origin of Figure 16.15b replication 3 5 5 RNA primer 3 3 Sliding clamp DNA pol III Parental DNA 5 3 5 5 3 3 5  To elongate the other new strand, called the lagging strand, DNA polymerase must work in the direction away from the replication fork The lagging strand is synthesized as a series of segments called Okazaki fragments, which are joined together by DNA ligase Animation: Lagging Strand © 2011 Pearson Education, Inc. Figure 16.16b-1 3 5 3 Template strand 5 Figure 16.16b-2 3 5 3 Template strand 5 3 RNA primer for fragment 1 5 1 3 5 Figure 16.16b-3 3 5 3 Template strand 5 3 RNA primer for fragment 1 5 1 3 5 3 Okazaki fragment 1 5 1 3 5 Figure 16.16b-4 3 5 3 Template strand 5 3 RNA primer for fragment 1 5 1 3 5 3 Okazaki fragment 1 5 1 RNA primer 3 for fragment 2 5 5 3 2 Okazaki fragment 2 1 3 5 Figure 16.16b-5 3 5 3 Template strand 5 3 RNA primer for fragment 1 5 1 3 5 3 Okazaki fragment 1 5 1 RNA primer 3 for fragment 2 5 5 3 2 Okazaki fragment 2 1 3 5 5 3 2 1 3 5 5 3 Figure 16.16b-6 3 5 3 Template strand 5 3 RNA primer for fragment 1 5 1 3 5 3 Okazaki fragment 1 5 1 RNA primer 3 for fragment 2 5 5 3 2 Okazaki fragment 2 1 3 5 5 3 2 1 3 5 5 3 2 1 3 5 Overall direction of replication Figure 16.17 Overview Leading Origin of replication Lagging strand strand Leading Lagging strand strand Overall directions Leading strand of replication 5 DNA pol III 3 Primer Primase 3 5 3 Parental DNA pol III Lagging strand DNA 5 4 DNA pol I DNA ligase 35 3 2 1 3 5 The DNA Replication Complex The proteins that participate in DNA replication form a large complex, a “DNA replication machine” The DNA replication machine may be stationary during the replication process Recent studies support a model in which DNA polymerase molecules “reel in” parental DNA and “ extrude” newly made daughter DNA molecules Animation: DNA Replication Review © 2011 Pearson Education, Inc. Figure 16.18 DNA pol III Parental DNA Leading strand 5 5 3 3 3 3 5 5 Connecting Helicase protein 3 5 Lagging DNA strand 3 Lagging strand template pol III 5 Proofreading and Repairing DNA DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides In mismatch repair of DNA, repair enzymes correct errors in base pairing DNA can be damaged by exposure to harmful chemical or physical agents such as cigarette smoke and X-rays; it can also undergo spontaneous changes In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DNA © 2011 Pearson Education, Inc. Figure 16.19 5 3 3 5 Nuclease 5 3 3 5 DNA polymerase 5 3 3 5 DNA ligase 5 3 3 5 Replicating the Ends of DNA Molecules Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes The usual replication machinery provides no way to complete the 5 ends, so repeated rounds of replication produce shorter DNA molecules with uneven ends This is not a problem for prokaryotes, most of which have circular chromosomes © 2011 Pearson Education, Inc. Figure 16.20 5 Ends of parental Leading strand DNA strands Lagging strand 3 Last fragment Next-to-last fragment Lagging strand RNA primer 5 3 Parental strand Removal of primers and replacement with DNA where a 3 end is available 5 3 Second round of replication 5 New leading strand 3 New lagging strand 5 3 Further rounds of replication Shorter and shorter daughter molecules  Eukaryotic chromosomal DNA molecules have special nucleotide sequences at their ends called telomeres Telomeres do not prevent the shortening of DNA molecules, but they do postpone the erosion of genes near the ends of DNA molecules It has been proposed that the shortening of telomeres is connected to aging © 2011 Pearson Education, Inc.  If chromosomes of germ cells became shorter in every cell cycle, essential genes would eventually be missing from the gametes they produce An enzyme called telomerase catalyzes the lengthening of telomeres in germ cells © 2011 Pearson Education, Inc.  The shortening of telomeres might protect cells from cancerous growth by limiting the number of cell divisions There is evidence of telomerase activity in cancer cells, which may allow cancer cells to persist © 2011 Pearson Education, Inc. Concept 16.3 A chromosome consists of a DNA molecule packed together with proteins The bacterial chromosome is a double-stranded, circular DNA molecule associated with a small amount of protein Eukaryotic chromosomes have linear DNA molecules associated with a large amount of protein In a bacterium, the DNA is “supercoiled” and found in a region of the cell called the nucleoid © 2011 Pearson Education, Inc.  Chromatin, a complex of DNA and protein, is found in the nucleus of eukaryotic cells Chromosomes fit into the nucleus through an elaborate, multilevel system of packing Animation: DNA Packing © 2011 Pearson Education, Inc. Figure 16.22a Nucleosome (10 nm in diameter) DNA double helix (2 nm in diameter) H1 Histone Histones tail Nucleosomes, or “beads on DNA, the double helix Histones a string” (10-nm fiber) Figure 16.22b Chromatid (700 nm) 30-nm fiber Loops Scaffold 300-nm fiber 30-nm fiber Replicated chromosome (1,400 nm) Looped domains (300-nm fiber) Metaphase chromosome  Histones can undergo chemical modifications that result in changes in chromatin organization © 2011 Pearson Education, Inc. Figure 16.UN03 DNA pol III synthesizes leading strand continuously 3 5 Parental DNA DNA pol III starts DNA synthesis at 3 end of primer, Origin of 5 continues in 5 3 direction replication 3 5 Lagging strand synthesized in short Okazaki fragments, Helicase later joined by DNA ligase Primase synthesizes 3 a short RNA primer 5 DNA pol I replaces the RNA primer with DNA nucleotides Osama Hussein 44  DNA can be changed in a number of ways, including - through spontaneous changes, - errors in the replication process, or - the action of radiation or particular chemicals. Another broad type of change in the genetic material is the point mutation, a change of one or a few base pairs. A point mutation may change the phenotype of the organism if it occurs within the coding region of a gene or in the sequences regulating the gene. Osama Hussein 45 Mutations Defined Mutation is the process by which the sequence of base pairs in a DNA molecule is altered. A mutation may result in a change to either a DNA base pair or a chromosome. A cell with a mutation is a mutant cell. If a mutation happens to occur in a somatic cell, it is a somatic mutation—the mutant characteristic affects only the individual in which the mutation occurs and is not passed on to the succeeding generation. A mutation in the germ line of sexually reproducing organisms - a germ-line mutation - Osama may be transmitted by the gametes to Hussein 46 the next generation, producing an individual with the mutation  Two terms are used to give a quantitative measure of the occurrence of mutations. The mutation rate is the probability of a particular kind of mutation as a function of time, such as the number of mutations per nucleotide pair per generation, or the number per gene per generation. The mutation frequency is the number of occurrences of a particular kind of mutation, expressed as the proportion of cells or individuals in a population, such as the number of mutations per 100,000 organisms or the number per 1 million gametes. Osama Hussein 47  Types of Point Mutations (1) Base-pair substitutions A transition mutation is a mutation from one purine–pyrimidine base pair to the other purine–pyrimidine base pair, such as A–T to G–C. A transversion mutation is a mutation from a purine–pyrimidine base pair to a pyrimidine–purine base pair, such as G–C to C–G, or A–T to C–G. Osama Hussein 48  Base-pair substitutions in protein-coding genes also are defined according to their effects on amino acid sequences in proteins. Depending on how a base-pair substitution is translated via the genetic code, the mutations can result in - no change to the protein, - an insignificant change, or - a noticeable change. Osama Hussein 49 Osama Hussein 50 Spontaneous and Induced Mutations Mutagenesis, the creation of mutations, can occur spontaneously or can be induced. Spontaneous mutations are naturally occurring mutations. Induced mutations occur when an organism is exposed either deliberately or accidentally to a physical or chemical agent, known as a mutagen, that interacts with DNA to cause a mutation. Induced mutations typically occur at a much higher frequency than do spontaneous mutations and hence have been useful in genetic studies. Osama Hussein 51  Repair of DNA Damage Mutation = DNA damage – DNA repair Both prokaryotic and eukaryotic cells have a number of enzyme- based systems that repair DNA damage. If the repair systems cannot correct all the lesions, the result is a mutant cell (or organism) or, if too many mutations remain, death of the cell (or organism). There are two general categories of repair systems, based on the way they function. - Direct reversal repair systems correct damaged areas by reversing the damage. - Excision repair systems cut out a damaged area and then repair the gap by new DNA synthesis. Mismatch Repair by DNA Polymerase Proofreading Occur by “backspacing” of the pol to remove the wrong nucleotide and then resuming synthesis in the forward direction. The mutator mutations in E. coli illustrate the importance of the 3’-to-5’ exonuclease activity of DNA pol for maintaining a low mutation rate. Repair of UV-Induced Pyrimidine Dimers Through photoreactivation, or light repair, UV light-induced thymine (or other pyrimidine) dimers are reverted directly to the original form by exposure to near-UV light in the wavelength range from 320 to 370 nm. Photoreactivation occurs when an enzyme called photolyase is activated by a photon of light and splits the dimers apart. Strains with mutations in the phr gene are defective in light repair. Photolyase has been found in bacteria and in simple eukaryotes, but not in humans.  Many mutations affect only one of the two strands. In such cases, the DNA damage can be excised and the normal strand used as a template for producing a corrected strand. Depending on the damage, excision may involve a single base or nucleotide, or two or more nucleotides. Each excision repair system involves a mechanism to recognize the specific DNA damage it repairs. Base Excision Repair Damaged single bases or nucleotides are most commonly repaired by removing the base or the nucleotide involved and then inserting the correct base or nucleotide. In base excision repair, a repair glycosylase enzyme removes the damaged base from the DNA by cleaving the bond between the base and the deoxyribose sugar. Other enzymes then cleave the sugar–phosphate backbone before and after the now baseless sugar, releasing the sugar and leaving a gap in the DNA chain.  The gap is filled with the correct nucleotide by a repair DNA polymerase and DNA ligase, with the opposite DNA strand used as the template. Mutations caused by depurination or deamination are examples of damage that may be repaired by base excision repair. Methyl-Directed Mismatch Repair Despite proofreading by DNA polymerase, a number of mismatched base pairs remain uncorrected after replication has been completed. Many mismatched base pairs left after DNA replication can be corrected by methyl-directed mismatch repair. This system recognizes mismatched base pairs, excises the incorrect bases, and then carries out repair synthesis. In E. coli, the products of three genes—mutS, mutL, and mutH —are involved in the initial stages of mismatch repair.  Mismatch repair also takes place in eukaryotes. However, it is unclear how the new DNA strand is distinguished from the parental DNA strand (no methylation is involved). In humans, four genes, respectively named hMSH2, hMLH1, hPMS1, and hPMS2, have been identified; - hMSH2 is homologous to E. coli mutS, and - the other three genes have homologies to E. coli mutL. Mutations in any one of the four human mismatch repair genes confer a phenotype of hereditary predisposition to a form of colon cancer called hereditary nonpolyposis colon cancer. Double-Strand Breaks Can Be Repaired by Homologous Recombination Repair and by Nonhomologous End Joining DSBs can be caused by - ionizing radiation (X-rays or gamma rays), - chemical mutagens, and - certain drugs used for chemotherapy. - In addition, reactive oxygen species that are the by-products of aerobic metabolism can cause double-strand breaks. Surprisingly, researchers estimate that naturally occurring double-strand breaks in a typical human cell occur at a rate of between 10 and 100 breaks per cell per day. DNA repair of DSBs via homologous recombination repair.  DNA repair of double-strand breaks via nonhomologous end joining