Lecture 12: DNA Replication and Restructuring (2023 PDF)

1 Lecture 12 DNA Replication and Restructuring: Repair, Recombination, Rearrangement, Amplification 2 EARLY INSIGHTS INTO DNA REPLICATION Three central features of DNA replication were predicted in 1953 from Watson and Crick’s model: 1.DNA replication is...

1 Lecture 12 DNA Replication and Restructuring: Repair, Recombination, Rearrangement, Amplification 2 EARLY INSIGHTS INTO DNA REPLICATION Three central features of DNA replication were predicted in 1953 from Watson and Crick’s model: 1.DNA replication is semiconservative—each of the two identical daughter DNA molecules contains one parental strand and one newly synthesized strand. This prediction was confirmed in 1957 by the elegant experiment of Meselson and Stahl. 2.Parental strand unwinding and synthesis of new DNA occur simultaneously, in the same microenvironment. In other words, replication occurs at a fork, in which parental strands are unwinding and both daughter strands are undergoing elongation, as suggested by the diagram. 3.Replication begins at one or more fixed sites—replication origins—on a chromosome. 3 EARLY INSIGHTS INTO DNA REPLICATION Biosynthesis of nucleic acids and proteins is carried out through the processes of replication, transcription, and translation. Most regulation occurs at the level of initiation.  DNA POLYMERASES: ENZYMES CATALYZING 4 POLYNUCLEOTIDE CHAIN ELONGATION The DNA polymerase reaction: Each incoming dNTP is positioned by base pairing with the appropriate template nucleotide, and a phosphodiester bond is created by nucleophilic attack of the primer strand 3’ hydroxyl group on the phosphate of the dNTP.  DNA POLYMERASES: ENZYMES CATALYZING 5 POLYNUCLEOTIDE CHAIN ELONGATION The DNA polymerase I molecule contains three active sites: o A polymerase o Two exonucleases  DNA POLYMERASES: ENZYMES CATALYZING 6 POLYNUCLEOTIDE CHAIN ELONGATION The Klenow fragment of E. coli DNA polymerase I: a-Carbon backbone representation of the Klenow fragment complexed with DNA, in an editing configuration. That is, the 3’ end of the growing strand (light blue) is bound at the 3’ exonuclease active site of the enzyme (yellow). The locations of the polymerase and 3’ exonuclease active sites have been identified by site-directed mutagenesis. 7 MULTIPLE DNA POLYMERASES  MULTIPLE DNA POLYMERASES 8 Comparison of DNA polymerase structures with primer and template bound: The four structures shown are oriented with respect to each other by superposition of the first two base pairs at the primer terminus. Palm, thumb, and fingers are shown for each structure. Taq polymerase is a thermostable enzyme used in PCR. HIV-1RT is the reverse transcriptase (RNA- directed DNA polymerase) from human immunodeficiency virus. RB69 gp43 is the replicative DNA polymerase encoded by RB69, a bacteriophage closely related to T4. pol b is a eukaryotic DNA polymerase involved in DNA repair.  OTHER PROTEINS AT THE REPLICATION FORK 9 Schematic view of a replication fork: Note that polymerases catalyzing leading- and lagging-strand replication are linked together.  OTHER PROTEINS AT THE REPLICATION 10 FORK Type I topoisomerases break and reseal one DNA strand Type II topoisomerases catalyze double-strand breakage and rejoining. Hence, type I and type II enzymes change DNA linking number in units of 1 and 2, respectively.  OTHER PROTEINS AT THE REPLICATION 11 FORK Action of type I and type II topoisomerases, as shown by gel electrophoresis: Lane 1 shows a relaxed circular DNA. Lane 2 shows the pattern from treatment of supercoiled DNA with type I topoisomerase. Lanes 3–5 show relaxed circles treated with DNA gyrase, a type II topoisomerase, for different lengths of time.  OTHER PROTEINS AT THE REPLICATION 12 FORK Action of a type I topoisomerase: The enzyme breaks one strand and immobilizes the 5’ end by a covalent bond between the DNA phosphate and a tyrosine residue (in E. coli topoisomerase I). Rotation of the 3’ end is followed by resealing. The linking number is increased by 1 in the example shown (an underwound DNA). Action of a type I topoisomerase on overwound DNA would decrease the linking number, by essentially the same mechanism.  OTHER PROTEINS AT THE REPLICATION 13 FORK Action of a type II topoisomerase: DNA gyrase of E. coli; the example shown is a tetrameric protein with two A and two B subunits. The enzyme is shown introducing two negative turns and changing the linking number from +1 to -1. The enzyme catalyzes a double-strand break, and the two DNA ends are bound by A subunits, which move the DNA ends apart so that the unbroken duplex can pass through the gap. Resealing converts the positive supertwist to a negative one, giving the overall molecule a DL of -2. Type II topoisomerases can relax underwound duplexes by the reverse of the above pathway.  OTHER PROTEINS AT THE REPLICATION 14 FORK The types of topological interconversions catalyzed by type II topoisomerases: a) Relaxation. b) Catenation and decatenation. c) Knotting and unknotting.  OTHER PROTEINS AT THE REPLICATION FORK 15 Helicases are multimeric proteins that bind preferentially to one strand of a DNA duplex and use energy of ATP hydrolysis to actively unwind the duplex. A model for helicase action: In this model a homodimeric enzyme, such as the E. coli Rep helicase, shows 3’à5’ polarity.  OTHER PROTEINS AT THE REPLICATION FORK 16 Structure and action of the T7 phage gene 4 helicase: (a,b) Comparison of T7 gp4 action with that of the ATP synthase rotary engine. Shaded subunits in both enzymes are noncatalytic sites.  OTHER PROTEINS AT THE REPLICATION 17 FORK A model of T7 gp4 helicase action: The protein is rotating along the blue DNA strand, excluding the red strand from the central channel.  OTHER PROTEINS AT THE REPLICATION 18 FORK Primase, a special class of RNA polymerase, synthesizes short RNA molecules as primers for lagging strand DNA replication.  OTHER PROTEINS AT THE REPLICATION 19 FORK The transfer experiment that demonstrated the existence of RNA primers in DNA replication: Each Okazaki fragment generated one radiolabeled ribonucleotide from its RNA-DNA junction after alkaline hydrolysis.  OTHER PROTEINS AT THE REPLICATION 20 FORK Subunit structure of the E. coli DNA polymerase III holoenzyme: DNA polymerase III holoenzyme, a complex bacterial enzyme containing at least 10 subunits, plays the predominant role in replicative chain elongation.  OTHER PROTEINS AT THE REPLICATION FORK 21 Structure of the sliding clamp: Each protein forms a “doughnut” that can completely surround double- stranded DNA and thus keep polymerase associated with its DNA templates. The a-helices on the inner surface of the subunit contact DNA but do not bind tightly enough to retard movement of the protein. The E. coli protein has two identical subunits with two DNA-associating domains, while the human and RB69 proteins have three subunits, each with two DNA-associating domains.  OTHER PROTEINS AT THE REPLICATION FORK 22 Scheme for action of the E. coli clamp loader (a) and structure of the g complex bound to DNA (b): The complex contains five protein subunits: three copies of g (B, C, and D in the figure) and one each of d and d’ (A and E, respectively).  OTHER PROTEINS AT THE REPLICATION FORK 23 The primase-polymerase switch during lagging strand synthesis: DnaB helicase encircles the lagging strand, and primase has synthesized an RNA primer. Primase must contact SSB to remain bound. Core polymerase on the lagging strand is forcing that strand and the daughter strand to loop out. The complex interacts with SSB, leading to primase displacement. The complex is opening the clamp, and a newly completed Okazaki fragment is being released, along with its template. Primase rebinds to single-strand template DNA to begin a new primer.  OTHER PROTEINS AT THE REPLICATION FORK 24 Discontinuous DNA Synthesis: Reiji Okazaki proposed that DNA replication could be discontinuous. In principle one parental strand (the leading strand) could be extended continuously, with polymerase moving from the 5’ terminus to the 3’ terminus in the same direction as fork movement. Synthesis on the lagging strand would be discontinuous; chain extension along the leading strand would expose single-strand template on the lagging strand. o This template could be copied in short fragments (later named Okazaki fragments), with polymerase moving opposite to the direction of fork movement. Thus, lagging strand synthesis would occur in short pieces. These could then be joined to high-molecular-weight DNA by the enzyme DNA ligase.  PROTEINS IN EUKARYOTIC DNA 25 REPLICATION  REPLICATION OF CHROMATIN 26 Structures of human DNA polymerase g (left) and T7 phage DNA polymerase (right) holoenzymes: Both enzymes are shown complexed with DNA. The pol g structure shows two unique domains (IP and AID, shown in gold), which are involved in DNA binding. Note the similarity in location of the polymerase and 3’ exonuclease catalytic domains.  OTHER PROTEINS AT THE REPLICATION 27 FORK The reaction catalyzed by DNA ligase:  OTHER PROTEINS AT THE REPLICATION 28 FORK Nick translation in removal of RNA primers by coordinated action of the 5’ exonuclease and polymerase activities of DNA polymerase I: The figure shows replacement of base paired UMP in the RNA primer by dTMP in the growing DNA chain. The template DNA is the lagging strand. 29 REPLICATION OF CHROMATIN  PROTEINS IN EUKARYOTIC DNA 30 REPLICATION Polarized replication termination in E. coli: Replication initiates bidirectionally from oriC. The orientation of Ter sites insures that both replisomes arrive at the same site (between TerA and TerC) before termination can begin.  INITIATION OF DNA REPLICATION 31 Model for chromatin replication: Nucleosomes on parental DNA are dissociated as the replication fork approaches and are reformed on newly synthesized daughter strands, with both old and newly synthesized histones being used. Maturation occurs slowly, with full organization not being re- established until many kilobases behind the moving fork. 32 REPLICATION OF LINEAR GENOMES The problem of completing the 5 end in copying a linear DNA molecule: Telomerase adds repeated short DNA segments to the ends of chromosomes. 33 FIDELITY OF DNA REPLICATION  FIDELITY OF DNA REPLICATION 34 Extension of telomeric DNA by telomerase: a)The overall reaction: telomerase adds simple repeat sequences to the 3’ end of telomeric DNA, by the mechanism shown in part (b). Addition of an RNA primer allows lagging-strand synthesis, followed by ligation and RNA removal. b)Proposed action of telomerase: the RNA carried by the telomerase matches the 3’ DNA end, and allows its extension. 35 FIDELITY OF DNA REPLICATION Crystal structure of telomerase catalytic subunit from the red flour beetle: DNA and telomerase RNA are modeled into the large central cleft. 36 DNA REPLICATION - A SUMMARY 37 DNA REPAIR A summary of the major processes in information restructuring: 38 DNA REPAIR DNA Replication: Does this process work without errors? 39 DNA REPAIR DNA Replication: Does this process work without errors? No What is the risk of error? 40 DNA REPAIR DNA Replication: Does this process work without errors? No What is the risk of error? 10-6 → 10-8 Are these errors corrected? 41 DNA REPAIR DNA Replication: Does this process work without errors? No What is the risk of error? 10-6 → 10-8 Are these errors corrected? DNA repair systems Do they do it effectively? 42 DNA REPAIR DNA Replication: Does this process work without errors? No What is the risk of error? 10-6 → 10-8 Are these errors corrected? DNA repair systems Do they do it effectively? In 99%. Actually 10-6 → 10-8 changes to 10-8 → 10-10 43 DNA REPAIR DNA Replication: Does this process work without errors? No What is the risk of error? 10-6 → 10-8 Are these errors corrected? DNA repair systems Do they do it effectively? In 99%. Actually 10-6 → 10-8 changes to 10-8 → 10-10 Is that even important? How often is our DNA damaged? 44 DNA REPAIR DNA Replication: Does this process work without errors? No What is the risk of error? 10-6 → 10-8 Are these errors corrected? DNA repair systems Do they do it effectively? In 99%. Actually 10-6 → 10-8 changes to 10-8 → 10-10 Is that even important? How often is our DNA damaged? It is estimated that the daily number of DNA damage in a human cell ranges from 100 to 500 spontaneous deaminations and from 20 000 to 40 000 single-strand breaks 45 DNA REPAIR Endogenous DNA-damaging reactions: The approximate frequency of each reaction, in number of lesions per mammalian cell per day, is indicated.  DNA REPAIR 46 Environmental DNA-damaging agents include: Chemical factors Physical factors Biological factors  DNA REPAIR 47 Environmental DNA-damaging agents include: o Ionizing radiation o Ultraviolet radiation DNA-methylating reagents: o N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG). DNA-cross-linking reagents: o Cisplatin, an anticancer drug. Bulky electrophilic agents: o Benzo[a]pyrene, one of the carcinogenic hydrocarbons in tobacco smoke. 48 DNA REPAIR Structures of pyrimidine dimer photoproducts: When one examines either UV- irradiated DNA or the DNA extracted from a UV-irradiated organism, one detects small amounts of many different altered DNA constituents, called photoproducts. Prominent among them are intrastrand dimers consisting of two pyrimidine bases joined by a cyclobutane ring structure involving carbons 5 and 6 called thymine dimers (a). 49 DNA REPAIR DNA can be repaired directly, by changing a damaged base to a normal one, or indirectly, by replacing a DNA segment containing the damaged nucleotide. 50 DNA REPAIR 1. Direct Photolysis - use of light energy to repair pyrimidine dimers Alkyltransferase - "Enzymes" inactivated after one catalytic cycle 2. Indirect Single-strand damages (BER, NER, MMR) Double-strand cords (NHEJ) 51 DNA REPAIR  DNA REPAIR 52 Mispairing of O6-methylguanine with thymine in a DNA duplex: The most highly mutagenic of these products, O6-alkylguanine, is mutagenic because the modified base has a very high probability of pairing with thymine when the modified strand replicates. 53 DNA REPAIR Thus, alkylation of a DNA-guanine stimulates a GC à AT transition. Repair of this type of damage involves an unusual enzyme, O6-alkylguanine alkyltransferase, which transfers a methyl or ethyl group from an O6– methylguanine or O6-ethylguanine residue to a cysteine residue in the active site of the protein. Having become alkylated, it cannot remove the alkyl group, and the protein molecule turns over. 54 DNA REPAIR The thymine dimer photolyase: Structure of the E. coli enzyme, showing distinct N-terminal (red) and C- terminal (green) domains, with a linker in orange and showing bound folate and flavin cofactors. 55 DNA REPAIR Direct repair enzymes include: o Photolyase - uses light energy to repair pyrimidine dimers o Alkyltransferases - “enzymes” that are inactivated after just one catalytic cycle.  DNA REPAIR 56 Excision repair of thymine dimers by the UvrABC excinuclease of E. coli: A complex of A and B proteins tracks along DNA until it reaches a thymine dimer or other damaged site, where it halts and forces the DNA to bend. UvrA (a “molecular matchmaker”) then dissociates, allowing UvrC to bind to B. The BC complex cuts on both sides of the dimer. Helicase, polymerase, and ligase remove the damaged undecamer and replace it with new DNA. This system may use DNA polymerase II as well as pol I.  DNA REPAIR 57 Action of the DNA uracil repair system: Uracil-DNA N-glycosylase (also called Ung) removes uracil, leaving an apyrimidinic site. A specific endonuclease recognizes this 5’ site and cleaves on the side. DNA polymerase I replaces the missing nucleotide, leaving deoxyribose-5- phosphate on the 5’ side of the nick. This is removed hydrolytically, and DNA ligase seals the nick. 58 DNA REPAIR A base excision repair process removes uracil residues in DNA, whether they arose through deamination of cytosine residues or incorporation of deoxyuridine nucleotides instead of thymidine nucleotides. 59 DNA REPAIR 60 DNA REPAIR One of the most abundant oxidation products resulting from DNA exposure to reactive oxygen species is 8- oxoguanine. This is a strongly mutagenic alteration because 8- oxoguanine pairs readily with adenine: 61 DNA REPAIR MutT has nothing to do with base excision repair. Instead, it is a nucleotidase, cleaving 8-oxo-dGTP, the dNTP of 8- oxoguanine, to the corresponding nucleoside monophosphate. This reaction is thought to “sanitize” cellular dNTP pools, by eliminating a nucleotide whose incorporation into DNA would be strongly mutagenic. 62 DNA REPAIR Actions of mutM, mutT, and mutY gene products in countering the mutagenic effect of 8-oxoguanine (oG): Depending upon the route of introduction of oG to DNA, the pathways shown can cause either GC à AT or AT à GC transversions. MutT hydrolyzes 8-oxo-dGTP and prevents its incorporation during DNA replication. MutM excises oG from a C-G base pair to begin BER. MutY excises A from an A-oG base pair in another BER process, allowing an additional possibility for correcting an error in the next round of replication.  DNA REPAIR 63 Structure of human OGG1 with either guanine or 8- oxoguanine bound flipped out and bound near the catalytic pocket: A combination of forces completely excludes guanine from the catalytic pocket. 64 DNA REPAIR Methyl-directed mismatch repair in E. coli: The newly replicated daughter strand (red) contains a T mismatched to G in the template strand (blue). The mismatch repair system identifies the daughter strand because it is not yet methylated. Thus, this system must function before the newly replicated daughter strand becomes methylated, through action of the Dam methylase on the A residue in the GATC sequence.  DNA REPAIR 65 Mutations in mismatch repair proteins were found in tumor cells from individuals with an inherited cancer predisposition called HNPCC (heritable nonpolyposis colon cancer). Germ-line mutations in the genes for five different mismatch repair proteins have been found to be associated with HNPCC. Tumor cells from those affected with HNPCC exhibit phenomenon called microsatellite instability—a large number of mutations in regions of the genome containing repeats of single-, double-, and triple-nucleotide sequences, usually with large increases in the numbers of repeating units in such sequences. These data suggest that the product and template strands can normally slip at such sites so that DNA polymerase copies a short repeating sequence more than once, or else skips a segment. This creates a heteroduplex with a short loop.  DNA REPAIR 66 DNA mismatch repair is a system for recognizing and repairing: erroneous insertion deletion mis-incorporation of bases that can arise during DNA replication and recombination  DNA REPAIR 67 Nucleotide excision repair (NER) 68 DNA REPAIR Replication fork regression as a likely response to a blocking DNA lesion in the leading strand template: (a), (b) Regression pairs the damaged site (shown as a triangle) with its undamaged template (blue). (c), (e) The daughter–strand complement of the damaged strand (black) can then be copied from the other daughter strand (green). The fork then reforms and replication can re-start, whether the damage has been repaired (f) or not (d). 69 DNA REPAIR Double-strand DNA breaks can be repaired either by homologous recombination or by nonhomologous end joining (NHEJ), a process that does not require DNA sequence homology at the ends being joined. A pathway for nonhomologous end joining.