DNA Replication - Bio 131 Lecture Notes PDF

Campbell Biology in Focus Third Edition Chapter 13 The Molecular Basis of Inheritance Part II...

Campbell Biology in Focus Third Edition Chapter 13 The Molecular Basis of Inheritance Part II Lecture Presentations by Kathleen Fitzpatrick and Nicole Tunbridge, Simon Fraser University Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved  DNA Replication: A Closer Look The copying of DNA is remarkable in its speed and accuracy – 3,000 nt/minute for human genome – 30,000 nt/ minute for bacterial genome More than a dozen enzymes and other proteins participate in DNA replication Much more is known about how this “replication machine” works in bacteria than in eukaryotes Most of the process is similar between prokaryotes and eukaryotes Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Getting Started (1 of 2) Replication begins at sites called origins of replication, where the two DNA strands are separated, opening up a replication “bubble” At each end of a bubble is a replication fork, a Y-shaped region where the parental strands of DNA are being unwound For the long DNA molecules in eukaryotes, multiple replication bubbles form and eventually fuse, speeding up the copying of D NA Replication is bidirectional Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Animation: Origins of Replication Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Figure 13.14 Origins of Replication in E. Coli and Eukaryotes Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Animation: DNA Replication Overview Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Getting Started (2 of 2) Several kinds of proteins participate in the unwinding – Helicases are enzymes that untwist the double helix at the replication forks – Single-strand binding proteins (SSB) bind to and stabilize single-stranded DNA – Topoisomerase relieves the strain caused by tight twisting ahead of the replication fork by breaking, swiveling, and rejoining DNA strands Topoisomerase SSB Helicase Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved DNA Topoisomerase I Cuts ONE side of the ds-DNA Opposite side spins around the phosphodiester bond Reseals nick No ATP required! – Stores energy of broken bond – Uses that to reseal 8 Figure 13.15 Some of the Proteins Involved in the Initiation of DNA Replication Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Primase makes a RNA primer DNA polymerase requires a primer – DNA polymerases cannot begin synthesis on a “bare” template strand – Primase puts down a few nucleotides that are bonded to the template strand – This provides a free 3 hydroxyl (OH) group that can combine with an incoming dNTP to form a phosphodiester bond The Primase – Is an RNA polymerase – Synthesizes a short RNA segment that serves as a primer (de novo) – DNA polymerase then adds bases to 3 end of the primer Synthesizing a New DNA Strand (1 of 3) Enzymes (DNA polymerases) that synthesize DNA cannot initiate synthesis of a polynucleotide; they can only add nucleotides to an already existing chain base-paired with the template The initial nucleotide strand is a short RNA primer The enzyme, PRIMASE, starts an RNA chain with a single RNA nucleotide and adds RNA nucleotides one at a time using the parental DNA as a template The primer is short (5–10 nucleotides long) RNA molecule Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Synthesizing a New DNA Strand (2 of 3) Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork They add nucleotides to the 3′ end of a preexisting chain using the free 3’OH 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 Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Synthesizing a New DNA Strand (3 of 3) Each Nucleotide that is added to a growing DNA strand consists of a sugar attached to a base and to three phosphate groups dATP is used to make DNA and is similar to the ATP of energy metabolism RNA DNA The difference is in the sugars: dATP has deoxyribose, whereas ATP has ribose As each monomer nucleotide joins the D NA strand, it loses two phosphate groups as a molecule of Pyrophosphate Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Figure 13.16 Addition of a Nucleotide to a DNA Strand Synthesized in the 5’ to 3’ direction Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Antiparallel Elongation (1,2 of 4) Newly replicated DNA strands must be formed ANTIPARALLEL to the template strand DNA polymerases add nucleotides only to the free 3′end of a growing strand, the strand can elongate only in the 5′to3′direction Along one template strand of DNA, the DNA polymerase synthesizes a leading strand continuously, moving toward the replication fork Only one primer is required to synthesize the leading strand Continuous synthesis Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Figure 13.17 Synthesis of the Leading Strand During DNA Replication Main “Replicase” Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Animation: Leading Strand Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Antiparallel Elongation (3 of 4) Can only synthesis in the 5’  3’ direction!! To elongate the other new strand, the lagging strand, D NA polymerase must work in the direction away from the replication fork  because of need for 3’ OH The lagging strand is synthesized One strand has free 5’-PO4 as a series of segments called One strand has Okazaki fragments free 3’-OH These are 100–200 nucleotides long in eukaryotes and 1,000–2,000 nucleotides long in E. coli POLARITY PARADOX!! Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved  Both strands synthesized in 5’-3’ direction – One strand CONTINUOUSLY synthesized Leading Strand Only one primer needed – Other strand DISCONTINUOUSLY synthesized Lagging Strand - Strand “lags” behind Short stretches synthesized in 5’-3’ direction Requires multiple primers! 19  Figure 13.18 Synthesis of the Lagging Strand Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved  Priming New DNA Synthesis DNA Pol III extends new DNA strand in prokaryotes On Lagging strand it stops when hits next Okazaki fragment Leading strand just goes Is there a problem here?? 21  RNA/DNA Heteroduplex RNA in the lagging strand DNA POL I comes in!! 5’ 3’ exonuclease activity to remove RNA primers 5’ 3’ polymerase activity to fill in the gap with DNA 22 DNA Polymerases DNA polymerase III – multiple subunits, responsible for majority of replication DNA polymerase I – a single subunit, rapidly removes RNA primers and fills in DNA, proofreading/repair function Both enzymes also have proofreading function Goes “backward” to proofread! Antiparallel Elongation (4 of 4) After formation of Okazaki fragments, DNA polymerase I removes the RNA primers and replaces the nucleotides with DNA The remaining gaps are joined together by DNA Ligase – Requires NAD or ATP – CANNOT fill gaps – CANNOT add nucleotides – ONLY fixes single nicks Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Figure 13.19 A Summary of Bacterial DNA Replication One RNA primer Topoisomerase Many RNA primers Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved 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 two DNA polymerase molecules “reel in” parental DNA and “extrude” newly made daughter DNA molecules The TROMBONE Model Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved  Figure 13.20 The “Trombone” Model of the DNA Replication Complex 2 catalytic cores of DNA pol III Primase Lagging strand DNA Pol I “loops” through Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Animation: DNA Replication Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Proofreading and Repairing DNA (1 of 2) Errors in the completed DNA molecule amount to only one in 10 billion DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides In Mismatch Repair of DNA, other enzymes correct errors in base pairing A hereditary defect in one such enzyme is associated with a form of colon cancer This defect allows cancer-causing errors to accumulate in DNA faster than normal Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Proofreading and Repairing DNA (2 of 2) DNA can be damaged by exposure to harmful chemical or physical agents, such as X-rays DNA bases can also undergo spontaneous changes In many cases a Nuclease cuts out and replaces damaged stretches of DNA One such DNA repair system is called Nucleotide Excision Repair DNA repair enzymes in our skin repair genetic damage caused by the ultraviolet light of sunlight Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Figure 13.21 Nucleotide Excision Repair of DNA Damage Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved  Evolutionary Significance of Altered DNA Nucleotides The error rate after proofreading repair is extremely low but not zero Sequence changes may become permanent and can be passed on to the next generation These changes (mutations) are the source of the genetic variation upon which natural selection operates Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved  Replicating the Ends of DNA Molecules (1 of 4) For linear DNA, the usual replication machinery cannot complete the 5′ ends of daughter strands Repeated rounds of replication produce shorter DNA molecules with uneven ends Figure 13.22 Telomeres Eukaryotic chromosomal DNA molecules have special nucleotide sequences at their ends called TELOMERES Problem with the lagging strand! Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Replicating the Ends of DNA Molecules (2 of 4) TELOMERES do not contain genes They typically consist of multiple repetitions of one short nucleotide sequence – Highly conserved short sequence 5’-T1-4A0-1G1-8-3’ in vertebrates – Human repeat sequence TTAGGG that is repeated 500-3300 times Telomeres do not prevent the shortening of D N A molecules, but they do postpone it It has been proposed that the shortening of telomeres is connected to aging Telomeres in Disease – Cancerous cells  no shortening with age – Increase in telomerase (immortal cells) Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Figure 15.12 DNA polymerase End of chromosome Leading strand Sliding clamp 1. DNA unwinding Lagging strand completed. Helicase 2. Leading strand completed. RNA primer Primase 3. Lagging strand completed. DNA polymerase Last Okazaki fragment 4. Lagging strand too short. Unreplicated end Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Replicating the Ends of DNA Molecules (3 of 4) 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 Telomerase brings in its own RNA template Uses “TTAGGG” as template and extends unreplicated end Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Replicating the Ends of DNA Molecules (4 of 4) Telomerase is not active in most human somatic cells However, it does show inappropriate activity in some cancer cells  increased telomerase Telomerase is currently under study as a target for cancer therapies Telomerase Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved Concept 13.3: A Chromosome Consists of a DNA Molecule Packed Together with Proteins (1 of 5) Copyright © 2020, 2016, 2014 Pearson Education, Inc. All Rights Reserved