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REPLICATION

1. REPLICATION. 2. INTRODUCTION TO MACROMOLECULAR SYNTHESIS Macromolecular synthesis involves Replication [the polymerization of deoxyribonucleoside triphosphates into DNA (as the

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REPLICATION

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  1. 1 REPLICATION

  2. 2 INTRODUCTION TO MACROMOLECULAR SYNTHESIS Macromolecular synthesis involves Replication [the polymerization of deoxyribonucleoside triphosphates into DNA (as the monophosphate)], Transcription [the polymerization of ribonucleoside triphosphates into RNA (as the monophosphates)], and Translation [the polymerization of amino acids into proteins]. A.Genetic information. Genetic information stored in chromosomes (as nucleotide sequences in genes and sites) directs its own replication by: 1. Serving as template for DNA synthesis, and 2. Serving as template for messenger, transfer, and ribosomal RNA synthesis. The messenger RNA will be translated into proteins, the functions of which will be to catalyze all the reactions needed for growth, including DNA synthesis. In replication, two complementary strands of DNA are unwound (separated) and the nucleotide sequence of each serves to direct the synthesis of complementary strands. The overall result is the production of two daughter chromosomes, each identical to the other and each identical to the parental chromosome. In transcription, the nucleotide sequence of one strand of DNA is used to direct the polymerization of one complementary strand of RNA. Messenger RNA sequences, as codons, are translated into proteins, the functions of which are determined by their amino acid sequences. These proteins then function to catalyze all biochemical reactions of growth, including, for example, glycolysis and energy production, synthesis of low molecular weight precursors of macromolecules, and polymerization, of macromolecules, including the chromosome itself. Thus, in cellular organisms, genetic information flows from DNA into RNA into Protein and the proteins then catalyze all the reactions necessary to duplicate the DNA which is to be passed on to the next generation. B. Biochemistry. 1. Chromosomes are replicated once per cell cycle by activation of the replication origin, formation of two replication forks which move in opposite directions around the chromosome, and termination of replication. DNA polymerase III catalyzes the incorporation of deoxyribonucleoside triphosphates [dATP, dGTP, dCTP, and dTTP] onto growing 3’ ends of leading and lagging strands in nucleotidyl transfer reactions. DNA chains are extended in the 5’ to 3’ direction. The anhydride bonds of the substrates yield the energy required to form the phosphodiesters which link mononucleotides into DNA. 2. RNA polymerase recognizes promoters and synthesizes single strands of RNA, using the nucleotide sequence of one strand of DNA as the template from which to synthesize a complementary RNA sequence. RNA polymerase catalyzes the polymerization of ribonucleoside triphosphates, ATP, GTP, CTP, and UTP in the 5’ to 3’ direction. RNA polymerase has the ability to initiate new chains of RNA. That is, transcription does not require a primer, unlike replication. 3. Translation requires activated amino acids, messenger RNA, ribosomes, and energy in the form of GTP for polymerization of the amino acids. This polymerization is in the direction from the N terminus to the C terminus. The sequenced of amino acids is dictated by the sequence of codons in the messenger, messengers being read 5’ to 3’. Transfer RNAs serve two functions (I) to carry activated amino acids and (II) to serve as adapters linking the amino acid to the codon (by codon-anticodon interaction). Peptidyl transfer reactions link the carboxyl of an amino acid or growing polypeptide to the amino group of the next amino acid to be polymerized forming an amide bond. Amide bonds between amino acids are called peptide bonds, In prokaryotes, transcription and translation can be coupled; that is, messengers can begin to be translated while they are in the process of being transcribed. In other words -messengers which are in the process of being synthesized can be simultaneously translated.

  3. 3 CHROMOSOME REPLICATION I. DNA PRECURSORS AND DNA STRUCTURE Mononucleotides (Purines, Pyrimidines, Deoxyribose, Phosphates) Complementary Nature of Double Stranded DNA Antiparallel Structure; 5' > 3' and 3' > 5' Hydrogen Bonds DNA POLYMERIZATION Nucleotidyl Transfer Reactions as General Reactions 5' > 3' Chain Elongation Primer, Template CHROMOSOME REPLICATION Initiation; Origin Replication Fork Supercoils; Topoisomerase Leading, Lagging Strands Helicase Primase DNA Polymerase III DNA Polymerase I DNA Ligase DNA POLYMERASE III SUBUNITS Trimeric Enzyme; Looping Model for Coordination of Leading/Lagging Strands Processivity Clamp Processivity Clamp Loading; Unloading Proofreading Subunit REPLICATING CHROMOSOME - ORGANIZATION AND DYNAMICS VI. AZT MECHANISM

  4. 4 BINARY FISSION- REPLICATION 3-5 x 106 BP 2-3 x109 MW 1 x 3 µm CELL oriC ter 1100 µm CHROMOSOME DOUBLE-STRANDED REPLICATION FORKS ORIGIN ACTIVATION (MELTED) ONCE/CYCLE COMPLEMENTARY DAUGHTER STRANDS TERMINUS DAUGHTER CHROMOSOMES SEMI-CONSERVATIVE

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  6. 6 NUCLEOTIDES ESTER NUCLEIC ACID BASE N-GLYCOSIDIC BOND DEOXYRIBOSE DEOXYRIBOSE DEOXYRIBONUCLEOSIDE DEOXYRIBONUCLEOSIDE MONOPHOSPHATE OR DEOXYRIBONUCLEOTIDE

  7. 7 NUCLEIC ACID BASE ANHYDRIDE BONDS DEOXYRIBONUCLEOSIDE TRIPHOSPHATE = DEOXYRIBONUCLEOTIDE

  8. 8 NUCLEIC ACID BASES PYRIMIDINES CYTOSINE THYMINE URACIL

  9. 9 PURINES ADENINE GUANINE

  10. 10 NOBEL WATSON, CRICK, WILKINS

  11. 11 PAIRS PAIRS 5' 3' 3' 5' 2 3 DNACHARACTERISTICS DOUBLE STRANDED [CELLULAR DNA IS DOUBLE STRANDED] COMPLEMENTARY STRANDS ANTI-PARALLEL ORIENTATION HYDROGEN BONDS DENATURATION; MELT HEAT, MELT TEMP a GC CONTENT GC CONTENT [G+C / G+C+A+T] VARIES ~ 30% - 70%

  12. 12 BASE PAIRS - HYDROGEN BONDS CYTOSINE GUANINE C G PAIR THYMINE ADENINE T A PAIR

  13. 13 OR OTHER REACTIVE OXYGEN NUCLEOTIDYL TRANSFER REACTION [SYNTHESIS - COENZYMES; FATTY ACIDS; PROTEIN; RNA; DNA] ATP NUCLEOPHILIC ATTACK PYROPHOSPHATE (PPi)

  14. 14 NMN NICOTINAMIDE ADENINE DINUCLEOTIDE NMN+ + ATP NAD+ + PPi NICOTINAMIDE MONONUCLEOTIDE ATP + APPRECIATE !!! NAD+ + PPi

  15. 13 DNA POLYMERIZATION 3' 5' DNA TEMPLATE: SINGLE STRAND REGION SERVES AS TEMPLATE 3' OH SERVES AS PRIMER 5' 3' OH dATP, dGTP, dTTP, dCTP- LOW MOLECULAR WEIGHT BUILDING BLOCKS + ENZYME DNA POLYMERASE III 3' 5' + PPi OH 5' 3' NOBEL DNA POLYMERASE I

  16. 14 PARENTAL/TEMPLATE STRAND (OLD) 3' 5' NEW STRAND PRIMER 5' 3' dTTP deoxyTTP PARENTAL STRAND 3' 5' DAUGHTER STRAND + PPi 5' 3'

  17. 15 CHROMOSOME REPLICATION STAGES • INITIATION - SPECIFIC SITE ORIGIN - ORI C • - ESTABLISHES REPLICATION FORKS • POLYMERIZATION - • MOVEMENT OF REPLICATION FORKS AROUND CIRCULAR CHROMOSOME • SYNTHESIS OF NEW DAUGHTER STRANDS • LEADING - CONTINUOUS • LAGGING - DISCONTINUOUS • TERMINATION - INCLUDES SEPARATION OF NEW DAUGHTER CHROMOSOMES

  18. 18 INITIATION ter POLYMERIZATION REPLICATION FORKS CLOCKWISE COUNTERCLOCKWISE ORIGIN TERMINATION NEW SYNTHESIS: LEADING LAGGING

  19. 19 INITIATION SUPERCOILED DNA - TOPOISOMERASES ORIGIN SEQUENCE ORIGIN BINDING PROTEIN: MELTS ORIGIN GUIDE HELICASE TO ORIGIN HELICASE - UNWINDS DUPLEX PRIMASE - SYNTHESIZES PRIMERS REPLICATION FORK MOVEMENT LEADING STRAND - CONTINUOUS LAGGING STRAND - DISCONTINUOUS (MULTIPLE PRIMERS) 5' PRIMER [PRIMASE] 3' 5' 3' LAGGING [DISCONTINUOUS] 3' 5' HELICASE LEADING [CONTINUOUS] 5' 3'

  20. 20 SINGLE STRAND BINDING PROTEIN OMITTED LEADING AND LAGGING STRANDS: 5' DNA POLYMERASE III OKAZAKI FRAGMENT PRIMER [RNA] PRIMASE 1 LAGGING STRAND 2 3' 5' HELICASE 3' 5' DNA POLYMERASE III 3' TEMPLATE

  21. A WORD ABOUT NUCLEASES: HYDROLYZE NUCLEOTIDES ENDONUCLEASES: BREAK BONDS WITHIN CHAINS EXONUCLEASES: BREAK BONDS FROM AN END 5’ > 3’ 3’ > 5’ DNA POLYMERASE I HAS THREE ACTIVITIES: (SOME PROTEINS HAVE MORE THAN ONE ENZYME ACTIVITY) DNA POLYMERIZATION 5’ > 3’ 5’ > 3’ EXONUCLEASE 3’ > 5’ EXONUCLEASE

  22. 21 LAGGING STRAND ONLY: 5' 3' 5' 1 3' 5' 3' 2 POLYMERIZATION - DNA POLYMERASE III 5' 3' 5' 3' PRIMER REMOVAL ONE NUCLEOTIDE AT A TIME [DNA POLYMERASE I] PRIMER

  23. 22 5' 3' 5' 3' 5' 3' 5' 3' GAP FILLING [DNA POLYMERASE I] dNTP

  24. 23 PRIMER REMOVAL; GAP FILLING PROCEED UNTIL: THE GROWING END OF THE OKAZAKI PIECE BUMPS INTO OKAZAKI PIECE [LEAVES 5' PHOSPHATE ADJACENT TO 3' OH] 3' END GAP FILLING-OKZAKI 5' END OKZAKI PIECE

  25. 24 SEALED BY DNA LIGASE [REQUIRES ENERGY] PHOSPHODIESTER FORMED TWO OKAZAKI PIECES JOINED LAGGING STRAND IS NOW INTACT [OVER THIS REGION]

  26. 25 DNA LIGASE JOINS ADJACENT 5' PHOSPHATES AND 3' HYDROXYLS IN SERIES OF STEPS (REQUIRES ATP) 5' TEMPLATE 3' OKAZAKI PIECE ONE OKAZAKI PIECE TWO 3' 5' 3' 5' ENZYME + ATP OR NAD AS SOURCE OF AMP

  27. 26 3' + AMP + ENZYME

  28. 27 REPLISOME – ALL ASSOCIATED PROTEINS WHICH MOVE REPLICATION FORK AND POLYMERIZE DNA DNA HELICASE UNWINDS DUPLEX DNA PRIMASE SYNTHESIZES PRIMERS DNA POLYMERASE III POLYMERIZES DNA; PROOF READ SINGLE-STRAND DNA BINDING PROTEIN BINDS SINGLE-STRANDED DNA; PREVENTS REFORMING THE DUPLEX

  29. 28 DNA POLYMERASE III - A COMPLEX ENZYME WHICH: • POLYMERIZES DNA STRANDS COMPLEMENTARY TO TEMPLATE – POLYMERIZING SUBUNIT OF CORE RECOGNIZES COMPLIMENTARY H BONDING BETWEEN BASES • COORDINATES LEADING AND LAGGING STRAND REPLICATION- IT'S A TRIMER • POLYMERIZES >105 NUCLEOTIDES WITHOUT DISSOCIATING FROM TEMPLATE - A PROCESSIVITY CLAMP HOLDS IT ON • CORRECTS ITS OWN MISTAKES– CONTAINS A PROOFREADING SUBUINT

  30. 29 LOOP MODEL PART 5' COORDINATING LEADING AND LAGGING STRAND SYNTHESIS BY LOOPING THE LAGGING STRAND TEMPLATE [SHOWN WITH DIMERIC Pol III FOR SIMPLICITY] 1 3' PRIMASE 3' 5' Pol III Pol III 5' • LAGGING STRAND TEMPLATE PULLED BACKWARDS THROUGH Pol III UNTIL OKAZAKI PIECE IS COMPLETE • Pol III THEN CYCLES TO PRIMER 3 3'

  31. 30 LOOP MODEL PART 5' 3' 2 3' Pol III 5' Pol III 5' • OKAZAKI PIECE 2 COMPLETE • Pol III CYCLES TOWARD PRIMER 3 3'

  32. 31 LOOP MODEL PART 5' 3' 3 3' 5' Pol III Pol III 5' 3' • Pol III EXTENDS PRIMER 3 • PRIMER 4 HAS BEEN SYNTHESIZED

  33. REPLISOME WITH TRIMERIC DNA POLYMERASE 32 POLYMERASE CORE WITH POLYMERIZING ENZYME AND PROOFREADING SUBUNIT CLAMP LOADER – REPLISOME ORGANIZER; BINDS THREE CORES; BINDS HELICASE AND PRIMASE TO COORDINATE FORK MOVEMENT 5’ 3’ 5’ HELICASE PRIMASE PRIMERS PROCESSIVITY CLAMP LEADING STRAND 3’ LAGGING STRAND

  34. PROCESSIVITY CLAMP – HOLDS POLYMERIZING SUBUNIT ON TEMPLATE 33 CLAMP = PROCESSIVITY SUBUNIT- HOLLOW RINGS; ENCIRCLE NEWLY SYNTHESIZED DUPLEX; SLIDE FREELY OVER DUPLEX DNA; BINDS TIGHTLY TO POLYMERIZING ENZYME OF CORE 5’ 3’ 5’ PROCESSIVITY CLAMP 3’

  35. HOW DO CLAMPS GET ON DUPLEX? 34 A CLAMP LOADER, OF COURSE, LOADS AND ALSO UNLOADS FOR LAGGING STRAND LOADS ONCE FOR EACH OKAZAKI PIECE UNLOADS CLAMP WHEN OKAZAKI PIECE HAS BEEN COMPLETED FOR LEADING STRAND LOADS ONCE NEAR ORIGIN LOADING AND UNLOADING REQUIRES OPENING AND CLOSING RING, REQUIRES ATP 5’ 3’ 5’ 3’

  36. PROOFREADING – CORRECTING LAST NUCLEOTIDE INCORPORATED IF IT IS NOT COMPLEMENTARY TO TEMPLATE 35 3’ TO 5’ EXONUCLEASE WHICH CLIPS OUT 3’ NUCLEOTIDE IF INCORRECT 5’ 3’ 5’ PROOFREADING ENZYME 3’

  37. 36 PROOFREADING BY DNA POLYMERASE III 3' 5' EXONUCLEASE ACTIVITY TEMPLATE 3' 5' GROWING NEW STRAND 3' 3' 5' ERROR! 3' 5' EXONUCLEASE dCMP THEN, ADDITION OF dTTP 3' 5'

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  41. TAKE HOME: CHROMOSOMES ARE DUPLICATED (ACCURATELY) BY SEPARATING AND COPYING THE NUCLEOTIDE SEQUENCES INTO COMPLEMENTARY STRANDS IN REGULATED WAY (ONCE AND ONLY ONCE/CELL CYCLE). WHY? CHROMOSOMES CONSIST OF THE GENETIC PROGRAM OF EACH ORGANISM; EXECUTING THIS PROGRAM PERMITS LIFE. THAT IS, THE PROGRAM ALLOWS ALL REACTIONS AND SYNTHESIS OF ALL FACTORS WHICH ALLOW LIFE TO GO ON. REPLICATING GENETIC INFORMATION ALLOWS DESCENDANTS TO BE FORMED. REPLICATING GENETIC INFORMATION IS THE MOST BASIC, IMPORTANT EVENT IN LIFE.

  42. 40 • RETROVIRUSES – • CHROMOSOME IS SINGLE-STRANDED RNA • AFTER INFECTION, RNA CHROMOSOME MUST BE • COPIED INTO DOUBLE STRANDED DNA. • NUCLEOTIDE SEQUENCE IN RNA IS REVERSE- • TRANSCRIBED INTO DNA • ENZYME IS REVERSE TRANSCRIPTASE • e.g., HUMAN IMMUNODEFICIENCY VIRUS - HIV

  43. 41 SS RNA REVERSE TRANSCRIPTASE RNA : DNA RNaseH – REMOVES RNA REVERSE TRANSCRIPTASE RNaseH REVERSE TRANSCRIPTASE DNA : DNA SS RNA REVERSE TRANSCRIPTASE + AZT AZT ACTION RETROVIRUS REPLICATION AZT INHIBITION

  44. 42 NORMAL REVERSE TRANSCRIPTION REVERSE TRANSCRIPTASE USES RNA TEMPLATE TO SYNTHESIZE DNA COPY PARENT RNA GROWING DNA STRAND EXTENDED BY ADDING ONE NUCLEOTIDE AT A TIME

  45. 43 AZT INHIBITS REVERSE TRANSCRIPTION REVERSE TRANSCRIPTASE INCORPORATES AZT INTO GROWING DNA STRAND PARENT RNA (+) (-) GROWING DNA STRAND AZIDO GROUP IONIC STRUCTURE GROWING STRAND CANNOT BE EXTENDED DNA SYNTHESIS STOPS

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