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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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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.

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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

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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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NUCLEOTIDES

ESTER

NUCLEIC ACID BASE

N-GLYCOSIDIC BOND

DEOXYRIBOSE

DEOXYRIBOSE

DEOXYRIBONUCLEOSIDE

DEOXYRIBONUCLEOSIDE MONOPHOSPHATE

OR DEOXYRIBONUCLEOTIDE

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NUCLEIC ACID BASE

ANHYDRIDE BONDS

DEOXYRIBONUCLEOSIDE TRIPHOSPHATE

= DEOXYRIBONUCLEOTIDE

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NUCLEIC ACID BASES

PYRIMIDINES

CYTOSINE

THYMINE

URACIL

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PURINES

ADENINE

GUANINE

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NOBEL

WATSON, CRICK, WILKINS

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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%

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BASE PAIRS - HYDROGEN BONDS

CYTOSINE GUANINE

C G PAIR

THYMINE ADENINE

T A PAIR

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OR OTHER REACTIVE OXYGEN

NUCLEOTIDYL TRANSFER REACTION

[SYNTHESIS - COENZYMES; FATTY ACIDS; PROTEIN; RNA; DNA]

ATP

NUCLEOPHILIC ATTACK

PYROPHOSPHATE (PPi)

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NMN

NICOTINAMIDE ADENINE DINUCLEOTIDE

NMN+ + ATP NAD+ + PPi

NICOTINAMIDE MONONUCLEOTIDE

ATP

+

APPRECIATE !!!

NAD+

+ PPi

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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

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PARENTAL/TEMPLATE STRAND (OLD)

3'

5'

NEW STRAND PRIMER

5' 3'

dTTP

deoxyTTP

PARENTAL STRAND

3'

5'

DAUGHTER

STRAND

+ PPi

5' 3'

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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
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INITIATION

ter

POLYMERIZATION

REPLICATION FORKS

CLOCKWISE

COUNTERCLOCKWISE

ORIGIN

TERMINATION

NEW SYNTHESIS:

LEADING

LAGGING

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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'

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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

slide23

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

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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

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5'

3'

5'

3'

5'

3'

5'

3'

GAP FILLING

[DNA POLYMERASE I]

dNTP

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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

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SEALED BY DNA LIGASE

[REQUIRES ENERGY]

PHOSPHODIESTER FORMED

TWO OKAZAKI PIECES JOINED

LAGGING STRAND IS NOW INTACT [OVER THIS REGION]

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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

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3'

+ AMP + ENZYME

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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

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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
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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'

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LOOP MODEL PART

5'

3'

2

3'

Pol III

5'

Pol III

5'

  • OKAZAKI PIECE 2 COMPLETE
  • Pol III CYCLES TOWARD PRIMER 3

3'

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LOOP MODEL PART

5'

3'

3

3'

5'

Pol III

Pol III

5'

3'

  • Pol III EXTENDS PRIMER 3
  • PRIMER 4 HAS BEEN SYNTHESIZED
slide35

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

slide36

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’

slide37

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’

slide38

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’

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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'

slide43

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.

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  • 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
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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

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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

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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