Replication PDF - Lecture Notes

COLLEGE OF MEDICINE AND HEALTH SCIENCES SCHOOL OF MEDINCE AND PHARMACY DEPARTMENT OF MEDICAL BIOCHEMISTRY, MOLECULAR BIOLOGY AND GENETICS BIOTECHNOLOGY AND DRUG DESIGN (FARM L227) LECTURE NOTE FOR YEAR 4...

COLLEGE OF MEDICINE AND HEALTH SCIENCES SCHOOL OF MEDINCE AND PHARMACY DEPARTMENT OF MEDICAL BIOCHEMISTRY, MOLECULAR BIOLOGY AND GENETICS BIOTECHNOLOGY AND DRUG DESIGN (FARM L227) LECTURE NOTE FOR YEAR 4 PHARMACY STUDENTS UNIT: ONE PRINCIPLES OF MOLECULAR BIOLOGY REPLICATION by Assoc. Prof. Abiola Stephanie Tijani  Objectives At the end of this lecture, student will be able to  Outline the important components in DNA replication  Discuss the mechanism involved in DNA replication  Compare and contrast the DNA replication in eukaryotes and prokaryotes  Describe the activities of enzymes required for replication 2  MOLECULAR BIOLOGY  Molecular biology is a branch of science that explains the biological processes in terms of molecular interactions.  It is best described as ‘ the biochemistry of genes and their products’  It is a combination of three basic streams of bioscience Cell biology Genetics Biochemistry 3  INTRODUCTION  Genetics deals with the identification and characterization of gene products and inheritance of genes  Cell biology is deals with the structural functioning of cells organelles  Biochemistry involves the finding out of three dimensional structures and actions of different macromolecules  The term Molecular biology was coined by Warren Weaver, the director of natural science division of Rockefeller Foundation in USA and his team in 1938 to describe a programme for the application of tools of physical sciences to biology, biochemistry, cell biology and genetics.  Although it was coined in 1938, it was first used in 1945 by William Astbury to study the chemical and physical structure of biological macromolecules. 4  INTRODUCTION Various fields of science have contributed to molecular biology. For example: Biochemistry Genetics INSTRUMENTATION and PHYSICS Archibaid Gerrod (1902) was 1st to BIOLOGICAL TECHNIQUES 1st biochemical experiment Szilard, a physicsist, describe the precise relationship Theodor Svedberg (1920) in 1897 by Edward Buchner, developed technique between genes and metabolism developed analytical centrifuge sugar fermentation in-vitro that is used to determine the for the analysis of George Beadle and Edward Tatum using cell free-extract. molecular weight of gene regulation in demonstrated that genes control Decoding of metabolic enzyme synthesis in Neurospora. macromolecules and small cell bacteria. pathways like Glycolysis, They proposed one gene one organelles or in parts. George Gamow Knoll and Ruska (1930) TCA cycle etc in the 1st half enzyme theory. (1954) interpreted Avery, Macleod and McCarty invented the electron of 20th century the genetic code to (1944) discovered the chemical microscope to study the The term “macromolecule” explain relationship nature of gene. structural details of viruses, was introduced by Hermann between sequences Watson and Crick (1953) described macromolecules and various cell Staudinger, a German organelles. of nitrogenous the double helical structure of DNA chemist in 1922 to describe bases and amino based on X-ray diffraction studies Mikhall Tswett (1906) developed biomolecules of the cell. made by M.H. F Wilkins and simple column acids in the 5 Rosalind Franklin chromatography. polypeptide chain.  INTRODUCTION  These techniques have immensely help in the separation and study of molecular structure of biological macromolecules, cell organelles and cells. The use of techniques of spectrophotometry, auto radiography, and isolation and determination of role of various enzymes in DNA replication, RNA transcription and translation of RNA to proteins have revolutionized the knowledge in the field of molecular biology.  Molecular biology can be classified into: NEW MOLECULAR BIOLOGY CLASSICAL MOLECULAR BIOLOGY Science of intervention and action Science of observation Adopts an experimental approach Centered on proteins Concerned with determining the sequences of Proteins with their three dimensional structure were considered nucleotides in genes and from that deducing to be the specific agents of biological processes. the sequence of amino acids in proteins  After a lot of experiments and verification the new molecular biology emerged.  Simple organisms like bacteria, viruses, bacteriophages, unicellular green algae yeast Neurospora and other fungi, nematodes fruitflies and plants Arabidopsis were used for molecular studies. 6  INTRODUCTION Landmarks In The Field Of Molecular Biology  Though the history of molecular biology dated back to 1865 when Gregor Mendel proposed that characters are controlled by factors and Johanson pointed out that genes are the factors in 1909  The beginning of molecular biology is considered to be in:  1928: F. Griffith discovered transformation in Diplococcus pneumoniae  1934: M. Schlesinger demonstrated that bacteriophages are composed of DAN and proteins.  1941: Beadle and Tatum published the results of biochemical genetics of Neurospora and established one gene one enzyme hypothesis.  1944: Avery, Macleod and McCarty identified tthat he transforming principle of Diplococcus bacteria was DNA  1950: Erwin Chargaff demonstrated that in DNA, the number of adenine molecules is equal to the number of thymine and the number of cytosine is equal to guanine.  1953: James Dewey Watson and Fracis Harry Compto Crick proposed the double helical model of DNA based on the studies of Maurice Wilkins and Rosalind Francklin  1957: Meselson and Stahl confirmed Watson and Crick’s semiconservative model of DNA replication.  1958: Kornberg isolated DNA poklymerase from E. coli and got Nobel price in 1959 7  INTRODUCTION Landmarks In The Field Of Molecular Biology  1958: F.H.C Crick proposed the ‘Central Dogman’ of molecular biology  1961: Nirenberg and Matthaei cracked the genetic code present on mRNA.  1961: Jacob and Monod proposed the ‘Operon concept for regulation of gene expression’ and got the Nobel prize in 1965.  1965: F.H.C Crick proposed the wobble hypothesis.  1966: Khorana, Nirenberg and Holley worked out the complete genetic code fore enzyme and got the nobel prize in 1968  1975: discovered reverse transcriptase (Temin and Baltimore)  1977: Maxam, Gilbert and Coulson described DNA sequencing techniques  1977: Sanger et al worked on the complete nucleotide sequence of Ѱ x 174 and received the nobel prize in 1980.  1981: invented DNA sequencing method (Gilbert and Sanger)  1985: invented PCR technique (Mullis)  1987: launched the human genome project  2000: chromosomes consist of DNA and protein, concluded DNA is genetic material  2001: accomplished the draft map of human genome. 8  April 2003: Human Genome Project Completed  INTRODUCTION Landmarks In The Field Of Molecular Biology ▪ Mouse genome is sequenced. ▪ April 2004 Rat genome sequenced. ▪ Next-generation sequencing – genomes being sequenced by the dozen 2016: Yoshinori Ohsumi discovered the mechanisms for autophagy and received the Nobel prize. 2019:: William G. Kaelin Jr, Sir Peter J. Ratcliffe and Gregg L. Semenza discovered how cells sense and adapt to oxygen availability and received the Nobel prize 9  COMMON TERMS IN MOLECULAR BIOLOGY  Gene: Segments of the chromosome that code for a specific product (usually a protein) and it is located within chromosomes (linear or circular)  Chromosomes : The structures that are composed of DNA that carry the hereditary information  Genome: Complete genetic information of the cell.  all genetic instructions (genes) for development of cellular structures, metabolic functions, and their regulation  a complete set of DNA, including all of its genes.  the sum of all genetic material in the cell  Genomics: is field of study that focus on evolution, structure, function, mapping and gene manipulation.  examine the function and composition of all genes in the organism  their interaction to identify their effect on the growth and development of the organism. 10  COMMON TERMS IN MOLECULAR BIOLOGY (cont’d)  Genotype : Organisms complete set of heritable genes (Genetic make-up)  genes that can be passed down from parents to offspring  combination of alleles that an individual possesses for a specific gene  the information in the DNA that control the phenotype  Phenotype: observable traits of an organisms that results from interaction of its genotype with the environment  all the heritable physical characters of the organism  observable physical properties of an organism  organism's appearance, development, and behavior.  Exons: are the various bits of DNA that actually code for protein  Introns: (“junk” DNA) are the intervening sequences that separate exons. 11  COMMON TERMS IN MOLECULAR BIOLOGY (cont’d) The field of molecular biology is expanding exponentially and has given birth to various branches : Proteomics Genomics ppr Bioinformatics Molecular Biology These three branches use the computer knowledge and biological processes  Bioinformatics: use of computer knowledge in genetics referred to genomics. 12  Proteomics: use of computer knowledge and the study of proteins.  GENETIC MATERIALS  They are substances that store information about structure, function and development of various characteristics of the living organisms.  They are responsible for the transmission of genetic information for all characteristics of living beings from parents to progeny  After the rediscovery of Mendel’s laws, geneticists were able to conclude that  Organism’s characters are controlled by genes  Genes are able to reproduce themselves or replicate without losing their information  Genes are arrange bon the chromosomes in a linear fashion and  Genes are transmitted from parents to offspring from one generation to another un altered. 13  CHARACTERISTICS OF GENETIC MATERIALS  Storage of genetic information: the ability to store genetic information about structure, function, development and reproduction in all the living organisms.  Accurate replication: the capability of accurate replication so that the same genetic information as in parent cell are passed on to the daughter cells.  Transcription: ability to accurately transcript so that the stored genetic information’s aretransmitted to the cell as when needed.  Stability: chemical and physical stability so that the store information’s are not lost  Variation: capable of undergoing modifications by mutations and recombination so that it can contribute to the variation and adaptations leading to the evolution of living organisms. 14  NUCLEIC ACID  The genetic information in all cells is carried by nucleic acids.  The information is decoded through series of processes into the amino acids’ sequence into protein molecules.  Nucleic acids are biopolymer composed of nucleotides linked in a linear sequential order through 3’,5’ phosphodiester bonds  polymer is a chemical compound that is made of small molecules that are arranged in a simple repeating structure to form a larger molecule.  nucleotides are the basic structural units of nucleic acids (genetic material) that composed of sugar (ribose or deoxyribose), nitrogenous bases and phosphate groups.  is monomer subunits that make up the nucleic acids  Genetic material or nucleic acids consists of DNA and RNA.  DNA and RNA are macromolecular structures composed of regular repeating polymers formed from nucleotides  The primary structure of DNA and RNA is defined as the nucleotide sequence in the 5’ – 3’ direction 15  NUCLEIC ACID (cont’d) Functions of nucleotides in the cell; 1. They are the subunits that make the DNA structure 2. They are part of certain enzymes 3. Their bonds are carriers of chemical energy 4. They serve as specific signaling molecule 16  NUCLEIC ACID CLASSIFICATION Nucleic acids (DNA and RNA) are assembled from nucleotides, which consist of three components: a nitrogenous base, a five-carbon sugar (pentose), and phosphate group.  The nucleic acids is classified according to the pentose sugar they contain. If the pentose sugar is a deoxyribose, the nucleic acid is deoxyribonucleic acid (DNA) and if the pentose sugar is a ribose, the nucleic acid is ribonucleic acid (RNA).  There are two types of nitrogen-containing bases commonly found in nucleotides: purines and Pyrimidines.  Purines contain two rings in their structure. The two purines commonly found in nucleic acids are adenine (A) and guanine (G); both are found in DNA and RNA.  Other purine metabolites, not usually found in nucleic acids, include xanthine, hypoxanthine and uric acid.  Pyrimidines have only one ring. Cytosine (C) is present in both DNA and RNA.  Thymine (T) is usually found only in DNA, whereas uracil (U) is found only in RNA. 17  NUCLEIC ACID CLASSIFICATION Purines and Pyrimidines  Adenine and guanine are structural to the parent molecule purine  Cytosine, thymine and uracil are structural to the parent molecule pyrimidine Nitrogenous bases 18  CHEMICAL COMPONENTS OF NUCLEIC ACIDS Pentose sugars 5´ 4´ 1´ 3´ 2´ β-D-ribose β-D-2-deoxyribose 19  CHEMICAL COMPONENTS OF NUCLEIC ACIDS  The nucleotides are identical except that each contains a different nitrogen-containing base.  Each nucleotide is made up of a phosphate group, pentose sugar (ribose for RNA or deoxyribose type for DNA), and one of the four bases (Uracil in RNA instead of Thymine in DNA).  Nucleosides are formed by covalently linking a base to the number 1 carbon (C1) of a sugar. The numbers identifying the carbons of the sugar are labeled with "primes" in nucleosides and nucleotides.  Nucleotides contain phosphoric acid  Nucleosides lack the phosphoric acid.  Nucleotides are the building stones of DNA Phosphate Nucleic acid Nucleotides Pentose Nucleotide = a phosphate group + nucleoside Nucleosides Bases The four bases are adenine (A), guanine (G) (found in purines) and cytosine (C) and thymine (T), Uracil (U) (found in pyrimidines) 20  CHEMICAL COMPONENTS OF NUCLEIC ACIDS Nucleosides and Nucleotides Nitrogenous base Phosphate Group Sugar 21  CHEMICAL COMPONENTS OF NUCLEIC ACIDS a) Ribonucleic acid (RNA) is composed of ribonucleotides.  found in nuclei and cytoplasm  participate in the gene expression  an important class of molecules in the flow of genetic information  Pentose sugar in RNA is Ribose sugar  All other organisms that use DNA as the genetic material must first transcribe their genetic information into RNA, in order to render the information accessible and functional a) Deoxyribonucleic acid (DNA) is composed of deoxyribonucleotides.  90% in nuclei and the rest in mitochondria  DNA store and carry genetic information; determine the genotype of cells  Pentose sugar in DNA is deoxyribose hence deoxyribonucleic acid  DNA is double stranded while RNA is single stranded 22  CHEMICAL COMPONENTS OF NUCLEIC ACIDS Deoxyribonucleic acid (DNA)  DNA stands for deoxyribonucleic acid.  DNA is an extremely long molecule that forms a double-helix made up of repeated units of nucleotides.  Each nucleotides consists of equal quantity of nitrogenous bases, pentose sugar and phosphate group  The double-helix backbone of the molecule consists of sugars and phosphates, and there is one base attached to each sugar.  There are four types of bases:  cytosine (C), guanine (G), adenine (A) and thymine (T).  The DNA consists of two strands, and each base attached to one strand forms a bond with a corresponding base on the other strand. (A only links with T and C links with G).  A triplet of bases encodes an amino acid.  Protein is a sequence of amino acids, and the functional subunit of DNA that encodes a protein is called a gene.  The base composition of DNA generally varies from one species organism to another.  DNA isolated from different tissues of the same species have the same base composition.  The base composition of DNA in a given species does not change with its age, nutritional state, and environmental variations. 23  CHEMICAL COMPONENTS OF NUCLEIC ACIDS Deoxyribonucleic acid (DNA)  The amount of T always equals the amount of A, and the amount of C always equals the amount of G. But the amount of A + T is not necessarily equal to the amount of G + C.  The total amount of pyrimidine nucleotides (T + C) always equals the total number of purine nucleotides (A + G).  The nucleotide monomers are joined together by a covalent bond between the pentose sugar of one nucleotide and the phosphate of the adjacent nucleotide.  Hence, the phosphate is a bridge that connect the number 5 carbon atom (termed 5’) of one deoxyribose sugar to the number 3 carbon atom of another (termed 3’).  The resulting molecules from this bonding is one with a backbone of alternating phosphate and deoxyribose sugar molecules.  The two ends of such molecule are different. The 3’ end has an hydroxyl group and the 5’ end has a phosphate molecule attached to the number 5 carbon atom of the deoxyribose sugar (3’ end is always the end of the molecules that keeps growing by addition of more nucleotides).  DNA is formed by coupling the nucleotides between the phosphate group from a nucleotide (which is positioned on the 5th C- atom of the sugar molecule) with the hydroxyl on the 3rd C-atom on the sugar molecule of the previous nucleotide.  To accomplish this, a diphosphate molecule is split off (and releases energy). This means that new nucleotides are always 24 added on the 3’ side of the chain.  CHEMICAL COMPONENTS OF NUCLEIC ACIDS Deoxyribonucleic acid (DNA) 25  CHEMICAL COMPONENTS OF NUCLEIC ACIDS Ribonucleic acid (RNA)  In RNA nitrogenous base Thymine is replaced Uracil  Pentose sugar in RNA is Ribose sugar  RNA is single stranded.  RNA is susceptible to hydrolysis Differences between DNA and RNA Nucleic acid Base Sugar DNA A, G, C, T Deoxyribose RNA A, G,C, U Ribose 26  CHEMICAL COMPONENTS OF NUCLEIC ACIDS Nucleosides and Nucleotides in DNA Base Nucleoside Nucleotide Guanine Deoxyguanosine Deoxyguanosine monophosphate dGMP) Cytosine Deoxycytidine Deoxycytidine monophosphate (dCMP) Adenine Deoxyadenosine Deoxyadenosine monophosphate (dAMP) Thymine Deoxythymidine Deoxythymidine monophosphate (dTMP) Nucleotides can be mono, di or triphosphate in DNA; for example There are 4 different nucleotides:  dATP : deoxyadenosine triphosphate  dGTP : deoxyguanosine triphosphate  dTTP : deoxythymidine triphosphate  dCTP : deoxycytidine triphosphate 27  CHEMICAL COMPONENTS OF NUCLEIC ACIDS Nucleosides and Nucleotides in RNA Base Nucleoside Nucleotide Guanine Guanosine Guanosine monophosphate (GMP) Cytosine Cytidine Cytidine monophosphate (CMP) Adenine Adenosine Adenosine monophosphate (AMP) Uracil Uridine Uridine monophosphate (UMP) Nucleotides can be mono, di or triphosphate in RNA; for example There are 4 different nucleotides:  dATP : deoxyadenosine triphosphate  dGTP : deoxyguanosine triphosphate  dTTP : deoxythymidine triphosphate  dCTP : deoxycytidine triphosphate For convenience, these 4 nucleotides are called dNTP's (deoxynucleoside triphosphates). A nucleotide is made of three major parts a nitrogen base, a sugar molecule and a triphosphate. Only the nitrogen base is different in the 4 nucleotides. 28  THE CENTRAL DOGMA  Coined by Crick in 1958  States that DNA contains instructions for making a protein which are copied by RNA. RNA then uses their instructions to make a protein.  In 1970, H. Temin and D. Baltimore discovered reverse transcriptase, a RNA-directed DNA polymerase.  Thus , the dogma was revised by addition of reverse transcription.  The central dogma explains the flow of genetic information from DNA to RNA, to make a functional product, a protein. Replication Transcription Translation 29 CENTRAL DOGMA  DNA contains the complete genetic information that defines  the structure and function of an organism. Proteins are formed using the genetic code of the DNA.  Three different processes are responsible for the inheritance of genetic information and for its conversion from one form to another : 1. Replication: a double stranded nucleic acid is duplicated to give identical copies. This process perpetuates the genetic information. 2. Transcription: a DNA segment that constitutes a gene is read and transcribed into a single stranded sequence of RNA. The RNA moves from the nucleus into the cytoplasm. 30 CENTRAL DOGMA 3. Translation: the RNA sequence is translated into a sequence of amino acids as the protein is formed. During translation, the ribosome reads three bases (a codon) at a time from the RNA and translates them into one amino acid.  In eukaryotic cells, the second step (transcription) is necessary because the genetic material in the nucleus is physically separated from the site of protein synthesis in the cytoplasm in the cell.  Therefore, it is not possible to translate DNA directly into protein, but an intermediary must be made to carry the information from one compartment to an other. 31  DNA REPLICATION  DNA replication it is a fundamental process in all living organisms to copy their DNA exactly.  It is the basis for biological inheritance  Replication begins at one or more origins of replication along DNA.  It is the process in which each strand of the original double-stranded DNA serves as a template for the reproduction of the complementary strand.  It is a process in which daughter DNAs are synthesized using parental DNAs as template  It is the transfer of genetic information to the descendant generation with a high fidelity.  Many enzymes and accessory proteins are required for in vivo replication, which begins at a region of the DNA termed the origins of replication  DNA has to be unwound by the enzyme before any of the proteins and enzymes needed for replication can act.  It occurs once in a cell and it is quick and accurate at the correct time. 32  DNA REPLICATION  The following are required and involved in DNA replication systems Template: double stranded DNA Substrate: dNTP Primer: short RNA fragment with a free 3´-OH end Enzyme: DNA-dependent DNA polymerase (DDDP), other enzymes, protein factor  Key replication proteins and their functions are listed in Table below: 33  DNA REPLICATION Steps involved in DNA replication  DNA replication is a sequence of repeated condensation (dehydration synthesis) reactions linking nucleotide monomers into a DNA polymer.  Replication, like all biological polymerizations, proceeds in three enzymatically catalyzed and coordinated steps: 1. Initiation 2. Elongation and 3. Termination  Replication begins at one or more origins of replication along DNA. Helicase enzymes catalyze unwinding of the double helix, creating replicating bubbles known as replicons, with replication forks at either end. 34  DNA REPLICATION Initiation:  DNA synthesis starts at one or more origins of replication. These are DNA sequences targeted by initiation proteins.  After these proteins break the hydrogen (H-) bonds at the origin of replication, the DNA double helix is progressively unwind or unzipped in both directions (i.e., by bidirectional replication).  The separated DNA strands serve as templates for new DNA synthesis (i.e on each exposed single strand, a short complementary RNA chain termed a primer is first produced, using the DNA as a template).  The primer is synthesized by an RNA polymerase enzyme known as a primase  Sequences at replication origins that bind to initiation proteins tend to be rich in adenine and thymine bases (because A-T base pairs have two H-bonds, which require less energy to break than the three H-bonds that hold G-C pairs together).  The eukaryotic replication origin are called Autonomously Replicating Sequences or Replicator (ARS) and shorter than the origin of Replication or ori-site in prokaryotes.  Initiation of replication in eukaryotic cells requires DNA-pol α (have primase activity) and DNA-pol-δ (with polymerase and helicase activities). DNA-pol-δ requires a protein Proliferating Cell Nuclear Antigen (PCNA) for its activity.  Initiation of replication in eukaryotic cells also needs topoisomerase and Replication factors (RF). 35  DNA REPLICATION Initiation:  Once initiation proteins loosen H-bonds at a replication origin, DNA helicase uses the energy of ATP hydrolysis to further unwind the double helix.  To prevent the single strands from re-annealing small proteins termed single-stranded DNA binding proteins (SSBs) attach to the single DNA strands.  But as the double helix of DNA separates from one side and super coils are formed ahead of the replication fork due to the massive length of the DNA; this super coil problems is solved by a group of enzymes known as DNA topoisomerase.  DNA polymerase III is the main enzyme that then elongates new DNA. Once initiated, a replication bubble (replicon) forms as repeated cycles of elongation proceed at opposite replication forks.  Replication fork is the junction between the newly separated stands known as the Leading and Lagging strands and the double stranded DNA (i. e the parental dsDNA and two newly formed dsDNA form a Y-shape structure called replication fork) 36  DNA REPLICATION Initiation:  DNA REPLICATION Elongation:  Looking at elongation at one replication fork, we see another problem (one of the two new DNA strands will grow continuously (leading strand) toward the replication fork as the double helix unwinds; but what about the other strand? Either this other strand must grow in pieces in the opposite (lagging strand) direction, or it must wait to begin synthesis until the double helix is fully unwound). In replication, the 5'-to-3' strand elongation catalyzed by all DNA polymerases presents a problem at the RF: only one new DNA strand (the leading strand) can be made continuously along its parental template strand. How does replication progress along the opposite template strand? 38  DNA REPLICATION Elongation:  If one strand of DNA must be replicated in fragments, then those fragments would have to be stitched (i.e., ligated) together, as hypothesized in the figure.  Giving to this hypothesis, a new leading strand of DNA grows (is lengthened) continuously by sequential addition of nucleotides to its 3′ end, against its leading-strand template. The other strand (lagging strand), however, would be made in pieces that would be joined in phosphodiester linkages in a subsequent reaction (discontinuous replication). Because joining these new DNA fragments should take extra time, this new DNA is called the lagging strand, making its template the lagging-strand template. The hypothesis proposed here is that during the elongation of DNA strands, at least one DNA strand at a replication fork (the lower, so-called lagging strand) must be synthesized discontinuously, i.e., in pieces. Each new “piece” would begin with an RNA primer. And these pieces would have to be correctly stitched together into a continuous DNA strand. 39  DNA REPLICATION Elongation:  But, for elongation to proceed normally; DNA polymerase III (DNApolIII) binds to one strand of the DNA and begins moving along it in the 3' to 5' direction, using it as a template for assembling a leading strand of nucleotides and reforming a double helix.  Note that DNA polymerase III can only add new nucleotides to the 3’ end.  But in the lagging strand DNA synthesis can only occur 5' to 3', a molecule of a second type of DNA polymerase (epsilon, ε, in eukaryote) binds to the other template strand as the double helix opens. This enzyme must synthesize discontinuous segments of polynucleotides (called Okazaki fragments). That is, Okazaki fragments synthesis occur in 5' to 3‘ direction of the RNA primer, therefore: 1. The primer must be first removed before the fragments will be joined together. Steps in the synthesis of DNA against the lagging 2. Removal of RNA primer nucleotides from Okazaki fragments requires template strand 40 the action of DNA polymerase I.  DNA REPLICATION Elongation:  DNA polymerase I has the unique ability to catalyze hydrolysis of the phosphodiester linkages between the RNA (or DNA) nucleotides and the 5’ end of a nucleic acid strand.  Another enzyme, DNA ligase I then stitches these together into the lagging strand 41  DNA REPLICATION Termination:  In eukaryotes, many replicons fuse to become larger replicons, eventually reaching the ends of the chromosomes. And now there is still another problem, illustrated in Figure. Problems arising in lagging strand replication when replication reaches the ends (telomeres) of linear chromosomal DNA. After a final Okazaki strand is primed and replicated (panel 1) the primer is removed from the penultimate fragment (panel 2). Then primer on the last strand is removed (panel 3). But then, there would be no DNA strand 3' end at which to add replacement DNA nucleotides. 42 This would cause chromosomal shortening at each cell division.  DNA REPLICATION Termination:  When a replicon nears the end of a double-stranded DNA molecule (i.e., the end of a chromosome), the new continuously synthesized strand stops when it reaches the 5’ end of its template DNA, and the primer is removed, having completed its replication (for leading stand).  But what about lagging-strand replication?  From the Figure (previous slide), The illustration shows primer removal from an Okazaki fragment primed near the end of the chromosome, and replacement with DNA nucleotides catalyzed by DNA polymerase I. The question marks in the Figure (previous slide) the DNA point to a dilemma which is:  if a final Okazaki fragment is primed and synthesized, DNA polymerase I has no free 3’ end to begin RNA nucleotide replacement with DNA nucleotides.  The problem then would be that every time a cell replicates, at least one strand of new DNA would get shorter. But, this is not true—and doesn’t happen!  Eukaryotic replication undergoes a termination process that extends the length of one of the two strands using the enzyme telomerase, as illustrated in Figure (Next slide). 43  DNA REPLICATION Termination:  Telomerase consists of several proteins and an RNA molecule. From the Figure:  the RNA component serves as a template for 5′→ 3’ extension of the problematic DNA strand. The protein with the requisite reverse transcriptase activity is called Telomerase Reverse Transcriptase, or TERT. The Telomerase RNA Component is called TERC. Following removal of the primer from a telomeric Okazaki fragment, the ribonucleoprotein enzyme telomerase prevents chromosome-shortening. Its RNA serves as a template to generate repeats at the 3’ telomeric end of lagging-strand DNA. When the extended repeated sequences on the 3’ end of the template DNA are long enough, they then serve as templates for new primer and Okazaki fragment synthesis, maintaining chromosome44 length.  DNA REPLICATION Termination: Generally, in termination step:  there is a need to remove the primer. The primers are removed by the exonuclease activity of DNA pol I, and fill the gaps. This is seen in prokaryotes  Then DNA ligase fills the gaps on the lagging strand, that is joining of the Okazaki fragments and also closes nicks in double-stranded DNA.  DNA REPLICATION Features of Eukaryotic DNA Replication  Replication is bi-directional and originates at multiple origins of replication (Ori C) in eukaryotes.  DNA replication uses a semi-conservative method that results in a double-stranded DNA with one parental strand and a new daughter strand.  It occurs only in the S phase and at many chromosomal origins.  Takes place in the cell nucleus.  Synthesis occurs only in the 5′to 3′direction.  Individual strands of DNA are manufactured in different directions, producing a leading and a lagging strand.  Lagging strands are created by the production of small DNA fragments called Okazaki fragments that are eventually joined together.  Eukaryotic cells possess five types of polymerases involved in the replication process.  DNA REPLICATION Enzymes Involved In Prokaryotic DNA Replication And The Functions. Enzyme/protein Specific Function DNA pol I Exonuclease activity removes RNA primer and replaces with newly synthesized DNA DNA pol II Repair function DNA pol III Main enzyme that adds nucleotides in the 5'-3' direction Helicase Opens the DNA helix by breaking hydrogen bonds between the nitrogenous bases Ligase Seals the gaps between the Okazaki fragments to create one continuous DNA strand Primase Synthesizes RNA primers needed to start replication Sliding Clamp Helps to hold the DNA polymerase in place when nucleotides are being added Topoisomerase Helps relieve the stress on DNA when unwinding by causing breaks and then resealing the DNA Single-strand binding proteins (SSB) Binds to single-stranded DNA to avoid DNA rewinding back.  DNA REPLICATION Summary of the Difference between Prokaryotic and Eukaryotic Replication  Replication in eukaryotes starts at multiple origins of replication. The mechanism is quite similar to prokaryotes.  A primer is required to initiate synthesis, which is then Difference between Prokaryotic and Eukaryotic Replication extended by DNA polymerase as it adds nucleotides one by Property Prokaryotes Eukaryotes one to the growing chain.  The leading strand is synthesized continuously, whereas the Origin of replication Single Multiple lagging strand is synthesized in short stretches called Okazaki fragments. 50 to 100 Rate of replication 1000 nucleotides/s  The RNA primers are replaced with DNA nucleotides; the nucleotides/s DNA remains one continuous strand by linking the DNA DNA polymerase fragments with DNA ligase. 5 14 types  The ends of the chromosomes pose a problem as polymerase is unable to extend them without a primer. Telomerase Not present Present  Telomerase, an enzyme with an inbuilt RNA template, extends the ends by copying the RNA template and extending one end RNA primer removal DNA pol I RNase H of the chromosome. Strand elongation DNA pol III Pol δ, pol ε  DNA polymerase can then extend the DNA using the primer. In this way, the ends of the chromosomes are protected. Sliding clamp Sliding clamp PCNA DNA REPLICATION  DNA REPLICATION Models for DNA replication:  From the complementary strands model of DNA, proposed by Watson and Crick in 1953, there are three straightforward possible replication patterns of DNA: (1) semi-conservative replication (2) Conservative replication (3) dispersive replication 1. Semi-conservative model: This model proposes that:  the two strands of a DNA molecule separate during replication  each strand acts as a template for synthesis of a new, complementary strand.  Daughter DNA molecules contain one parental strand and one newly synthesized strand.  base pairing allows each strand to serve as a template for a new strand  new strand is 1/2 parent template and 1/2 new DNA.  DNA replication is said to be Semi-conservative because:  One strand is the original (conserved)  One strand is freshly assembled (semi-half)  DNA REPLICATION Models for DNA replication: 1. Semi-conservative model:  DNA REPLICATION Models for DNA replication: 2. Conservative Model proposed that:  each strand of the DNA duplex is replicated.  the two newly synthesized strands join to form one DNA double helix, while the two parental strands remain associated with each other.  the products are one completely new DNA duplex and the original DNA duplex.  DNA REPLICATION Models for DNA replication: 3. Dispersive model proposed that:  parent helix is broken into fragments, dispersed, copied then assembled into two new helices.  each of the four strands in the two daughter DNA duplexes contains both newly-synthesized segments and segments from the parental strands.  DNA REPLICATION Features of DNA Replication 1. DNA replication is semiconservative  the two parental strands separate and each makes a copy of itself  separation of the two strands provides two templates  each carries all the information of the original molecule  after one round of replication, every new DNA double helix would be a hybrid that consisted of one strand of old DNA bound to one strand of newly synthesized DNA.  DNA REPLICATION Features of DNA Replication 2. Bidirectional Replication can be Uni- or Bidirectional Bidirectional replication Origin 5’ 3ʹ Origin 3’ 5ʹ 5ʹ 3ʹ ’3ʹ 5ʹ Unidirectional replication  DNA REPLICATION Models for DNA replication: 3. Semi discontinuous  A new strand of DNA is always synthesized in the 5’→ 3’ direction.  one daughter strand at the replication fork is synthesized continuously called leading strand  the other synthesized discontinuously called leading strand.  Continuous synthesis of the leading strand and discontinuous synthesis of the lagging strand represent a unique feature of DNA replication.  it is referred to as the semi-continuous replication 🗶 5 ′ 3 5 5 3 3 5 ′ ′ ′ ′ ′ 3 5 ′ Lagging strand ′ ′ ligase growing replication fork 3 5 ′ ✔ ′ Leading strand 3 5 ′ ′ 3 ′ ▪ Okazaki fragments joined by ligase DNA polymerase III  DNA REPLICATION Models for DNA replication: 3. Semi discontinuous DNA polymerase III lagging strands DNA polymerase I primase 3ʹ Okazaki fragments 5ʹ 5ʹ ligase SSB 3ʹ 5ʹ 3ʹ helicase ʹ DNA polymerase III 5’ leading strand 3ʹ direction of replication SSB = single-stranded binding proteins  DNA REPLICATION Enzymes involved in DNA Replication 1. DNA polymerase  The enzyme that replicate DNA are called DNA polymerases  DNA polymerases select the incoming base according to Watson and Crick’s base-pairing rules  DNA polymerases synthesize daughter chains in the 5’→3’ direction.  DNA polymerases cannot initiate DNA synthesis de novo—all require a primer , oligonucleotides with a free 3’-OH to build upon. a. DNA-pol I  Mainly responsible for proofreading and filling the gaps, repairing DNA damage.  It identifies the mismatched nucleotide, removes it using the 3´- 5´ exonuclease activity, add a correct base, and continues the replication. b. DNA-pol II  Temporary functional when DNA-pol I and DNA-pol III are not functional.  Still capable for doing synthesis on the damaged template.  Participating in DNA repair process  DNA REPLICATION Enzymes involved in DNA Replication c. DNA-pol III  A heterodimer enzyme composed of 10 different kinds of subunits  Having the highest polymerization activity (105 nt/min)  The true enzyme responsible for the elongation process Eukaryotic cells contain five different DNA polymerases; α, β, γ, δ and ε.  DNA polymerases α and δ replicate chromosomal DNA, DNA polymerases β and ε repair DNA, and DNA polymerase γ replicates mitochondrial DNA.  DNA polymerases α (have primase activity) synthesizes the primer  DNA polymerase α synthesize the lagging strand.  DNA polymerase δ synthesizes the rest of the Okazaki fragment.  The RNA primers are synthesized by DNA polymerase α which carries a primase subunit.  DNA polymerase ε synthesizes the leading strand.  Telomerase, a DNA polymerase that contains an integral RNA that acts as its own primer, is used to replicate DNA at the ends of chromosomes (telomeres).  DNA REPLICATION Enzymes involved in DNA Replication 2. DNA helicase  The helicase molecule requires a single-strand region for binding and then moves along the single strand to unwind the dsDNA.  Helicases cleave the hydrogen bonds between the two strands.  The helicases consume ATP to separate the strands. 3. Topoisomerase  During replication, opening of the dsDNA will create supercoil ahead of replication forks  The supercoil constraint needs to be released  is an enzyme used unwind the supercoiled DNA strand  The enzymes cut a dsDNA molecule first; pass another portion of the duplex through the cut and reseal the cut.  This process needs ATP for energy  DNA REPLICATION Enzymes involved in DNA Replication 4. Primase ▪ Primase is able to synthesize primers using free NTPs as the substrate and the ssDNA as the template. ▪ Primers are short RNA fragments of a several decades of nucleotides long. ▪ Primers provide free 3´-OH groups to react with the α-P atom of dNTP to form phosphodiester bonds 5. Ligase ▪ It seals nicks in double stranded DNA (dsDNA) where a 3’-OH and a 5’-phosphate stand side by side. ▪ It is responsible for joining Okazaki fragments together to make the lagging strand a contiguous chain ▪ It can only ligate a nick at one of double strands of DNA, but cannot ligate two single strands to one strand of DNA ▪ Connect two adjacent ssDNA strands by joining the 3´-OH of one DNA strand to the 5´-P of another DNA strand. ▪ Sealing the nick in the process of replication, repairing, recombination, and splicing.  DNA REPLICATION Enzymes involved in DNA Replication 6. Single Stranded Binding (SSB) Protein SSB protein maintains the DNA template in the single strand form in order to:  Prevent the unwound DNA strands at replication forks from re-annealing during replication.  Protect the vulnerable ssDNA from nucleases. 7. DNA Gyrase  Gyrase cut phosphodiester bonds on both strands of the double stranded DNA (dsDNA), release the supercoil constraint and reforms the phosphodiester bonds  Can change the dsDNA into negative supercoil state with consumption of ATP.  DNA REPLICATION ENZYME FUNCTIONIN DNA REPLICATION DNA helicase A helix destabilizing protein that unwinds a double helix at replication forks DNA polymerase Builds new double-stranded DNA, adding deoxy-nucleotides 5’-3’; some can proofread and correct errors DNA clamp protein Prevents DNA pol III from separating from the parent template strand. Single-strand binding proteins (SSBs) Keep unwound DNA strands at replication forks from re-annealing during replication topoisomerases Relax supercoiled DNA caused by DNA unwinding during replication DNA gyrases A specific kind of topoisomerase DNA ligase Joins Okazaki Fragments to growing DNA strands during replication primase Initiates replication using nucleotides to synthesize an RNA primer required for DNA polymerases to then add deoxynucleotides telomerase Enzyme that adds repetitive DNA sequences to telomeric DNA to maintain the length of eukaryotic chromosomal DNA  DNA REPLICATION Steps and Proteins Involved in Prokaryotic and Eukaryotic DNA Replication Step in Replication Prokaryotic Cells Eukaryotic Cells Bridge to Pharmacology Recognition of origin of replication dna A protein Unknown  Quinolones and DNA Gyrase Unwinding of DNA double helix Helicase (requires ATP) Helicase (requires ATP)  Quinolones and Fluoroquinolones inhibits DNA Stabilization of unwound template Single-stranded DNA- Single-stranded DNA- gyrase(prokaryotic strands binding protein (SSB) binding protein (SSB) Topoisomerase II), preventing DNA replication and Synthesis of RNA primers Primase Primase Transcription.  These drugs, which are most Synthesis of DNA: DNA polymerase III DNA polymerase δ active against aerobic gram- Leading strand Lagging strand polymerase III DNA polymerase α negative bacteria include: (Okazaki fragments). Nalidixic acid. Ciprofloxacin.Norfloxacin Removal of RNA primers DNA polymerase I (5ʹ→ 3ʹ Unknown exonuclease)  Resistance to the drugs has Replacement of RNA with DNA DNA polymerase I Unknown developed overtime; current Joining of Okazaki fragments DNA ligase (requires NAD) DNA ligase (requires ATP) uses include treatment of gonorrhea and upper and Lower urinary tract infections in Removal of positive supercoils DNA topoisomerase II DNA topoisomerase II both sexes. ahead (DNA gyrase) of advancing replication forks Synthesis of telomeres Not required Telomerase  DNA REPLICATION Summary:  DNA polymerases catalyze the synthesis of DNA.  The reaction involves a nucleophilic attack by the 3'-hydroxyl group on the innermost phosphorous atom of the nucleotide triphosphate. Pyrophosphate is the leaving group.  The synthesis reaction occurs in the 5'→3‘ direction - new bases are added at the 3' end of the growing chain.  DNA polymerase I has three activities:  5‘→3' DNA synthesis  3‘→5' exonuclease (Used as an error corrector to check the last base of the chain to ensure accuracy).  5‘→3' nuclease (used to remove bases (especially the RNA primer) ahead of synthesis occurring in the same direction).  DNA synthesis occurs at replication forks. Synthesis begins at an origin of replication and proceeds in a bidirectional manner. 65  DNA REPLICATION Summary:  DNA polymerase III is the primary enzyme for DNA replication in E. coli.  Leading the synthesis is the helicase enzyme, which unwinds the DNA strands. This introduces positive supercoils into the DNA which must be relieved by DNA gyrase. The single stranded DNA is protected by binding to a single stranded binding protein.  A primase synthesizes a short strand of RNA (about 5 nucleotides), because DNA polymerase requires a primer annealed to the template strand.  The polymerase proceeds down the helix, directly synthesizing one strand in the 5'→3‘ direction - the leading strand. The other strand loops around and through the polymerase, and is synthesized in short, Okazaki fragments in the 5'→3' direction - the lagging strand.  DNA polymerase I removes the RNA primers from the Okazaki fragments, replacing them with DNA.  DNA ligase seals the breaks that are left after DNA polymerase I finishes. 66 References  Janet Iwasa and Wallace Marshall. 2016. Karp’s Cell and Molecular Biology: Concepts and Experiments, 8th Edn., Wiley.  Madigan, M. T., Martinko, J. M., Bender, K. S., Buckley, D. H., & Stahl, D. A. (2015). Brock biology of microorganisms (Fourteenth edition.). Boston: Pearson.  https://en.wikipedia.org/wiki/Proofreading_(biology)  https://sciencing.com/comparing-contrasting-dna-replication-prokaryotes-eukaryotes-13739.html  Reverse transcriptase (RT)-PCR: Principles, Applications Microbe Online  Reverse Transcription PCR: Principle, Procedure, Protocol, Advantages, Limitations, Applications (geneticeducation.co.in) THANK YOU 68