DNA Replication in Eukaryotes Lecture 4 PDF

Molecular Biology I BIO316 Lecture 4 DNA Replication in Eukaryotes Prepared by Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics ...

Molecular Biology I BIO316 Lecture 4 DNA Replication in Eukaryotes Prepared by Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli Theory of Replication Associate Professor of Biochemistry and Molecular Genetics  Most prokaryotes have a single circular chromosome, where DNA replication begins at a single origin of replication.  Eukaryotes typically contain linear chromosomes, where DNA replication begins at multiple origins of replication along each chromosome.  Eukaryotic cells generally have more DNA than prokaryotic cells.  Prokaryotic cells have a single circular chromosome, while eukaryotic cells typically contain multiple linear chromosomes.  DNA replication in prokaryotic cells starts at a single origin of replication.  DNA replication in eukaryotic cells starts at multiple origins.  Dr. Rami Elshazli Prokaryotic chromosome structure Associate Professor of Biochemistry and Molecular Genetics  Prokaryotic chromosomes are found in the nucleoid of prokaryotic cells.  They are circular in shape.  Prokaryotic cells don’t have a membrane-bound nucleus.  The genetic material of prokaryotes could be found in a region of the cytoplasm called the nucleoid.  A prokaryotic cell typically has only a single, coiled, circular chromosome.  There are a few prokaryotes that have more than one—Vibrio cholerae, the bacterium that causes cholera, has two circular chromosomes.  The nucleoid (meaning nucleus-like) is an irregularly- shaped region within the prokaryotic cells that contains most of the genetic material.  It is not surrounded by a nuclear membrane.  Prokaryotic chromosome structure  Prokaryotic cells have only one chromosome, but this chromosome must be condensed and supercoiled to fit inside a tiny space.  Most prokaryotic cells don’t use histones with DNA packing.  Prokaryotic DNA undergoes supercoiling, but it is not wound around histone clusters.  The folding of prokaryotic DNA is facilitated by nucleoid-associated proteins (NAPs) instead of histones.  NAPs are proteins within the nucleoid that can bind to the DNA molecule.  Genophore is the DNA of prokaryotic cells and usually referred to as the prokaryotic chromosome.  The term “chromosome” is misleading, because the genophore lacks chromatin. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Prokaryotic chromosome structure  Prokaryotic cells have only one chromosome, so they are classified as haploid cells (without paired chromosomes).  Many prokaryotes, such as bacteria, reproduce via binary fission; a method of asexual reproduction.  Prokaryotic cells also carry small molecules of DNA called plasmids.  Plasmids are small, circular DNA molecules that contain the cell’s nonessential genes.  Plasmids can occur in a variety of sizes and usually have a small number of genes.  The genetic material of plasmids is separate from that of the cell’s main chromosome, and they can replicate independently of the cell's chromosome. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Summary of prokaryotic replication Characteristic Eukaryotic DNA Prokaryotic DNA Shape Linear double helix Circular double helix  DNA replication is fundamental process occurring in all Size Large Small living organism and consists of three major steps: Number Multiple Single  Initiation step. Location Nucleus Nucleoid  Elongation step.  Termination step. Packed proteins Histones Nucleoid-associated proteins (A) Initiation step  DNA replication begins from origin.  In E coli, replication origin is called OriC which consists of 245 base pairs.  The binding of helicase is key step in replication initiation.  The activity of helicase causes the unwinding strand to form supercoiled DNA.  This supercoiling is relieved by the DNA topoisomerase (DNA gyrase). Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  (A) Initiation step  Single stranded binding protein binds to the separated strands to prevent reannealing of separated strands and stabilize the separated strands.  The DNA polymerase cannot initiate DNA replication.  DNA primase synthesize RNA primers along the 5’-3’ direction. (B) Elongation step  Leading strand synthesis:  The synthesis of leading strand is a straightforward process which begins with the synthesis of RNA primer by primase at replication origin.  DNA polymerase III then adds the nucleotides at the 3’end.  The synthesis of leading strand proceed continuously along the replication fork until it encounters the termination sequences. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  (B) Elongation step  Lagging strand synthesis:  The lagging strand synthesized in short fragments called Okazaki fragments.  RNA primer is synthesized by primase, then DNA polymerase III binds to RNA primer and adds nucleotides at the 5’-3’ direction.  Helicase and primase constitute a functional unit within replication complex called primosome.  DNA pol III uses one set of core subunit to synthesize leading strand and other set of core subunit to synthesize lagging strand.  When the Okazaki fragments synthesis is completed, the replication halted, and the core subunit dissociates from their sliding clamps and associates with new clamp.  This initiates the synthesis of new Okazaki fragments. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  (B) Elongation step  Both leading and lagging strand are synthesized within the 5’-3’ direction.  Both leading and lagging strand can be replicated at same time by two subunits of DNA polymerase III that move in the same direction.  Lagging strand synthesis is not completes until the RNA primer has been removed and the gap between adjacent Okazaki fragments are sealed.  The RNA primer are removed by exonuclease activity (5’-3’) of DNA polymerase I and replaced by DNA.  The gap is then sealed by DNA ligase using NAD+ as cofactor. (C) Termination step  The two-replication fork of circular E. coli chromosome meet at termination recognizing sequences (ter). Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli Summary of prokaryotic replication Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli  Eukaryotic Replication Associate Professor of Biochemistry and Molecular Genetics  Eukaryotic replication is a complicated process.  DNA replication in eukaryotes occur only in S-phase of cell cycle.  However, pre-initiation occur in G1 phase of the cell cycle.  Dr. Rami Elshazli  Eukaryotic Replication Associate Professor of Biochemistry and Molecular Genetics Prokaryotic Replication vs Eukaryotic Replication  Eukaryotic replication is a complicated process. Prokaryotic Replication Eukaryotic Replication  Multiple origins of replication for each chromosome It is a continuous process It is a discontinuous process at S phase were established. Circular, double-stranded DNA Linear, double-stranded DNA with ends  This process requires new enzymatic activities only for dealing with the ends of chromosomes. The DNA replicates in the cytoplasm The DNA replicates in the nucleus Single origin of replication Multiple origins of replication  The replication machinery of eukaryotes resemble that DNA polymerase I and III are involved DNA polymerase α, δ and ε are involved found in bacteria, but it is larger and more complex. β subunit sliding clamp PCNA sliding clamp  The initiation phase of replication requires more factors Large Okazaki fragments Small Okazaki fragments to assemble both helicase and primase complexes onto The process is rapid, 2000 base pairs per second The process is slow, 100 base pairs per second the template, then load the polymerase with its sliding clamp unit. Two circular chromosomes are obtained Two sister chromatids are obtained DNA gyrase is required Topoisomerase I & II are required  The eukaryotic primase makes short RNA primers, then extends these with DNA to produce the final primer.  The sliding clamp subunit that allows the enzyme  The main replication polymerase is also a complex of complex to stay attached to the template is called PCNA two different enzymes that work together. (proliferating cell nuclear antigen).  One is called DNA polymerase epsilon (Pol ε) and the  The structure of PCNA sliding clamp resembles the β other DNA polymerase delta (Pol δ). subunit sliding clamp. Features of Eukaryotic DNA Replication  Eukaryotic replication is bidirectional process and originates at multiple origins of each chromosome.  DNA replication represents a semiconservative model 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.  Eukaryotic replication takes place in the cell nucleus.  Synthesis occurs only in the 5′ to 3′direction.  DNA replication occurs in different directions, DNA polymerase Functions producing a leading strand and a lagging strand. DNA pol α (alpha) RNA primers – initiation of DNA synthesis  Lagging strands are created by the production of small DNA pol δ (delta) Lagging strand synthesis – repair - proofreading DNA fragments called Okazaki fragments that are DNA pol ε (epsilon) Leading strand synthesis – repair - proofreading eventually joined together. DNA pol γ (gamma) Mitochondrial DNA replication and repair DNA pol β (beta) Base-excision repair Dr. Rami Elshazli DNA pol η (eta), ζ (zeta), κ (kappa) Translesion DNA synthesis Associate Professor of Biochemistry and Molecular Genetics DNA pol θ (theta), λ (lambda), μ (mu) DNA repair  Dr. Rami Elshazli Enzymology for Eukaryotic DNA Replication Associate Professor of Biochemistry and Molecular Genetics  Helicases: Unwind the DNA helix at the start of Pol ε replication process.  SSB proteins: Bind to the single strands of unwound DNA to prevent reformation of the DNA helix during replication. Pol δ  DNA Polymerases:  Eukaryotic cells contain several different DNA polymerases; α, β, γ, δ and ε.  DNA replication is accomplished by a suite of three polymerases (Pol α, Pol δ, Pol ε).  Primase DNA polymerase α  DNA polymerase α carries a primase subunit and  DNA polymerase ε synthesizes RNA primers = DNA primase  DNA polymerase ε synthesizes the leading strand.  DNA polymerase δ  DNA polymerase γ replicates mitochondrial DNA.  DNA polymerase δ synthesizes the lagging strand, via Okazaki fragments  DNA polymerase β involved in base  It is the primary lagging strand replicase. excision of DNA nucleotides. Enzymology for Eukaryotic DNA Replication  RNase H and FEN1:  Ribonuclease H (RNase H) and Flap Endonuclease 1 (FEN1) are two enzymes that play critical roles in the removal of RNA primers during DNA replication in eukaryotes.  RNase H recognizes and removes the RNA primers that are paired with the DNA strand, leaving a small 5’ flap of RNA.  FEN1 is a 5’-endonuclease that cuts the "flap" structures of nucleic acids.  FEN1 cleaves the RNA flap structure left by RNase H, ensuring that only DNA is left in the newly synthesized strand.  DNA ligase I:  FEN1 has other roles in DNA repair and Okazaki  This enzyme has a role in sealing nicks in double-stranded fragment maturation on the lagging strand. DNA during DNA replication.  Then, DNA polymerase δ extends the DNA strand by  it joins short Okazaki fragments and forms a 3′-5′ adding nucleotides to the 3' end. phosphodiester bond between adjacent fragments of DNA. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics Enzymology for Eukaryotic DNA Replication  DNA topoisomerase:  In eukaryotes, topoisomerase I and II enzymes handle the relaxation of supercoils that form during replication.  These enzymes cut and re-ligate the DNA to relieve tension.  DNA topoisomerase I: Relaxes the DNA helix during replication through creation of a nick in one of the DNA strands.  DNA topoisomerase II: Relieves the strain on the DNA helix during replication through creation of nicks in both strands of DNA.  Telomerases:  This DNA polymerase contains an integral RNA that acts as its own primer.  It is used to replicate DNA at the ends of chromosomes Dr. Rami Elshazli (telomeres). Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli Steps of Eukaryotic Replication Associate Professor of Biochemistry and Molecular Genetics Pol ε  Replication of each linear DNA molecule starts bidirectionally and discontinuously at many origins.  At each origin, a replication bubble forms consisting of two replication forks. Pol δ  The helix is unwound by helicase to form a pair of replication forks.  The unwound helix is stabilized by SSB proteins and DNA topoisomerases.  The RNA primers are made by DNA polymerase α which carries a primase subunit.  The leading strand is synthesized continuously in the 5′  DNA polymerase α initiates synthesis of the lagging to 3′ direction while the lagging strand is synthesized strand, making first the RNA primer and then extending discontinuously in the 5′ to 3′ direction through the it with a short region of DNA. formation of Okazaki fragments.  DNA polymerase δ then synthesizes the rest of the  DNA ligase I seals the breaks between the Okazaki Okazaki fragment. fragments as well as around the primers to form  The leading strand is synthesized by DNA polymerase ε. continuous strands.  Dr. Rami Elshazli Summary of DNA replication Associate Professor of Biochemistry and Molecular Genetics Characteristic Prokaryotic DNA Eukaryotic DNA Location Found freely in the cytoplasm. Found within the nucleus. Occurrence Occurs as a covalent closed circular form of Occurs as a linear form of DNA with two ends. DNA. Introns Introns are absent in the coding region of DNA. Introns are present in the coding region of DNA. Nucleosome There is no formation of nucleosomes. There is a formation of nucleosomes. DNA Replication DNA replication occurs in the cytoplasm of the DNA replication occurs within the nucleus of the cell. cell. Number of Genes Prokaryotic DNA contains a small number of Eukaryotic DNA contains large number of genes. genes. Number of Chromosomes Prokaryotic DNA is organized into a single Eukaryotic DNA is organized into many chromosome. chromosomes. Histone Proteins Associated with nucleoid-associated proteins. Associated with histone proteins.  Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics