DNA Packaging and Replication Lecture Notes PDF

DNA packaging and Replication 1 Central Dogma DNA ---------→ RNA---------→Protein. This unidirectional flow equation represents the Central Dogma (fundamental law) of molecular biology. This is the mechanism whereby inherited information is used to create actual o...

DNA packaging and Replication 1 Central Dogma DNA ---------→ RNA---------→Protein. This unidirectional flow equation represents the Central Dogma (fundamental law) of molecular biology. This is the mechanism whereby inherited information is used to create actual objects, namely enzymes and structural proteins. DNA packaging 2 m/cell Very thin (2.0 nm), extremely fragile Diameter of nucleus = 5-10 mm DNA must be packaged to protect it, but must still be accessible to allow gene expression and cellular responsiveness Chromosomes Single DNA Molecule and associated proteins Karyotype Chromatin vs. Chromosomes The Normal Human Chromosomes Normal human cells contain 23 pairs of homologous chromosomes: i. 22 pairs of autosomes. ii. 1 pair of sex chromosomes. Autosomes are the same in males and females Sex chromosomes are: i. XX in females ii. XY in males. Both X are homologous. Y is much smaller than X and has only a few genes. ◼ To fit within a living cell, the DNA double helix must be extensively compacted into a 3-D conformation ◼ This is aided by DNA- binding proteins (as histone proteins) Chromatin is the complex of DNA and proteins that comprise eukaryotic chromosomes. 2 classes of chromatin proteins: A) Histones and B) Non-histone proteins 1) Chromosome structure 4 core histones found in 2) Gene regulation proteins Nucleosomes - 146 bp of DNA wrapped around an octomer of 2 (H2A, H2B, H3, H4) HISTONES Main packaging proteins 5 classes: H1, H2A, H2B, H3, H4. Rich in Lysine and Arginine  Fig. 9 Orders of chromatin structure from naked DNA to chromatin to fully condensed chromosomes...  The chromosomal DNA is complexed with five types of histone. H1, H2A, H2B, H3 and H4. Histons are very basic proteins, rich in Arginine and Lysine. Nucleosomes: regular association of DNA with histones to form a structure effectively compacting DNA. ”beads” 11  Chromosome is a compact form of the DNA that readily fits inside the cell To protect DNA from damage DNA in a chromosome can be transmitted efficiently to both daughter cells during cell division Chromosome confers an overall organization to each molecule of DNA, which facilitates gene expression. 13 DNA Packaging DNA compaction must be dynamic, because changes in the degree of condensation must occur quickly and when needed, as the cell passes through the stages of the cell cycle. Furthermore, when in its most highly compacted form, DNA is not accessible to transcription or replication enzymes, so it must be able to rapidly expose regions containing genes that are required at any given moment, and then condense again. Modification enzymes that alter the state of DNA condensation, and can target their activity to specific regions of the chromosome that must be transcribed or replicated.  Two Major States of Chromatin from Dawson, Science 355, 1147 (2017) Heterochromatin is more compact than euchromatin and is largely transcriptionally silent Epigenetic regulators bind chromatin modifications to facilitate DNA templated processes  DNA Replication Process of duplication of the entire genome prior to cell division Biological significance Extreme accuracy of DNA replication is necessary in order to preserve the integrity of the genome in successive generations In eukaryotes , replication only occurs during the S phase of the cell cycle. Replication rate in eukaryotes is slower resulting in a higher fidelity/accuracy of replication in eukaryotes How Fast? Prokaryotic DNA polymerase can work at about 1000 bases per second. Eukaryotic DNA polymerase can work at about 50 bases per second. Basic rules of replication A. Semi-conservative B. Starts at the ‘origin’ C. Synthesis always in the 5-3’ direction D. Semi-discontinuous E. RNA primers required A.Semi-conservative replication: One strand of duplex passed on unchanged to each of the daughter cells. This 'conserved' strand acts as a template for the synthesis of a new, complementary strand by the enzyme DNA polymerase  B) Starts at origin Initiator proteins identify specific base sequences on DNA called sites of origin Prokaryotes – single origin site E.g E.coli - oriC Eukaryotes – multiple sites of origin (replicator) E.g. yeast - ARS (autonomously replicating sequences) Prokaryotes Eukaryotes DNA replication is initiated at many points in eukaryotic chromosomes. Called Replication Bubbles They will eventually all meet to form whole replicated strand  C)Direction of replication DNA polymerase add nucleotides only to the 3′ end of a growing strand. The replication can only go 5′→3′. In what direction does DNA replication occur? C) Synthesis is ALWAYS in the 5’-3’ direction What happens if a base mismatch occurs? Where does energy for addition of nucleotide come from? Why does DNA replication only occur in the 5’ to 3’ direction? Should be PPP here D) Semi-discontinuous replication Anti parallel strands replicated simultaneously ❑Leading strand synthesis continuously in 5’– 3’ ❑Lagging strand synthesis in fragments in 5’-3’ Semi-discontinuous replication New strand synthesis always in the 5’-3’ direction E) RNA primers required  Eukaryotic Replication Direction of replication: Leading strand: undergoes continuous replication Lagging strand: undergoes discontinuous replication Okazaki fragment: the discontinuously synthesized short DNA fragments forming the lagging strand DNA Replication Replication of the DNA molecule is semi-conservative, which means that each parent strand serves as a template for a new strand and that the two (2) new DNA molecules each have one old and one new strand. DNA replication requires: A strand of DNA to serve as a template Substrates - deoxyribonucleoside triphosphates (dATP, dGTP, dCTP, dTTP). DNA polymerase - an enzyme that brings the substrates to the DNA strand template A source of chemical energy to drive this synthesis reaction. Core proteins at the replication fork Topoisomerases - Prevents torsion by DNA breaks Helicases - separates 2 strands Primase - RNA primer synthesis Single strand - prevent reannealing binding proteins of single strands DNA polymerase - synthesis of new strand DNA ligase - seals nick via phosphodiester linkage The mechanism of DNA replication Arthur Kornberg, a Nobel prize winner and other biochemists deduced steps of replication Initiation Proteins bind to DNA and open up double helix Prepare DNA for complementary base pairing Elongation Proteins connect the correct sequences of nucleotides into a continuous new strand of DNA Termination Proteins release the replication complex When and where does DNA Replication take place? Synthesis Phase (S phase) S phase in interphase of the cell cycle. Nucleus of eukaryotes S DNA replication takes phase place in the S phase. G1 interphase G2 Mitosis -prophase -metaphase -anaphase -telophase  Enzyme Topoisomerase also called DNA gyrase Unwinds double helix Enzyme Enzyme DNA Enzyme Helicase: separates (breaking H-bonds) double helix at the replication fork Helicase DNA Helicase The enzyme is unwinding the chain and breaking the H-bonds between the complementary base pairs (A-T, G-C). Single strand binding proteins Stabilize the DNA strands as they are being replicated Prevents rejoining of DNA strands  Enzyme: Primase = the enzyme that makes RNA nucleotides into a primer RNA Primer Nucleotides for the starting point for DNA replication Short strands of RNA  Replication Forks Y-shaped regions of replicating DNA molecules where new strands are growing. DNA Polymerases DNA Polymerase I Cuts off RNA primers and fills in with DNA (between Okazaki fragments) –lagging strand Can proofread DNA Polymerase III Elongates the strand by adding DNA nucleotides on leading strand Also proofreads and corrects the DNA strand  Leading Strand Lagging Strand Template strand of DNA Other DNA strand Continuous addition of Forms short strands of Okazaki nitrogenous bases fragments (that will be joined in 5’ to 3’ direction later) in the 5’ to 3’ direction OKAZAKI FRAGMENTS The short strands of newly made DNA fragments on the lagging strand are called Okazaki fragments after the Japanese Biochemist Reiji Okazaki.  Enzyme: DNA Ligase a linking enzyme joins the strands Example: joining two Okazaki fragments together. DNA ligase Okazaki Fragment 1 Okazaki Fragment 2 5’ 3’ 3’ Lagging Strand 5’ All Together Now DNA Replication Nucleotides are always added to the growing strand at the 3' end (end with free -OH group). The hydroxyl group reacts with the phosphate group on the 5' C of the deoxyribose so the chain grows Energy is released when the bound linking 2 of the 3 phosphate groups to the deoxyribonucleoside triphosphate breaks Remaining phosphate group becomes part of the sugar-phosphate backbone Step 1 - Unwinding and Exposing Strands DNA strands are unwound and opened by enzymes called HELICASES Helicases act at specific places called ORIGINS OF REPLICATION Synthesis of new DNA strands proceeds in both directions from an origin of replication resulting in a bubble with REPLICATION FORKS at each growing point. Step 2 - Priming the Strand In order to begin making a new strand, a helper strand called a PRIMER is needed to start the strand. DNA polymerase, an enzyme, can then add nucleotides to the 3' end of the primer. Primer is a short, single strand of RNA (ribonucleic acid) and is complimentary to the DNA template strand. Primers are formed by enzymes called PRIMASES. Step 3 - Strand Elongation DNA Polymerase III catalyses elongation of new DNA strands in prokaryotes Two molecules of DNA polymerase III clamp together at the replication forks, each acting on 1 of the strands One strand exposed at its 3' end produces a daughter strand which elongates from its 5' to 3' end and is called the LEADING STRAND. This strand is synthesized continuously and grows from 5' to 3'. Step 3 - Strand Elongation The second daughter strand is called the LAGGING STRAND and is antiparallel to the leading strand. It’s template is exposed from the 5' to 3' end but it must direct the 5' to 3' synthesis of the lagging strands, since nucleotides are added at the 3' end of the chain. The lagging strand is constructed in small, backward directed bits consisting of discontinuous sections of 100-200 nucleotides in eukaryotes and 1000-2000 nucleotides in prokaryotes, called OKAZAKI FRAGMENTS. Step 3 - Strand Elongation When an Okazaki fragment forms: DNA polymerase I removes the RNA primer and replaces it with DNA adjacent to the fragment. leaving 1 bond between adjacent fragments missing. A second enzyme called a DNA LIGASE catalyses the formation of the final bond. Thank you