DNA REPLICATION

DNA copying Each cell division cell must copy its entire DNA So each daughter cell gets a complete copy Rate of synthesis Bacteria = 1000 bases per second Mammals = 100 bases per second Problem - with a single replication origin in DNA Bacteria genome is 4 x 10E6. Takes 20 minutes to copy. Human is 3.2 x 10E9. Would take 10,000 times longer.

Chromosomes Chromosomes Genes Strands of DNA that contain all of the genes an organism needs to survive and reproduce Genes Segments of DNA that specify how to build a protein genes may specify more than one protein in eukaryotes Chromosome maps are used to show the locus (location) of genes on a chromosome The E. Coli genome includes approximately 4,000 genes

Chromosomes Genetic Variation Phenotypic variation among organisms is due to genotypic variation (differences in the sequence of their DNA bases) Differences exist between species and within a species Different genes (genomes)  different proteins (proteomes) Different versions of the same gene (alleles) Differences in gene expression (epigenetics)

DNA copying solutions Double helix has to be copied Semi-conservation solution exists – Each daughter cells gets one of the original copies

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DNA copying solutions Double helix has to be copied Semi-conservation solution exists – Each daughter cells gets one of the original copies Unwind at one point and use that as the origin of replication

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DNA copying solutions Double helix has to be copied Semi-conservation solution exists – Each daughter cells gets one of the original copies Unwind at one point and use that as the origin of replication Region is AT rich to allow easy separation Eukaryotes have multiple replication origins (Humans = 10,000)

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Schematic 1

Schematic 2 The addition of dNTPs to the growing DNA strand

Schematic 3

DNA Replication The DNA is copied by a replication machine that travels along each replication fork. One of the most important members of this complex is DNA POLYMERASE It is able to add DNA subunits, making new DNA

DNA Polymerase First discovered in 1956 by Kornberg Bacteria E.coli Bacteria have 3 types DNA Pol I, II, and III DNA Pol III involved in replication of DNA DNA Pol I involved in repair Humans have 4 types (you need to know, now) DNA Pol alpha, beta, delta - nuclear DNA DNA Pol gamma - mitochondrial DNA

In prokaryotes, there are three main types of DNA polymerase Polymerase Polymerization (5’-3’) Exonuclease (3’-5’) Exonuclease (5’-3’) #Copies I Yes 400 II No ? III 10-20 •3’ to 5’ exonuclease activity = ability to remove nucleotides from the 3’ end of the chain •Important proofreading ability •Without proofreading error rate (mutation rate) is 1 x 10-6 •With proofreading error rate is 1 x 10-9 (1000-fold decrease) •5’ to 3’ exonuclease activity functions in DNA replication & repair.

Eukaryotic enzymes: Five common DNA polymerases from mammals. Polymerase  (alpha): nuclear, DNA replication, no proofreading Polymerase  (beta): nuclear, DNA repair, no proofreading Polymerase  (gamma): mitochondria, DNA repl., proofreading Polymerase  (delta): nuclear, DNA replication, proofreading Polymerase  (epsilon): nuclear, DNA repair (?), proofreading Different polymerases for the nucleus and mtDNA Some polymerases proofread; others do not. Some polymerases used for replication; others for repair. Polymerases vary by species.

DNA Polymerase… ALL DNA Pol’s have 2 properties Only synthesize DNA in one direction 5’ to 3’ Only add to the end of existing double stranded DNA Therefore they CANNOT start synthesis of DNA from scratch. RNA polymerases can, but not DNA polymerases

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What happens at the other strand? 3’ to 5’ strand replication Solved by Reiji Okazaki He saw - one strand made in a continuous manner (leading strand) and the other from short discontinuous pieces (lagging strand) The discontinuous pieces are known as Okazaki fragments

Trombone model

DNA ligase seals the gaps between Okazaki fragments with a phosphodiester bond (Fig. 3.7)

Okazaki Growth RNA is added by Primase (3 - 10 bases) DNA is added by DNA Pol alpha Primase can start RNA synthesis de novo We have a RNA/DNA joint, so RNA is involved in eukaryote DNA replication

Closing the gap between Okazaki fragments RNA primer removed by RNase H DNA Pol delta fills the gap DNA Ligase - the gap closer

Stabilization of the replication machine DNA Pol delta and epsilon are attached to the DNA and held in place by other proteins Sliding-clamp proteins (proliferating cell nuclear antigen - PCNA), allow the stable binding of DNA Pol and strand synthesis Clamp-loading proteins (replication factor C - RFC), aid in attaching the sliding-clamp proteins.

DNA Pol loading

DNA Pol… …more facts Has a proofreading mechanism built in Checks for base matching Removes mismatched bases by going backwards Reason why it is not able to build DNA in the 3’ to 5’ direction. No energy from ATP hydrolysis. Makes just one error in 10E8 or 10E9 (billion) bases added

Other helpers Unwinding of DNA strands by Helicases Require ATP Single-stranded DNA-binding proteins Bind to single stranded DNA to stabilize structure RPA (replication protein A - in eukaryotes) Topoisomerase - helps with prevention of DNA strand twisting - ‘swivels’ Two types Type I - Break one strand only and then rejoin Type II - Break both strands and then rejoin

Protein aids

Overview

DNA replication in eukaryotes: Copying each eukaryotic chromosome during the S phase of the cell cycle presents some challenges: Major checkpoints in the system Cells must be large enough, and the environment favorable. Cell will not enter the mitotic phase unless all the DNA has replicated. Chromosomes also must be attached to the mitotic spindle for mitosis to complete. Checkpoints in the system include proteins call cyclins and enzymes called cyclin-dependent kinases (Cdks). Kinases are enzymes that transfer phosphate groups from donor molecules such as ATP to specific substrates by the process of phophorylation. Important for cell signaling and protein regulation.

Each eukaryotic chromosome is one linear DNA double helix Average ~108 base pairs long With a replication rate of 2 kb/minute, replicating one human chromosome would require ~35 days. Solution ---> DNA replication initiates at many different sites simultaneously. Rates are cell specific! Fig. 3.14

Fig. 3.13 - Replication forks visible in Drosophila

Replication of circular DNA in E. coli (3.10): Two replication forks result in a theta-like () structure. As strands separate, positive supercoils form elsewhere in the molecule. Topoisomerases relieve tensions in the supercoils, allowing the DNA to continue to separate.

Rolling circle model of DNA replication (3.11): Common in several bacteriophages including . Begins with a nick at the origin of replication. 5’ end of the molecule is displaced and acts as primer for DNA synthesis. Can result in a DNA molecule many multiples of the genome length (and make multiple copies quickly). During viral assembly the DNA is cut into individual viral chromosomes.

What about the ends (or telomeres) of linear chromosomes? DNA polymerase/ligase cannot fill gap at end of chromosome after RNA primer is removed. If this gap is not filled, chromosomes would become shorter each round of replication! Solution: Eukaryotes have tandemly repeated sequences at the ends of their chromosomes. Telomerase (composed of protein and RNA complementary to the telomere repeat) binds to the terminal telomere repeat and catalyzes the addition of of new repeats. Compensates by lengthening the chromosome. Absence or mutation of telomerase activity results in chromosome shortening and limited cell division.