Decoding the Genetic Code Replication RNA Synthesis Decoding the Genetic Code

DNA – an emblem of the 20th century. A simple yet elegant structure – a double helix with a sugar phosphate “backbone” linked to 4 types of nucleotide on the inside that are paired according to basic rules. Amazingly this simple molecule has the capacity to specify Earth’s incredible biological diversity. The double-stranded structure suggests a mode of copying (replication) and the long “strings” of the 4 bases encode biological life. The human genome is just 3.5 billion base pairs and greater than 95% is considered to be non-coding (or “junk”). Consider the human genome as a 3.5 gigabyte hard drive filled with information of which greater than 95% is rubbish, corrupted etc.

These lectures cover the research that led to the elucidation of the replication (copying/ reproduction) of DNA, how DNA can generate protein products and the code that lies within DNA to generate proteins. Summary of Lecture 1 DNA replication is semi-conservative (Meselson-Stahl, 1958). Replication requires a DNA polymerase, a template, a primer and the 4 nucleotides and proceeds in a 5’ to 3’ direction (Kornberg, 1957). Replication of the Escherichia coli genome (a single circular DNA) starts at a specific site (ori) and is bi-directional (Cairns, 1963). Replication is semi-discontinuous (continuous on leading strand and discontinuous on lagging strand) and requires RNA primers (Okazaki’s, 1968). Lagging strand synthesis involves Okazaki fragments. DNA polymerase III is the replicative enzyme of E. coli (Cairns and deLucia, 1969).

Replication as a Process 1. Double-stranded DNA unwinds. 2. The junction of the unwound molecules is a replication fork. 3. A new strand is formed by pairing complementary bases with the old strand. 4. Two molecules are made. Each has one new and one old DNA strand.

DNA Replication is Semi-discontinuous Continuous synthesis Discontinuous synthesis

Lecture 2 Outline The replicative enzyme, DNA polymerase III Evidence that RNA primers are required for replication. Additional features of the replication process. The central dogma in biology: DNA RNA Protein. Transcription.

DNA SYNTHEIS REACTION products 5' end of strand P P CH2 Base CH2 Base + P 3' P P Phosphodiester bonds OH P P Synthesis reaction CH2 Base P O 5' CH2 Base O OH 3' 3' end of strand OH

The subunits of E. coli DNA polymerase III Function Holoenzyme a e q t b g d d’ c y 5’ to 3’ polymerizing activity 3’ to 5’ exonuclease activity a and e assembly (scaffold) Assembly of holoenzyme on DNA Sliding clamp = processivity factor Clamp-loading complex Core Enzyme dimer

DNA Must Be “Primed” Before DNA Polymerase Can Replicate It DNA polymerase cannot initiate polymerisation de novo. Okazaki and colleagues provided evidence for short stretches of RNA linked to nascent chains of DNA during replication. Sugino et al., (1972) isolated Okazaki fragments after pulsing with 3H-U (incorporates into RNA and not DNA) and found it associated with newly replicated DNA. Initiation of DNA replication, but not continuation, was shown to be sensitive to rifampicin (an antibiotic that inhibits RNA polymerases). In follow up experiments Sugino et al., (1973) isolated Okazaki fragments after a short pulse (3H-dT) by banding on a CsCl gradient. Treatment of the Okazaki fragments with alkali (hydrolyses RNA but not DNA) or ribonuclease resulted in a small shift in density (size).

Conclusions There is a covalent linkage between ribonucleotides and deoxyribonucleotides in the newly synthesised DNA. RNA fragments (10 to 20 nt) are located at the 5’ end of the nascent fragments and are required for priming de novo DNA synthesis. How is the DNA primed?

Primase: Makes initial nucleotide (RNA primer) to which DNA polymerase attaches New strand initiated by adding nucleotides to RNA primer RNA primer later replaced with DNA

Proteins Involved in DNA Replication in E. coli

Enzymes in DNA replication Primase adds short primer to template strand Helicase unwinds parental double helix Binding proteins stabilise separate strands DNA polymerase binds nucleotides to form new strands DNA polymerase I (Exonuclease) removes RNA primer and inserts the correct bases Ligase joins Okazaki fragments and seals other nicks in sugar-phosphate backbone

Replication Helicase protein binds to DNA sequences called Primase protein makes a short segment of RNA complementary to the DNA, a primer. 5’ 3’ Binding proteins prevent single strands from rewinding. 3’ 5’ Helicase protein binds to DNA sequences called origins and unwinds DNA strands.

Replication DNA polymerase enzyme adds DNA nucleotides Overall direction of replication 5’ 3’ DNA polymerase enzyme adds DNA nucleotides to the RNA primer.

Replication DNA polymerase enzyme adds DNA nucleotides 5’ Overall direction of replication 3’ DNA polymerase enzyme adds DNA nucleotides to the RNA primer. DNA polymerase proofreads bases added and replaces incorrect nucleotides.

Replication Leading strand synthesis continues in a 5’ 3’ Overall direction of replication Leading strand synthesis continues in a 5’ to 3’ direction.

Replication Leading strand synthesis continues in a 3’ 5’ Overall direction of replication Okazaki fragment Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

Replication Leading strand synthesis continues in a Overall direction of replication 3’ 3’ 5’ 5’ Okazaki fragment 3’ 5’ 3’ 5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

Replication Leading strand synthesis continues in a 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

Replication Leading strand synthesis continues in a 5’ 3’ 3’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

Replication 3’ 5’ 5’ 5’ 3’ Exonuclease activity of DNA polymerase I removes RNA primers.

Replication Polymerase activity of DNA polymerase I fills the gaps. 3’ 5’ Polymerase activity of DNA polymerase I fills the gaps. Ligase forms bonds between sugar-phosphate backbone.

DNA REPLICATION 3 Pol III synthesises leading strand 2 1 Helicase opens helix Topoisomerase nicks DNA to relieve tension from unwinding 4 Primase synthesises RNA primer 5 Pol I excises RNA primer; fills gap 6 7 Pol III elongates primer; produces Okazaki fragment DNA ligase links Okazaki fragments to form continuous strand

DNA Synthesis •Synthesis on leading and lagging strands •Proofreading and error correction during DNA replication •Simultaneous replication occurs via looping of lagging strand

Simultaneous Replication Occurs via Looping of the Lagging Strand •Helicase unwinds helix •SSBPs prevent closure •DNA gyrase reduces tension •Association of core polymerase with template •DNA synthesis •Not shown: pol I, ligase

Replication Termination of the Bacterial Chromosome ori ter Origin 5’ 3’ BIDIRECTIONAL REPLICATION

Procaryotic (Bacterial) Chromosome Replication ori ter Replication Forks Bidirectional Replication Produces a Theta Intermediate

Replication Termination of the Bacterial Chromosome Termination: meeting of two replication forks and the completion of daughter chromosomes Region 180o from ori contains replication fork traps: ori Ter sites Chromosome

Replication Termination of the Bacterial Chromosome TerA TerB One set of Ter sites arrest DNA forks progressing in the clockwise direction, a second set arrests forks in the counterclockwise direction: Chromosome

Replication Termination of the Bacterial Chromosome Ter sites are binding sites for the Tus protein Tus: 35.8 kD DNA binding at Ter Monomer Tus DNA Ter Replication fork arrested in polar manner Tus may inhibit replication fork progression by directly contacting DnaB helicase, inhibiting DNA unwinding

Summary DNA replication proteins: DNA PolIII DNA PolI DNA Ligase Primase (DnaG) Helicase (DnaB) SSB Replication termination Replication fork traps opposite oriC Ter sites Tus protein