DNA Repair and Viruses

Repair of Damaged DNA

• Modified nucleobases lead to base substitutions

  • Glycosylase removes oxidized nucleobase.

  • Another enzyme cuts DNA at this site

  • DNA polymerase removes short section and synthesizes replacement.

  • DNA ligase seals gap.

Repair of Thymine Dimers

• Several methods to repair damage from UV light.

  • Photoreactivation (light repair):

    • Enzyme uses energy from light.

    • Breaks covalent bonds of thymine dimer.

    • Not found in placental mammals.

  • Excision repair (dark repair):

    • Enzyme removes damage.

    • DNA polymerase and DNA ligase repair.

SOS Repair

• Last-ditch repair mechanism found in some prokaryotes.

  • Induced following extensive DNA damage.

  • Photoreactivation and excision repair are unable to correct.

  • DNA and RNA polymerases stall at unrepaired sites.

  • Several dozen genes in SOS system activated.

  • Includes a DNA polymerase that synthesizes even in extensively damaged regions.

Repair Mechanisms (Table 8.2 Summary)

Cancer and DNA Repair

• Cancers are often the result of mutations in DNA repair genes.

  • Xeroderma pigmentosum (XP): Autosomal recessive disease characterized by susceptibility to UV-induced skin cancer. Genes XPA through XPG are involved in nucleotide excision repair.

  • BRCA1 and BRCA2: Genes associated with human breast cancer, involved in the repair of double-stranded breaks in DNA.

Nobel Prize in Chemistry (2015)

• "For mechanistic studies of DNA repair."

  • Base excision repair

  • Nucleotide excision repair

  • Mismatch repair

• Lindahl: Funded by the MRC in the UK.

• Paul Modrich: NIH and Hughes funding.

• Aziz Sancar: NIH and NATO.

General Characteristics of Viruses

• Why should you care about viruses?

  • Leads to significant mortality worldwide.

  • Every major pandemic since 1900 (excluding cholera) is caused by a virus.

  • Viruses are ubiquitous: Estimated 10^{31} viruses on the planet, mostly infecting prokaryotes.

  • Viruses have shaped life: About 8% of the human genome is viral in origin.

Composition and Structure

• Viruses consist of genetic information (DNA or RNA), proteins, and optionally a membrane.

  • Genetic information (DNA or RNA) contained within a protective coat.

  • May optionally contain an enzyme or be enclosed within a membrane.

  • Inert particles: No metabolism, replication, or motility.

  • Infectious agents, not organisms.

  • Classified based on the type of cell they infect (eukaryotic or prokaryotic).

  • Bacteriophages (phages) infect prokaryotes.

Size

• Most viruses are notable for their small size.

  • Smallest: ~10 nm, ~10 genes

  • Largest: ~800 nm

  • Typical: 10s of nm, handfuls to a few 100 genes

Virion Structure

• Virion: An infectious virus particle.

  • Contains nucleic acid inside a protein coat and optionally a membrane.

  • Protein coat is the capsid, protects nucleic acids, composed of capsomeres, and carries enzymes when required.

  • Capsid plus nucleic acids is called the nucleocapsid.

Enveloped vs. Non-Enveloped Viruses

• Virion (viral particle) is nucleic acid, protein coat + membrane.

  • Enveloped viruses have lipid bilayer envelope; matrix protein is usually present between nucleocapsid and envelope.

  • Non-enveloped (naked) viruses lack envelope; more resistant to disinfectants.

Viral Genomes

• Viral genome is either DNA or RNA, never both.

  • Is part of the classification (DNA or RNA viruses).

  • Genomes may be linear or circular.

  • Double- or single-stranded.

Viral Attachment Mechanisms

• Viruses have protein components for attachment.

  • Phages have tail fibers.

  • Many animal viruses have spikes.

  • Allow virion to attach to specific receptor sites.

Viral Shapes

• General shapes:

  1. Icosahedral (20-faced polyhedron)

  2. Helical

  3. Complex (everything else)

Viral Nomenclature

• Virus families end in suffix -viridae.

  • Names follow no consistent pattern.

    • Some indicate appearance (e.g., Coronaviridae from corona, meaning “crown”).

    • Others named for geographic area (e.g., Bunyaviridae from Bunyamwera in Uganda, Africa; Norovirus after an outbreak in Norwalk, Ohio).

  • Genus ends in -virus (e.g., Enterovirus).

  • Species name often name of disease (e.g., poliovirus causes poliomyelitis).

  • Viruses commonly referred to only by species name.

Viral Replication

• Viruses lack their own "replication machine."

  • Require host cell to replicate.

  • Viruses are a piece of genetic code surrounded by proteins and optionally a membrane and/or required enzymes inside.

General Virus Life Cycle

• Viral particle (virion) interacts with host cell.

  • Nucleic acid enters host, host machinery copies it, capsid proteins are made, and virus is assembled.

  • If an enveloped virus, progeny are enveloped, the membrane comes from the host cell.

  • Release of many viral particles.

  • Enveloped viruses are more susceptible to drying and disinfectants since the envelope is damaged, and the envelope is required for attachment to the host.

  • Can result in productive infection vs. latent state.

  • Some viruses can integrate into the host genome.

Bacteriophages

• Bacteriophages: Viruses of bacteria.

  • Three general types based on relationship with host:

    • Lytic phages: Always have a lytic life cycle.

    • Temperate phages: Have both a lytic and a lysogenic life cycle.

    • Filamentous phages: chronic infection secreted from host cell without killing cell

Lytic Phage Infections

• T4 phage (dsDNA) as model:

  • The T4 phage genome encodes 289 proteins.

  • The phage is about 90nm wide by about 200nm long.

  • The T4 phage initiates an Escherichia coli infection by binding OmpC porin proteins and Lipopolysaccharide (LPS) on the surface of E. coli cells with its long tail fibers.

• Genome Entry

  • The tail contracts and phage DNA is injected into the bacterial cell, leaving the phage coat outside.

• During this phase:

  • The virus turns off host cell gene expression.

  • The virus takes over the host cell’s transcription and translation machinery.

  • Enzymes from the virus help to replicate the viral DNA; the host DNA is broken down for "supplies."

  • Virion components are synthesized.

  • The structural and assembly proteins are made after other proteins are made.

  • The average “burst size” is ~ 130 phages.

Lytic Phage Infections - Process

• Five-step process:

  1. Attachment

  2. Genome entry

  3. Synthesis

  4. Assembly

  5. Release

• Entire process takes ~30 minutes.

Temperate Phages

• Option of lytic infection or incorporation of DNA into host cell genome.

  • Lysogenic infection

  • Infected cell is lysogen

  • Lambda (λ) phage as model

    • The lytic life cycle is identical to what we just discussed assembled together.

    • In the lysogenic life cycle, viral DNA integrates into the host genome to form a prophage.

• Cell division copies the prophage as part of the bacterial genome during growth in high nutrient conditions. The prophage is excised about 1/10,000 cellular divisions.

Lysogenic Conversion

• Change in phenotype of lysogen caused by prophage.

  • e.g., toxins encoded by phage genes; only strains carrying prophage produce the toxins.

  • Repressor maintaining integrated prophage also binds to the operator on incoming phage DNA and prevents gene expression: immunity to superinfection.

Methods to study Bacteriophages

• Viruses multiply only inside living cells; must cultivate suitable host cells to grow viruses.

• Plaque assays used to count phage particles in samples: sewage, seawater, soil.

  • Soft agar inoculated with bacterial host and phage-containing specimen

  • Bacterial lawn forms

  • Zones of clearing from bacterial lysis are plaques

  • Plaque-forming unit (PFU) represents single phage

  • Counting plaques yields the titer, which is the concentration of phage in the original sample.

Bacterial Defense

• Bacterial protection from infection

  • Preventing phage attachment

  • Restriction enzymes and DNA modification systems

  • CRISPR-system

Preventing Phage Attachment

• Alter or cover specific receptors on the surface.

  • Lose receptor entirely.

  • Staphylococcus aureus produces protein A, which masks phage receptors and also protects against certain human host defenses.

  • Capsules, slime layers, and biofilms also mask receptors.

Restriction-Modification Systems

• Consist of two enzymes:

  • Restriction enzymes: Recognize and cut fairly short, specific DNA sequences (the restriction site). Some bacteria have multiple restriction-modification systems. Large numbers of systems across all bacterial species.

  • Modification enzymes: Methylate host (bacteria) sequences at the restriction site. Restriction enzymes don’t cut the methylated DNA (the host is protected). Occasionally, the DNA modification enzyme will methylate the phage DNA, and then it survives after entry.

Restriction-Modification outcome

• No phage Restriction enzyme degrades unmethylated DNA. Phage DNA not methylated = Phage DNA degraded, no phage replication.

• Restriction enzyme does not degrade methylated DNA. Phage DNA methylated after entry = Phage replication.

• Recognition of self vs non-self.

CRISPR System

• Phage spacer DNA inserted into CRISPR provides a record of infection.

• Transcribed, cut.

• Small RNAs bind to Cas (CRISPR-associated sequences) proteins.

• Binding of spacer RNA to the phage genome targets the phage genome for destruction.

• CRISPR system: Clusters of Regularly Interspersed Short Palindromic Repeats.

Animal Viruses Infection Cycle

• Five-step infection cycle – Animal viruses

  1. Attachment: *Viruses bind to receptors *Usually glycoproteins on cytoplasmic membrane *Often more than one required (for example, HIV binds to two)

     • The normal function of the receptors is unrelated to viral infection

     • Specific receptors required (tropism); limits the range of the virus

     • For example, dogs do not contract measles from humans

  2. Penetration and uncoating: fusion or endocytosis

     • Non-enveloped viruses cannot fuse

  3. Synthesis

     • Expression of viral genes to produce viral structural and catalytic genes (for example, capsid proteins, enzymes required for replication)

     • Synthesis of multiple copies of genome

     • Most DNA viruses multiply in the nucleus

     • Enter through nuclear pores following penetration

     • Three general replication strategies depending on type of genome of virus:

       • DNA viruses

       • RNA viruses

       • Reverse transcribing viruses

  4. Assembly

     • Protein capsid forms; genome, enzymes packaged

     • Takes place in the nucleus or in organelles of the cytoplasm

  5. Release

     • Most via budding

     • Viral protein spikes insert into the host cell membrane; matrix proteins accumulate; nucleocapsids extruded

     • Covered with matrix protein and lipid envelope

     • Some obtain envelope from organelles

     • Non-enveloped viruses released when the host cell dies, often by apoptosis initiated by virus or host

Viral Genomes - forms

• Viral genomes come in many forms

  • DNA

    • ssDNA

    • dsDNA

  • RNA

    • Single-stranded +RNA

    • Single-stranded –RNA

    • dsRNA

Replication of viruses

Replication of DNA Viruses

• Usually in the nucleus

  • Poxviruses are the exception: Replicate in the cytoplasm, encode all enzymes for DNA, RNA synthesis

  • dsDNA replication straightforward

  • ssDNA similar except complement first synthesized

• DNA-dependent DNA polymerase (usually the host cell’s DNA polymerase)

• Transcriptase – DNA-dependent RNA polymerase

Replication of RNA Viruses

• Majority single-stranded; replicate in cytoplasm

  • Require virally encoded RNA polymerase (replicase), which lacks proofreading, allows antigenic drift

  • ss (+) RNA used as mRNA

  • ss (–) RNA and dsRNA viruses carry replicase to synthesize (+) strand

• Replicase - RNA-dependent RNA polymerase

• Replicase comes with the virus

Replication of dsRNA Viruses

• Replicase enters host cell with dsRNA because host cell cannot translate dsRNA

• Replicase uses (−) RNA strand to produce (+) RNA strand

• (+) RNA strand can serve as mRNA to make viral proteins

• Replicase comes with the virus

Replication of Reverse-Transcribing Viruses (Retroviruses)

• Encodes reverse transcriptase (RT): makes DNA from RNA

• Can direct productive infection or remain latent

• Once genome is integrated, it cannot be eliminated
  *Reverse transcriptase – RNA-dependent DNA polymerase

Virus Replication Summary

• All types of viruses must replicate their genomes AND produce mRNA

  • Type I: dsDNA uses host transcriptase to make mRNA and host replication machinery to make the genome

  • Type II: ssDNA uses host replication machinery to make dsDNA and then host transcriptase to make mRNA

  • Type III: dsRNA uses viral RDRP to make +mRNA and to make –RNA ½ of the genome

  • Type IV: ss+RNA uses viral RDRP to make -RNA and then RDRP to make +mRNA genome

  • Type V: ss-RNA uses viral RDRP to make +mRNA and then RDRP to make -RNA genome

  • Type VI: ss+RNA uses viral reverse transcriptase to make -DNA, and the host replication machinery to make dsDNA

Review

• Viruses are obligate, intracellular parasites.

  • They require the host to replicate their genomes

  • Make their proteins

  • Provide an envelope (if enveloped)

• Viruses can be RNA or DNA viruses and ss or ds.

• The form of the genome is related to the mechanism of genome replication and what enzymes the viral genomes MUST code.

• You should know about all the various enzymes that replicate viral genomes

  • e.g., if I say RNA-dependent RNA polymerase, you should know what that means.