BIO417 Term test 2

Lectures 7-11

Semiconservative replication

New DNA has one parental strand (conserved) and one new strand

Polymerase ε

Continuous replication on the leading strand; 5’ → 3’

Polymerase δ

Replicates the lagging strand one short fragment at a time

Polymerase ⍺

Synthesizes RNA-DNA hybrid primers (contains a primase)

Single stranded DNA binding protein (SSBP)

Binds to unwound DNA to stabilize it during replication (replication protein A)

Sliding clamp

A ring-shaped protein complex that anchors DNA polymerase to the DNA template, preventing it from detaching (Proliferating cell nuclear antigen, PCNA)

CMG helicase

Unwinds the double helix during DNA replication

Polymerase erros

• Wrong nucleotide incorporated

• Extra or missing nucleotides (repetitive sequences) from replication slippage

Polymerase error solutions

• Proofreading from pol ε and δ

• Mismatch repair

Simultaneous DNA replication and transcription erros

• Encounters between replisome and transcriptional complexes

• R-loops → ssDNA, mutations, nicks, fork stalling or collapse, genomic instability

R-loops

Three stranded nucleic acid structures that form when the RNA transcript anneals to DNA template (CG rich region)

Simultaneous DNA replication and transcription error solutions

• Temporal separation (early replication genes are transcribed later in the S phase

• If transcribed throughout the S phase (e.g. rRNA genes), genes alternate between replication and transcription

  • Both don’t happen at the same time/place

Mismatch repair (MMR)

Repairs mispairing of nucleotides from tautomeric shifts, wobble, and strand slippage

Mispairing/wobble

• Slight shifts in the position of nucleotides in space

  • e.g. causes wobble between a normal T & normal G

• Non-tautomeric forms of bases → an additional proton on adenine causes a wobble in C:A

Strand slippage

A strand can loop out, causing an addition (newly synthesized strand loops out) or deletion (template strand loops out)

MMR genome maintenance mechanism

• Recognition of replication errors

• Cleaves newly synthesized strand

• Exonuclease removes area around error

• DNA polymerase fills gap and ligase seals break

• Highly conserved

Key components of MMR

• Mut proteins

  • MSH → MutS homolog complex, dimer

    • Associate with PCNA (sliding ring) and recruits:

  • MHL → MutL homolog complex (with endonuclease activity), dimer

  • Exonuclease (EXO1)

  • DNA polymerase

  • Ligase

MMR process

1. MutS (mutator S) and MutL homologs (MSH, MLH) initiate MMR

2. MSH complex recognizes mismatch

   1. Associates with PCNA (proliferating cell nuclear antigen)

3. MSH recruits MLH, w/ endonuclease activity

4. Newly synthesized strand is cleaved, region around mismatch is removed by EXO1

5. Gap filled by pol δ, nick sealed

Role of epigenetic marks in MMR

• H3K23me3 (active chromatin) → recruits MSH complex (MutS 𝛂)

  • Recruitment of MSH to protect actively transcribed genes from mutation (prior to replication, followed by scan of newly synthesized strand)

Base excision repair (BER)

• Removal of damaged or altered bases (non-bulky)

• Altered by oxidation, methylation, deamination

• Or excised by hydrolysis

Nucleotide excision repair (NER)

Repairs damage (chemicals, UV, radiation) that distorts DNA helix (pyrimidine dimers, DNA adducts); removal and re-synthesis of segments

Reactive oxygen species (ROS)

• Byproducts of metabolism that can cause chemical modifications to bases

• High reactivity → can cause damage as soon as it comes in contact w/ DNA

• Can cause mispairing during replication if not repaired

BER mechanism

• Constantly scan for errors

• Glycosylases initiate excision of damage

  • Specific glycosylases for specific types of altered bases

• Flips nucleotide out of helic, cleavage of abnormal bases

  • Creates apurinic or apyrimidinic sites (AP sites)

• Recognized by AP endonucleases

  • Excise sugar phosphate groups

• Addition of nuclotides:

  • Short patch (1 nucleotide) = Pol β

  • Long patch (2-10 nucleotides) = Pol ε/δ

• Ligase seals nick

Glycosylases

Recognize and remove specific damaged or inappropriate bases

NER pathways

• GG-NER (global genome NER)

• TC-NER (transcription coupled NER)

Global genome NER (GG-NER)

• Checks entire genome (actively transcribed and silent)

• Ex) Xeroderma pigmentosum (XP)

Transcription coupled NER (TC-NER)

• Removes blocks - RNA pol elongation

• Ex) Cockayne syndrome (CS), XP

GG- and TC-NER…

Share components, but differ in the way damage is recognized

GG-NER damage recognition

XPC protein → recognizes damage anywhere in the genome

TC-NER damage recognition

Recognized by pol II stalling

NER pathway

• Recruitment of TFIIH complex (SU: XPB, XPD: helicases, associated XPA)

• Unwinding

• Cut by endonucleases (XPG, XPF):

  • 25-30 nucleotides excised (excises region around damage)

• DNA synthesis (pol δ/ε)

• Ligation to seal nick

• Role for DICER in chromatin decondenzation

Xeroderma pigmentosum (XP)

• Hypersensitivity to sunlight (UV, eyes, skin)

• Autosomal recessive

• Dry skin, freckles, skin coloring

• Predisposition to cancer (skin and other types of cancer)

• Development of neurological abnormalities

• XPA-G, V

Cockayne syndrome (CS)

• Neurodegenerative disease

• No predisposition to cancer

• Premature aging, growth deficiency

• Photosensitivity

• XPB (D, G), CSA-B

NER and chromatin

• DNA damage, histone mods, and NER are interlinked

  • Ubiquiten ligase recruited to DNA damage → H2AUb

  • Attracts ZRF1 (Zutoin-related factor 1)

  • HMT recruited to damage in DICER-dependent manner → H4K20me2

  • Recruitment of XPA

Double strand breaks

• Can arise from replication over SS DNA nicks

  • SS nicks are transient during replication or repair paths

• Deleterious consequences: cytotoxic damage

• Initiate damage response pathways

  • Can arrest cell cycle (G1/S, S, G2/M)

• Involve master protein kinases: ATR, DNA-PK, ATM

SSB induced by

• DNA replication errors → stalled replication forks

• Ionizing radiation

• ROS (reactive oxygen species)

• BER

SSB can lead to

• DSB (dividing cels)

• RNA pol stalling and transcriptional defects (non-dividing cells)

• Important to delay replication to allow time to repair before they become DSBs

Single strand break repair (SSBR)

• SSBs detected by poly (ADP ribose) polymerase 1 (PARP-1) - first responder

• Recruits a “scaffold” protein (X-ray cross complementing group 1 (XRCC1))

• XRCC1 interacts w/ various proteions

• Next steps: can use BER factors

Double strand break repair (DSBR) pathways

NHEJ and HR

Non-homologous end joining (NHEJ)

• Major pathway for DSBs

• Direct ligation of broken ends

• Mostly G1, but throughout cell cycle

• Faster and less accurate (error prone), straight forward re-ligation

Homologous recombination (HR)

• Uses sister chromatid or homologous chromosome as template

• Mostly S, G2 phase (sister chromatids available)

• Transcribed genes

• Usually error free

NHEJ steps

1. Break detection by Ku heterodimers

2. Processing of broken ends

   1. Ku recruits DNA-PK (DNA-dependent protein kinase)

   2. Complex protects and aligns broken ends

   3. Platform for DNA repair enzymes

   4. End processing enzymes (depending on damage), e.g.:

      1. DNA cross-link repair 1C (5’-3’ exonuclease), ss overhang cleavage

      2. DNA pol µ and λ (fill in gaps)

      3. Polynucleotide kinase (PNK) (3’ phosphatase, 5’ kinase activity)

3. End ligation

   1. Ku recruits XRCC4 (X-ray repair cross complementing 4) and XLF protein

   2. Targets DNA ligase VI to broke ends

If DSB recognized by MRN…

HR pathway intiated

Key characteristics of HR

• One strand is cut; MRN endonucleases function (and EXO1 and helicase)

• Long 3’ ssDNA (longer) → invade homologous SNA mediated by recombination proteins

• Formation of Holliday junction: need to be resolved or dissolved

• Involves BRCA1 and BRCA2

HR pathway

• MRN complex detects DSB

• MRN (+ EXO1 and helicase) → end procesing to generate ss tail: end resection

  • Processing of ends to create overhang

• ssDNA tail covered by strand exchange protein: Rad51 (recA-type): filament for strand invasion

  • Stimulated by BRCA1 and BRCA2

  • D-loop → to invade homologous DNA strand

• Formation of Holliday junction (4 connected helices, pol δ)

  • Or non-crossover synthesis-dependent strand annealing (SDSA)

• Resolvase, dissolution

BRCA2 mutations

• Germline mutations are heritable

• Large “founder” effect in well-defined population

  • Cna theoretically be traced back to common ancestor

• Iceland: single BRCA2 (999del5, frameshift, truncated form) accounts for almost all breast/ovarian cancer families

  • 0.6% of gen population

  • Incomplete penetrance

5-methylcytosine

• Transposable element silencing

• Genome integrity and stability

• Regulation of gene expression

• Gene silencing

• Protection of gene expression (gbM, moderately expressed, conserved genes)

Maintenance of DNA methylation

• Plants = MET1

• Mammals = DNMT1

• Enzymes associate with PCNA/UHRF1 (ubiquitin-like with PHD and RING finger domains 1)

• At replication fork

• High affinity for hemimethylated DNA

Nucleosome removal at replication fork

• H2A-H2B dimers removed before H3-H4 dimers

  • Replaced in opposite order

• Synthesis of new histones required

  • Old and new histones associate with both strands

• FACT (facilitates chromatin transcription) and ASF1 (anti-silencing function 1) help with disassembly

  • FACT re-establishes nucleosomes behind replication fork

  • + other factors (CAF-1, Nap-1)

BER overview

• Cause: ROS, X-rays, alkylating agents, spontaneous reactions

• DNA damage: Oxidation, uracil, abasic site, SSB

• Molecular components: DNA glycosylases, PARP 1, ATR kinase

MMR overview

• Cause: Replication errors

• DNA damage: A-G mismatch, T-C mismatch, insertion, deletion

• Molecular components: MSH, MSL, pol δ

NER (GG- and TC-NER) overview

• Cause: UV, light, polycyclic aromatic, hydrocarbons

• DNA damage: Bulky adducts, intrastrand crosslink

• Molecular components: XPC or pol II stalling; TFIIH

DSBR (NHEJ and NR) overview

• Cause: X-rays, ionizing radiation, anti-tumor agents

• DNA damage: DSB, interstrand crosslink

• Molecular components: NHEJ = Ku dimer + DNA-PK; HR = MRN complex, ATM kinase, Rad51, BRCA 1/2, Holliday junction

Repair pathways most active during G0/G1

NHEJ

Repair pathways most active during S phase

BER, MMR, TC-NER, HR

Repair pathways most active during G2

HR

Microirradiation experiments

• Cell line

• UV - micro laser beam to cause localized damage in G1

• Pulse labeling with ³ H-thymidine (radioactive isotope)

• Detect “unscheduled” DNA-synthesis

• Subsequent mitosis

• With autoradiography

• Determined that the interphase nucleus is organized instead of intermized

Hybrid cell line and in situ hybridization

• Provided further evidence of the organization of interphase nucleus

• Selective detection of X chromosome only

• Hybrid cell line (hamster x man hybrid; all human chrs usually lost)

• Contains active human X chromosome as the only free human chromosome

  • Cells with no X chromosome are lost

• Metaphase plate and two interphase nuclei

• In situ hybridization with ³H-labeled human genomic data

FISH as evidence for chromosome organization

• Pancentromeric probe (green)

• Telomeric probe (red)

• DNA counterstained (blue)

• HC11 (mouse mammary epithelium)

Chromosome territories (CTs)

• Positioning: non-random (size, gene density)

• Probabilistic, depending on tissue

• Predominant configuration maintained during cell cycle

• Relative position can strongly depend on cell types

• Space between = splicing, transport, diffusible factors

CT subdivisions

• Divided into active and inactive domains

• Polycomb-repressed regions

• Gradient

  • Heterochromatin towards outside, euchromatin towards middle

Topologically associated domains (TADs)

• Structural building blocks of chromosomes (spatial structures)

• Functional unites of gene regulation (co-expression)

• kb-Mb in size

• Separated by genetically defined boundary elements, recognized by architectural proteins

• Housekeeping genes common at boarders

Loop formation in the genome

• Human genome: 10,000 loops

  • Average loop size = 200,000 bp

• Often similar chromatin marks within loop domain (e.g. H3K36me3, H3K27me3)

  • Domains w/ similar marks located next to each other (subcompartments)

• Regulation of gene expression

• Not fixed structure, can be dynamically adjusted

CTCF

• Multidomain TF

  • 11 zinc fingers (ZF)

  • Each ZF of ZF 3-7 contact 3 bases each of 15bp consensus

• Shapes loops/subdomains

  • Fixes loop in place/stabilizes

• Not symmetric

  • N-terminus → interacts w/ cohesion (stabilizing interaction)

  • C-terminus → No stable interaction w/ cohesion

Cohesion

• Forms a ring like structure that topologically encircles the DNA

• Is an SMC complex (structural maintenance of chromosomes)

  • Uses ATP to translocate DNA and extrude loops (get larger and larger)

• Reels DNA from both sides until barrier is met

CTCF orientation and loop extrusion process

• If CTCF-bound to DNA encounters a cohesion ring, it pauses a bit (regardless of orientation)

  • If cohesion encounters N-terminus → forms a stable interaction and cohesion remains paused at the CTCF

  • If cohesion encounters C-terminus → no stable interaction is formed and the cohesion interface is partially opened/permissive and the DNA w/ CTCF passes through

• Extrusion continues until each side of the cohesion complex encounters a DNA bound CTCF whose N-terminus faces the cohesion complex (i.e. a convergent pair)

  • Allows for stabilization on both sides

Ways to study the organization of the interphase DNA

FISH, 3C, DAPI stain

Examples for CTCF-mediated loops in the regulation of gene expression:

• Human 𝛃-globulin locus: active chromatin hub

• IGF2-H19 system: enhancer blocking

• Hox-timer: Extrusion into sub TADs

Loops in human 𝛃-globulin locus

• Active chromatin hub

• Sequential activation of the β-globin gene cluster

• 5 gene, arranged in order of expression during development

• One shared Locus Control Region

  • Complex enhancers, modulate chromatin structure

• Recruitment of transcription factors

• Recruitment of HAT activities: hyperacetylation of H3

• Folding activates gene expression → ACH, loop

Loop formation and imprinting of IGF2-H19 system

• Enhancer blocking and sub TADs

• Gene is expressed based on whether the copy came from mom or dad

• CTCF forms a loop to block enhancer and Igf2 interaction

Loop formation and Hox gene clusters

• Subset of homeobox genes

  • Master regulators of development, control of body pain

• Chromosomal organization: anterior-posterior expression

• Co-linear gene activation: requires coordinated changes in higher order chromatin

• Gradual looping out from chromosome territory coincides with gene expression

CT reorganization

• Nocturnal animals use the physics of chromatin as a lens

• Inverted nuclei: “microlenses” → channel photons to photosensitive ganglion cells

  • Dense in the center (heterochromatin), funnels light

Nuclear envelope

• The interface between nucleus and cytoplasm

• Protects the genome

• Role in gene regulation

Structure of the nuclear membrane

• Double membrane

  • Outer membrane: connected with ER

  • Inner membrane: associated with lamina

• Nuclear core complex

Nuclear lamina

• Mesh of protein fibers

• Complex filamentous protein network associated with the inner nuclear membrane

  • Covers inner membrane

• Consists of Lamins (4 types in mammals) + Lamina associated proteins

  • Plants: Nuclear Matrix Constituent Proteins (NMCPs)

• Provides mechanical stability of the nucleus

  • Used as anchor proteins for chromatin

• Attachment of the genome (e.g. via HP1)

• Restricts CT movement; maintenance of chromosome positioning

  • Helps CTs stay in place

• Defects can lead to age related disorders

Genes coding for proteins of the nuclear lamina

• Most invertebrates: single lamin

• Most vertebrates: 4 genes

  • Lamin B1, B2, A, LIII (lost in most mammals)

Mislocalization of centromeres

• Mutation in human lamin A gene (LMNA)

  • Missense 433G>A (E145K)

• Mutation in a domain of the protein (𝛂-helical central rod) that is required for polymerization

• Abnormal localization of centromeres, mislocation of telomeres

Link between nuclear morphology and DNA repair

• Frequent DNA DSBs can induce nuclear envelope tubules: DSB-capturing nuclear envelope tubules (dsbNETs)

• Contribute to genome stability

• Nuclear structure-function relationships

• Human cell lines

Nuclear core complex

• Large multiprotein structure

• Ca. 30 different proteins, in repeated subunits

• transport requires nuclear localization sequence (import) and nuclear export seqeunce

Nuclear bodeis

Membraneless structures in the nucleus (i.e. Nucleolus, cajal bodies, PML bodies, speckles, transcription factories)

Nucleolus

Ribosome biogenesis

Cajal bodies

Telomerase biogenesis; RNA processing, small nuclear ribonucleoproteins snRNP

PML bodies

a.o. Promyelocytic Leukemia Protein genome maintenance

Speckles

Enriched in pre-mRNA splicing factors, CT borders

Biomolecular condensates

• Arise through phase separation for membraneless compartment

  • Driven by multivalent macromolecular interactions

  • Separation into two phases:

    • One with higher concentration and once with lower

  • Need macromolecules and charges/polarity → changes the interactions

• Can exist outside of nucleus

Liquid-liquid phase separation (LLPS) or condensation

• Formation of membraneless compartments (membraneless bodies, biomolecular condensates, liquid assemblies)

• Occurs in solutions of macromolecules automatically if conditions are right

• Can separate into a dense phase (resembles liquid droplets) and a dilute phase

Functional benefits of three features common to condensates:

• Compartmentalization → give substructure

• Selective partitioning → bring similar molecules in close proximity

• Concentration → act as reaction hubs/storage compartments

Transcription factories

• Transcriptional hotspots

• Pol II transcription organized into small structures

• Fewer sites of active transcription than active genes and RNA pol II molecules (grouping of resources)

• Brings co-expressed genes into direct contact

• Hyperphosphorylated elongation form of RNA pol II

• Ex) erythroid cells (𝛼- & β-globulin, Eraf [𝛼-globulin-stabilizing proteins]: closer together than other cell types)

Nucleolus

• FC/DFC → center; rRNA gene transcription by pol I (except for 5s rRNA: pol III)

• DFC → rRNA transcript processing

• GC → outer region of nucleolus

  • Associated with re-imported ribosomal proteins

  • Final stages of ribosome assembly

• Perinucleolar heterochromatin → silent rRNA genes loop out of nucleolus

• Other processes:

  • Assembly of telomerase (to be transported to Cajal bodies)

  • miRNA storage

  • tRNA transcription by pol III

Forward genetics

• Which gene(s) is/are necessary to support a phenomenon

• No prior knowledge of the gene involved required:

  • Unbiased and powerful

  • Don’t need to know mechanisms of genes/pathways

• Works well for single gene/phenotype

Things needed for forward genetics

• Phenotype

• Large data set or collection of random mutations

• Screen for mutant phenotypes

Outcome/tools of forward genetics

• Genetic linkage map

• Physical map

• Cytogenetic maps

How to get a mutant collection

• Induce mutagenesis (use mutagen on organism)

  • Chemically, radiation, molecularly (T-DNA insertion, transposon mutagenesis)

• Go out in field and collect different ecotypes (differ in different ways)

  • Spontaneous, varieties, true-to-type)

Sutton and Boveri

Found structure of metaphase chromosomes; Info comes in bundles (linked traits)

Back cross/test cross

Cross a heterozygous offspring with the homozygous recessive parent

Gamete ratio if characteristics are independent (on different chromosomes)

1:1:1:1

Gamete ratio if characteristics are coupled (on same chromosome)

2:0:0:2

Genetic/linkage map

• Based on linkage and recombination frequency

• 1% recomb frequency = 1cM

• Not a true physical distance

Physical maps

• Genomic/chromosome fragment library, cloning, sequencing

• Uses physical distance in bp, based on:

  • Restriction Fragment Length Polymorphisms (RFLPs)

  • Variable number of tandem repeats (VNTRs)

  • Single nucleotide polymorphisms (SNPs)

• Requires overlap (contig construction, chromosome walking)

• True physical representation of the genome

• Modern application = optical maps (genome assembly, scaffolding)

Restriction Fragment Length Polymorphisms (RFLPs)

Cut DNA with restriction enzymes to get different fragment lengths to ID marks in the DNA; used to determine overall structure