Eukaryotes Exam 4

self duplication (DNA replication)

• H bonds between bases hold complementary strands together

• each strand contains information required for construction of other strand

• can act as template to direct synthesis of complementary strand and restore double-stranded state

semiconservative

DNA replication is semiconservative because each daughter duplex contains one strand from the parent structure

Early progress in bacterial research was driven by 2 approaches:

• isolation of temp‐sensitive (ts) mutants used to study DNA synthesis for replication, repair, and genetic recombination

• In vitro studies with purified cellular components have shown activity of >30 proteins for E.coli chromosome replication

start of replication

starts at origin where proteins bind to initiate DNA replication, proceeds bidirectionally

Replication forks

• sites where parental dsDNA helix unwinding

• nucleotides being incorporated into newly synthesized strands

DNA Polymerases

• responsible for synthesizing new DNA from DNA template

• cannot initiate formation of DNA strand, requires primer

• short strand provides 3′ OH terminus

• adds nucleotides to 3′ hydroxyl terminus of existing strand

• strand synthesis occurs 5'→3'

Semidiscontinuous Replication

• both daughter strands synthesized simultaneously

• DNA pol moves 3′ → 5′

• 2 newly assembled DNA strands grow in opposite directions, one growing toward replication fork, other growing away

• major enzyme is DNA pol III

Primase

RNA polymerase that assembles short RNA primers

Okazaki fragments

• small DNA segments

• constructs lagging strand

• uses DNA ligase to join into continuous strand

leading vs lagging strand synthesis

• leading strand synthesized continuously, 5'→3'

• lagging strand synthesized discontinuously, 5'→3 (constructed by Okazaki fragments)

Helicase

• unwind a DNA duplex, uses energy from ATP hydrolysis

• moves along one of DNA strands

• breaks H bonds that hold 2 strands together

• exposes single-stranded DNA templates

• major helicase during replication in E. coli is DnaB

Single-stranded DNA-binding proteins (SSB)

stabilize single stranded DNA

DNA polymerase III holoenzyme

• synthesize successive fragments of lagging strand

• held to DNA by β clamp as it moves along template strand and synthesizes complementary strand

• after completion of 1 Okazaki fragment, it disengages from β clamp

• cycles to recently assembled clamp “waiting” at upstream RNA primer–DNA template junction, forms another Okazaki fragment

• original β clamp left behind for period on finished Okazaki fragment, eventually disassembled and reutilized

β clamp

keeps polymerase associated with DNA

DNA polymerase I

• involved in DNA repair

• removes RNA primers at 5′ end of Okazaki fragments during replication and replaces with DNA

• maximizes fidelity of DNA replication

• 5′ → 3′ and 3′ → 5′ exonucleases

• polymerase activity simultaneously fills gap with deoxyribonucleotides

• uses DNA ligase to seal gaps between fragments

5′ → 3′ exonuclease (DNA pol I)

• removes nucleotides from 5′ end of single-strand nick

• RNA primers removal

3′ → 5′ exonuclease

• removes mispaired nucleotides from 3′ end of growing DNA strand

• DNA repair

3 experimental systems that help understanding of replication in euks:

• isolation of mutant yeast and animal cells unable to produce specific gene products required for various aspects of replication

• analysis of structure and mechanism of action of homologous replication proteins from archaeal species

• development of in vitro systems using cell extracts or purified proteins

replicons

• small portions of eukaryotes genomes for replication

• has its own origin (and ARS) from which replication forks proceed outward in both directions

Autonomous replicating sequence (ARS)

associated with multiprotein complex called origin recognition complex (ORC)

5 polymerases of Eukaryotic Replication Fork

• α - initiates Okazaki fragment synthesis

• β - involved in DNA repair

• γ replicates mtDNA

• δ (delta) - lagging strand synthesis

• ε - leading strand synthesis

DNA is synthesized semidiscontinuously

Nucleotide Excision Repair (NER)

• cut- and-patch mechanism that removes bulky lesions

• generated by environmental mutagens such as UV irradiation and bulky chemical compounds

• two distinct pathways: transcription-coupled and global genomic paths

Base Excision Repair (BER)

• removes altered nucleotides generated by reactive chemicals from diet or metabolism

• corrects only the damaged bases

• initiated by DNA glycosylase which recognizes and removes altered bases by cleavage of glycosidic bond holding base to deoxyribose sugar

Mismatch repair (MMR)

• correction of mistakes that escape DNA polymerase proofreading activity

• repair enzymes recognize distortions caused by mismatched bases

Double Strand Break (DSB) repair

• nonhomologous end joining (NHEJ): complex of proteins binds to broken ends of DNA duplex and catalyzes reactions that rejoin broken strands

• homologous recombination: requires homologous chromosome to serve as template for repair of broken strand

Mitosis

• production of cells that are genetically identical to parent

• basis for producing new cells

• process of nuclear division in which two nuclei are produced to maintain chromosome number

• consists of Prophase, Prometaphase, Metaphase, Anaphase, Telophase which represents a segment of a continuous process

Meiosis (reduction)

• production of cells with half genetic content of parent

• basis for producing new sexually reproducing organisms

• ensures production of haploid phase in life cycle, and fertilization ensures diploid phase

• without this, chromosome numbers would double each generation, making sexual reproduction unsustainable

• gametic, zygotic, sporic

Cell Cycle Stages

• Interphase

  • G1

  • S

  • G2

• M phase

  • Mitosis

    • Prophase

    • Metaphase

    • Anaphase

    • Telophase

  • Cytokinesis

M phase

• consists of mitosis and cytokinesis

• Cell contents divide into two new cells

• Mitosis lasts about an hour

Interphase

• G1, S, G2

• period between cell divisions

• cell grows and engages in diverse metabolic activities

• lasts longer than M phase; (day-week+)

G1 (interphase)

• takes place between end of mitosis

• cell growth and carries out normal metabolism and organelles duplication

S phase (interphase)

DNA replication and chromosome duplication

G2 (interphase)

• occurs between end of S and beginning of mitosis

• cell grows and prepares for mitosis

3 cell types with capacity to grow and divide in vivo

1. Cells that are highly specialized and lack ability to divide. (nerve cells, muscle cells, or red blood cells)

2. Cells that normally don’t divide but can be induced to begin DNA synthesis and divide when given stimulus. (liver cells and lymphocytes)

3. Cells that normally possess relatively high level of mitotic activity. (stem cells for blood elements, skin and other epithelia, plant apical meristems)

maturation-promoting factor (MPF)

• activation triggers entry into M phase

• two subunits: kinase and cyclin (regulatory subunit)

• increased concentration of cyclin activates kinase

cyclin-dependent kinases (Cdks)

• MPF-like, studied in yeast Ts mutants

• products of cdc2 (fission yeast) and cdc28 (budding yeast) genes responsible for passage through G1 (START) and G2 control points

• START – cell committed to DNA replication (G1/ S cyclins)

• G2/M transition (Mitotic cyclins)

Checkpoints

• check if chromosomal DNA is damaged

• check if certain critical processes such as DNA replication during S phase or chromosome alignment during M phase, have not been properly completed

• progress through cell cycle can be arrested at checkpoint by sensors that detect chromosomal abnormalities, transmitters that signal the information, effectors that inhibit cell cycle machinery

prophase (mitosis)

• formation of mitotic chromosome which consists of two chromatids

• mitotic machinery assembled

• duplicated chromosomes prepared for segregation

• chromosome compaction/condensation occurs in early prophase

Cohesin

• with condensin, are proteins responsible for compaction

• associates with DNA of each chromosome prior to replication, forms ring to encircle 2 sister DNA molecules

Centromeres

occur at a primary constriction on chromosomes and serve as binding site for proteins

Kinetochores

• on outer surface of centromeres

• sites where chromosomes attach to microtubules of mitotic spindle

mitotic spindle

• entering mitosis, microtubules undergo disassembly before reassembly into microtubules

• in animal cells, microtubules are arranged in aster around each centrosome

Nuclear pore complexes

disassembled, nucleoporin subcomplexes disrupted and subcomplexes dissociate

Nuclear lamina

is disassembled - depolymerization of lamin filaments

Nuclear membranes

are disrupted mechanically - holes are torn into nuclear envelope by cytoplasmic dynein molecules

Membranous organelles (mitochondria, lysosomes, and peroxisomes)

remain relatively intact through mitosis

Golgi complex

may become incorporated into ER during prophase or become fragmented to form distinct population of small vesicles partitioned between daughter cells

Prometaphase (mitosis)

• mitotic spindle assembly completed

• chromosomes moved by microtubules into center of cell

• single kinetochore attached to microtubules form both spindle poles

Metaphase (mitosis) + 3 microtubule groups

1. Astral microtubules - radiate from centrosomes to regions outside spindle body, help position spindle apparatus and determine plane of cytokinesis

2. Chromosomal spindle fibers - exert pulling force on kinetochores maintaining chromosome in equatorial plane

3. Polar microtubules - maintain integrity of spindle

chromosomes aligned at spindle equator on metaphase plate

Anaphase (mitosis)

• begins when sister chromatids of each chromosome split apart and start their movement toward opposite poles

• Anaphase A and B: chromosomes split in synchrony

Anaphase A (mitosis)

• movement of chromosomes toward poles

• tubulin subunits lost from both ends of chromosomal microtubules, resulting in shortening and movement of chromosomal fibers

Anaphase B (mitosis)

• 2 spindle poles move in opposite directions due to elongation of microtubules

• tubulin subunits added to + ends of polar microtubules

Telophase (mitosis)

• final stage of mitosis, daughter cells return to interphase

• mitotic spindle disassembles

• nuclear envelopes of 2 nuclei reassembled

• chromosomes become dispersed

Cytokinesis (M phase)

process where 1 cell is divided into 2 daughter cells

contractile ring theory

• suggests that a thin band of actin and myosin filaments generates force to cleave cell

• site of filament assembly (plane of cytokinesis) determined by signal coming from spindle poles

Motor Proteins

• powered by microtubule motors (dynein and kinesin-related proteins)

• located at spindle poles and kinetochores, keeps poles apart

• bring chromosomes to metaphase plate and keep them there

• elongate the spindle during anaphase B

2 unique challenges plant cells face when undergoing division

rigid cell wall and little freedom to move

Preprophase band

• dense ring of cytoskeleton filaments and proteins

• microtubules, actin, organelles, accessory proteins

• band disappears by prometaphase, but cell remembers its original location

• division plane forms at former site

• molecular mark is unknown, membrane proteins recruited and retained at site during mitosis

cell plate

• precursor structure that allows plant cells to build new extracellular wall during division

• starts from cell center and growing outward

phragmoplast

• guides cell plate formation

• during cytokinesis, remnants of central spindle transport Golgi-derived vesicles to midzone

• made of vesicles, cytoskeletal proteins and membranes

plant cell wall formation

• vesicles fuse midzone to form tubular, disk-shaped membrane network

• as phragmoplast expands to cell edges, cell wall matures and fuses with parent plasma membrane, forming cell wall

• requires gradual deposition of polysaccharides, pectin, cellulose, and hemicellulose into cell wall lumen

meiosis stages

1. interphase

2. Prophase 1

   1. leptotene

   2. zygotene

   3. pachytene

   4. diplotene

   5. diakinesis

3. MAT 1

4. PMAT 2

5. 4 haploid cells

Meiosis I

• homologous chromosomes pair and then segregate, ensuring that daughter cells receive full haploid set of chromosomes

• Genetic recombination takes place

• Start with diploid parent cells and end with 2 haploid daughter cells

Meiosis II

• sister chromatids separated, simpler then meiosis 1

• start with 2 haploid parent cells and end with 4 haploid daughter cells, maintaining # of chromosomes and DNA in each cell

Gametic (terminal) meiosis

• in animals and protists

• meiosis occurs during gamete formation

• in males, spermatogonia become spermatocytes → meiosis → spermatids → sperm

• in females, oocytes enter extended meiotic prophase; meiosis completes only after fertilization

Zygotic (initial) meiosis

• found in protists and fungi

• meiosis happens immediately after fertilization, forming haploid spores

• spores undergo mitosis to produce haploid adults; diploid phase is brief

Sporic (intermediate) meiosis

• Occurs in plants and algae

• Meiosis occurs during sporogenesis in diploid sporophyte

• Spores grow into haploid gametophytes, which produce gametes via mitosis

Gametic Meiosis - Male

• meiosis occurs prior to differentiation of spermatozoa

• 2 divisions of meiosis produces 4 undifferentiated spermatids

• each spermatid undergoes complex differentiation to become highly specialized sperm cell

Gametic Meiosis - Female

• oogonia become primary oocytes, which then enter extended meiotic prophase

• vertebrate eggs fertilized before completion of meiosis (usually metaphase II)

• meiosis completed after fertilization, while sperm resides in egg cytoplasm

• only after differentiation of oocyte is complete does meiotic divisions occur

Prophase 1 (meiosis)

• DNA replicated prior to meiosis

• consists of leptotene, zygotene, pachytene, diplotene, diakinesis

Leptotene (Prophase 1 of meiosis)

chromosomal condensation starts

Zygotene (Prophase 1 of meiosis)

synapsis, homologous chromosomes pair

Pachytene (Prophase 1 of meiosis)

synapsis ends, synapsed chromosomes formed tetrads

Diplotene (Prophase 1 of meiosis)

Chiasmata occur (crossing over); point of genetic recombination

Diakinesis (Prophase 1 of meiosis)

• chromosomes prepared for attachment to spindle fibers

• ends with disappearance of nucleolus and disassembly of nuclear envelope

• triggered by increase in MPF activity

Metaphase I (meiosis)

• 2 homologous chromosomes aligned at metaphase plate

• homologous chromosomes held by one or several chiasmata

Anaphase I (meiosis)

• homologous chromosomes separate

• maternal and paternal chromosomes of each tetrad segregate into 2 daughter cells independent of other chromosomes

Telophase I (meiosis)

• produces less dramatic changes than telophase of mitosis

• nuclear envelope may or may not reform

• interkinesis: stage between 2 divisions

Cells communicate with one another through….

extracellular messenger molecules

Autocrine messengers

• cell has receptors on its surface that respond to messenger

• cells releasing message will stimulate/inhibit themselves

Paracrine messengers

• travel short distances through extracellular space

• limited in their ability to travel around body because they are inherently unstable, or they are degraded by enzymes, or they bind to extracellular matrix

Endocrine messengers (hormones)

• reach target cells through bloodstream

• also called hormone; act on target cells located at distant sites in body

First messenger/Ligand

• molecule that binds to receptor

• activate receptors that stimulate effectors to give rise to a physiological response

Receptors

in target cells, receive an extracellular message

2 different types of signal transduction pathways

1. activation by diffusible second messenger

2. recruitment of cytoplasmic proteins to plasma membrane

effectors

• generated when receptor transmits signal from cytoplasmic domain to nearby enzyme

• second messenger/enzyme

• small molecules that act as activators/inhibitors of specific proteins

• diffuses through cytosol or remains embedded in lipid bilayer of membrane

recruiting station

• a transformed cytoplasmic domain of a receptor that allows it to transmit it’s signal for cellular signaling proteins

• proteins interact with one another, or with components of cellular membrane

Signaling pathways

• a series of proteins

• each protein in pathway alters conformation of next protein

• protein conformation usually altered by phosphorylation

kinases vs phosphatases

• kinases add phosphate groups while phosphatases remove them

• human genome encodes >500 protein kinases and 150 protein phosphatases

• transfer phosphate groups to serine/threonine residues of their protein substrates or phosphorylates tyrosine residues

• soluble cytoplasmic proteins or integral membrane proteins.

Protein phosphorylation

• can change protein behavior in different ways

• activating or inactivating an enzyme

• increasing or decreasing protein-protein interactions

• changing subcellular location of protein

• triggering protein degradation

signal transduction

• transmitted signals reach target proteins that ultimately receive message to alter cell activity

• changes in cellular activities, gene expression, and ion permeability

• alteration of activity of metabolic enzymes

• reconfiguration of cytoskeleton

• increase/decrease in cell mobility

• activation of DNA synthesis

• cell death

5 extracellular messengers

• small molecules, AAs + derivatives

  • glutamate, glycine, acetylcholine, epinephrine, dopamine, and thyroid hormone act as neurotransmitters and hormones

• Gases such as NO and CO

• Steroids, derived from cholesterol

• Eicosanoids

• peptides and proteins

Steroid hormones (extracellular messenger)

regulates sexual differentiation, pregnancy, carb metabolism, and excretion of sodium and potassium ions

Eicosanoids

• nonpolar molecules derived from FA, arachidonic acid

• regulate variety of processes including pain, inflammation, blood pressure, and blood clotting.

5 types of receptor types

• G-protein coupled receptors (GPCRs)

• Receptor protein-tyrosine kinases (RTKs)

• Ligand gated channels

• Steroid hormone receptors

• Specific receptors

GPCR

• largest superfamily of proteins encoded by animal genomes (1000s)

• 7 α-helical transmembrane domains

• interact with heterotrimeric G Proteins

• capable of binding diverse array of ligands

• receptor isoforms have different affinities for ligand or interact with different types of G proteins

4 natural ligands that bind to GPCRs

• Hormones (both plant and animal)

• Neurotransmitters

• Opium derivatives

• Chemoattractants (odorants, tastants, and photons)

8 steps of Signal Transduction by G Protein-Coupled Receptors

1. ligand binds to receptor, altering conformation and increasing affinity for G protein to which it binds

2. Gα subunit releases its GDP, which is replaced by GTP

3. Gα subunit dissociates from Gβγ complex and binds to effector (adenylyl cyclase), activating effector

4. activated adenylyl cyclase produces cyclic AMP (cAMP)

5. Gα reassociates with Gβγ, reforming trimeric G protein, and effector ceases activity.

6. To prevent overstimulation, receptors must be blocked (desensitization) from continuing to activate G proteins

7. Receptor is phosphorylated by a G protein-coupled receptor kinase (GRK)

8. Arrestin molecule binds to phosphorylated receptor to inhibit ligand-bound receptor from activating additional G proteins. Receptor bound to arrestin is likely to be taken up by endocytosis

Desensitization

process that blocks active receptors from turning on additional G proteins

Termination of the Response in Signal Transduction by G Protein-Coupled Receptors

• Phosphorylation of GPCRs by G protein-coupled receptor kinase allows binding of arrestins (complete for binding with G proteins)

• Upon arrestin binding, GPCRs become desensitized

• If receptors are recycled and returned to cell surface, cells remain sensitive to ligand and are resensitized

• Arrestin-bound GPCRs internalized by clathrin-coated pits that bud into cytoplasm

• Clathrin-coated vesicles deliver their contents, including GPCRs, to endosomes.

• In endosomes, arrestins are scaffolds for assembly of signaling complexes (MAPK cascade and transcription factor ERK)

• GPCRs delivered to lysosomes for degradation OR returned to plasma membrane in recycling endosome, where they interact with new extracellular ligands