Lecture 4: DNA Packaging and Cell Division

What does DNA look like in prokaryotes

single circular DNA and non-chromosomal DNA/plasmids

gene

segment of DNA that codes for a protein or RNA product

sister chromatid

identical copies in replicated chromosome

replicated chromosome

a chromosome comprised of two identical DNA molecules, joined together

homologous chromosomes

different versions of same chromosome type in the same cell 

• maternal and paternal versions of each chromosome

chromatin

• complex of DNA and protein

• nucleosomes are further packed on top of one another to generate a more compact structure called chromatin fiber

human cells contain ___ copies of every chromosome

2

• one from dad and one from mom

supercoiling

• DNA more compact

• occurs in eukaryotes and prokaryotes

• negative supercoiling: opposite direction to helix

• positive supercoiling: same direction as helix

• supercoiling can occur spontaneously during DNA replication

• supercoiling controlled by topoisomerase

  • temporarily breaks and rejoins DNA strands to relieve stress

Detection of supercoiling

DNA gel, put DNA in each lane, some of it is relaxed, cut (linear) or supercoiled 

DNA travels at different speeds

• slowest to fastest: relaxed, linear, supercoiled

• supercoiled moves faster because more compact and denser 

Compaction of the Bacterial Chromosome

• relaxed DNA is circular in prokaryotes

• DNA-binding proteins can bind to circular chromosome

  • DNA becomes supercoiled into loops held in place by DNA-binding proteins which stabilize structure

Eukaryote Nucleus

• density is not uniform

• euchromatin

  • lighter area

  • less packed/dense

  • interphase

• heterochromatin

  • darker area

  • more packed/dense

  • mitosis

• nucleolus

  • special area where ribosomes are made

level of compaction controls _____

transcription 

genome size

number of base pairs

Prokaryotes genome

10⁶ base pairs

• one circular chromosome + non chromosome DNA (plasmids)

Eukaryotes genome

linear chromosomes + non-nuclear DNA (mitochondrial)

• 10⁷ bp fungi

• 10⁹ bp mammals

• 3 × 10⁹ bp humans

species with similar genome sizes can have ____

very different chromosome numbers and sizes 

interphase

• when chromosomes are duplicated = DNA replication

• cells express many genes (transcription and translation)

• cells are long thin threads of DNA that can’t easily be distinguished

• chromosomes are less condensed but still considered tightly packed

mitosis

• duplicated chromosomes are highly condensed

• DNA is compacted ≥ 15000 fold in mitotic human chromosomes

• theorized that chromosomes condense so don’t get tangled during metaphase

• gene expression ceases (transcription and translation)

• nuclear envelope breaks down 

Replication Origins

• multiple origins in eukaryotes

• one origin in prokaryotes 

telomeres

• repetitive nucleotide sequence that caps the ends of linear chromosomes

• helps chromosomes not shorten with each round of replication

• protective caps that keep chromosome from bring mistaken as broken DNA

• two telomeres

centromeres

specialized DNA sequence that allows duplicated chromosomes to be separated during M phase

nucleosome

• nucleosome consists of 8 histone proteins: two H2A, two H2B, two H3, and two H4 

• refers to both core particle and linker DNA

• DNA wrapped around histone two times

• ~200 bp of DNA 

• beads on a string

• first level of chromatin packing

histones

• highly conserved proteins around which DNA wraps to form nucleosomes

why do histones stick to DNA

high proportion of positively charged amino acids help bind to negative phosphate on DNA

how many bp of DNA around core particle 

146 bp 

how many base pairs of DNA around linker DNA?

~ 44 bp

• nucleosomes can slide together or apart so number changes

incorrect hierarchical model of Chromatin compaction

• said compaction was a regular ordered process

• in reality: disordered (not tangled) chromatin chains

  • different configurations and densities each time

  • width is thicker in mitotic chromosomes

Cell cycle

orderly sequence of events by which a cell duplicates and divides

G₁ (Interphase)

1) cell grows

2) organelles replicated

3) high rates of transcription and translation

• during transcription nucleosomes are broken apart to expose certain genes called “open” DNA

S (Interphase)

1) when chromosomes are duplicated

G₂ (Interphase) 

cell grows 

do transcription and translation occur during all of interphase

yes

mitosis length

• A briefer stage when duplicated chromosomes are distributed into two daughter nuclei 

cell cycle control system 

• Makes sure each process is complete before next begins 

Cell cycle checkpoints

1) Late G₁

• environment is favorable for proliferation

• can enter G₀ if not

2) End of G₂

• make sure DNA is fully replicated

3) Midway through Mitosis

• make sure chromosomes properly attached to mitotic spindle

What does the cell-cycle control system depend on?

• cyclically activated protein kinases (maturation-promoting factor)

  • phosphorylate and dephosphorylate proteins

    • turns proteins off and on

how does phosphorylation work?

phosphorylation done by kinases

• phosphate attached to side chain of amino acids

dephosphorylation done by phosphatases 

Cyclin 

• discovered by Tim hunt using SDS-Page on Sea Urchins

• regulatory protein whose concentration rises and falls at specific times 

• helps control progression from one state of cell cycle to next by binding to cyclin dependent kinase

• concentration varies in cyclical fashion

  • rise in concentration due to continued transcription of cyclin gene

  • decline in cyclin concentration due to targeted destruction of the protein

how does a cell progress through various stages of the cell cycle?

• controlled expression and degradation of various cyclin proteins 

is the same cyclin-cdk used for all steps in cell cycle

no, cyclin B-cdk complex promotes mitosis but there are others

Activated CDK phosphorylate different proteins that are responsible for…

1) nuclear membrane degradation

2) microtubule assembly

3) chromatin condensation

Do levels of CDK change?

no 

centrosomes

• are duplicated before mitosis

• principal microtubule-organizing center

  • house the centrioles

• when mitosis begins, two centrosomes separate

mitotic spindle

• form from microtubules and other proteins during prophase

• individual filaments alternate between growing and shrinking: dynamic instability

3 types of microtubules

1) kinetochore

• reach out from centrosome to center of chromosome

• multiple microtubules can attach here

2) aster

• spread out backwards from centrosome and will reattach to nuclear membrane when it reforms

3) polar

• don’t attach to chromosomes but to each other

kinetochore

a protein complex associated with centromere

• each duplicated chromosome has two kinetochores: one on each sister chromatid

Prophase (1st step of Mitosis)

• chromosomes condense

• mitotic spindle assembles outside nucleus between two centrosomes which have begun to move apart

Prometaphase (2nd step of Mitosis)

• breakdown of nuclear envelope

• chromosomes attach to spindle microtubules via kinetochore 

• motor proteins composed of kinesin or dynesin pull microtubules and chromosomes in position 

Metaphase (3rd step of Mitosis)

chromosomes align in equator

Anaphase (4th step of Mitosis)

• Sister chromatids separate and are pulled toward spindle pole 

• Pushing and pulling force

  • Push spinde poles from each other and pull spindle poles towards reforming cell membrane

Telophase (5th step of Mitosis)

• two sets of chromosomes arrive at poles of spindle 

• new nuclear membrane reassembles around each set

Cytokinesis (6th step of Mitosis)

cell divides

microtubules

• extend through cytoplasm

• can rapidly assemble and dissassemble

• form cilia or flagella 

microtubule structure

• polymers whose building blocks are tubulin alpha and beta dimers

• hollow and spiral tube

• + and - negative orientation ends

  • - end anchored to centrosome

    • where you subtract alphabeta subunits

  • + end attaches to centromere

    • where you add alphabeta subunits

• organize in centrosome