DNA organization , segregation & DNA recombination

End-to-end length of Human DNA

1.02 m

• Human DNA needs to be organized in less than 2000 micrometers cubed of space

DNA Packaging/Organization

Involves several levels

• supercoiling

  • The following mechanisms are required to efficiently and effectively organize the DNA into a usable form:

• Nucleosomes

• Chromatosomes

• Solenoids

• looping onto matrix

  • The following mechanism is required to produce a mitotic chromosome

• Higher order coiling

• Proteins that organize chromosomes are essential and provide a mechanism for condensation, segregation, and organization of chromosomes

Virus

A non-cellular infectious particle genome containing a small nucleic acid genome with a limited number of genes

• The nucleic acid can be single-stranded or double-stranded

• The nucleic acid can be DNA or RNA

• Physical size and genomic size varies a lot

Bacteriophage

A virus that infects (“eats”) bacteria

Capsid

Protein coat that contains viral genetic material

Non-enveloped virus

Contains genetic material in only a protein shell

Enveloped virus

Has an envelope of host cell cytoplasmic membrane surrounding the capsid

Bacteria

• Haploid genomes

• Most often have a single dsDNA chromosome

• Some have more than one chromosome

Nucleoid

• Bacterial chromosomes are densely packed to form a small region called the ____

• Organized into a series of tight loops

• Allow for efficient packaging of relatively long DNA molecules into very small spaces

Bacterial chromosome compaction

1. Proteins help organize the DNA into loops that pack the chromosome into the nucleoid

2. The circular DNA undergoes supercoiling

Small nucleoid-associated proteins

Participate in the DNA bending that contributes to folding and condensation of bacterial chromosomes

Structural maintenance of chromosome (SMC) proteins

Holds DNA in coils or V-shapes, maintains the shape facilitated by small nucleoid assisted proteins

Supercoiling

• Covalently closed circular chromosomes exist in various ___ forms

• The relaxed circle is the least twisted

• _____ compacts DNA as a result of over or under rotations of helical twisting

Chromatin

The DNA and associated proteins of eukaryotic chromosomes

Chromosomes

Composed of half DNA and half protein

Histone proteins

• Makeup half of the proteins in chromosomes, small basic proteins that tightly binds DNA

• 5 types in chromatin:

  • H1 - Linker DNA

  • H2A - Nucleosome

  • H2B - Nucleosome

  • H3 - Nucleosome

  • H4 - Nucleosome

• Tightly conserved among eukaryotes

• The remaining half of proteins, non-____ proteins, are very diverse and perform a variety of tasks in the nucleus

Nucleosome

• Most basic unit of DNA packaginf

• 8 histone proteins within the core

• 2 of each: H2A, H2B, H3, and H4

• Formed by a span of DNA that is wound around each protein octamer 1.65 times (~146 bp)

• In humans, there is ~50 bp space between core nucleosomes (linker region)

Nucleosome assembly

• Histones H2A and H2B assemble into H2A-H2B dimers, histones H3 and H4 assemble into H3-H4 dimers

• Two H3-H4 dimers form a tetramer, then two H2A-H2B dimers associate with it to form the octamer

First level of DNA condensation

The wrapping of DNA around the octamer - compacts the DNA about sevenfold. E.g. 7m → 1m

Chemical modification

• The ends of the histone proteins lie outside of the nucleosome and can be ___ _____

• E.g.

  • Methylation

  • acetylation

  • phosphorylation

• All associated with regulating gene expression

Electron micrographs of chromatin

In its least condensed state, it show a 10-nm fiber, or “beads-on-a-string” morphology-the “beads” are the nucleosomes

Kornberg

Proposed the nucleosome-based model of chromatin in 1974

• The variable-length “string” between nucleosomes is linker DNA

• H1 may associate with linker DNA

Chromatosome

H1 with a nucleosome may sometimes be called a ____

• Histone H1 binds to DNA at edges of histone particle

• covers another ~20 bp of DNA (total now ~166 bp) from 146 bp

• DNA now wrapped about 2x around the histones

Solenoid structure

• The 10-nm fiber is not observed under normal cellular conditions

• a 30-nm fiber (x6 more condensed) is observed and forms when the 10-nm fiber coils into a ____ ___, with six to eight nucleosomes per turn and histone H1 stabilizing the solenoid

• ______ chromatin is lopped and attached periodically to a nonhistone protein chromosome scaffold

• The loops on the scaffold form the 300-nm fiber

• This is the state of the chromatin in a functioning cell (interphase)

Matrix attachment regions (MARs)

• Chromatin loops of 20-100 kb are anchored to the chromosome scaffold by non-histone proteins at sites

Radial loop-scaffold model

Suggests that the loops gather into “rosettes” and are further compressed by non-histone proteins

• Metaphase chromatin is compacted 250-fold compared to the 300-nm fiber

Euchromatin

Regions that contain actively expressed genes are less condensed during interphase

• Open structure, DNA is accesible to enzymes (solenoidal form of chromatin) - expressed genes are found here

Heterochromatin

Regions that remain condensed in interphase and contain many fewer expressed genes

• Often associated with the methylation of histones

• Compact, inactive DNA common at centromeres and telomeres (no genes)

• Comes in two forms:

  • Facultative

  • Constitutive

Facultative heterochromatin

• Not always heterochromatin E.g. the sex chromosomes

• Exhibits variable levels of condensation, related to levels of transcription of resident genes

• Inactivation is reversible

Constitutive heterochromatin

• Permanently condensed

• Found prominently in centromeres and telomeres

• composed primarily of repetitive DNA sequences

Dynamic chromatin structure

Changes in level of compaction regulate access to DNA by proteins for replication, transcription, recombination, or repair

• E.g. chromosome centromeres have constitutive heterochromatin

  • But when they replicate in S phase, heterochromatin dissipate

  • Nucleosome core particles dissociate from DNA ahead of replication fork and re-form centromeric heterochromatin after the fork passes

  • Variable borders for reestablishing centromeres and typically no impact on gene expression

Gene expression

• Can be controlled by the state of chromatin in which a gene is located

• Can be dictated by chromatin structure, which is transmissible from one cell generation to the next

Hermann Muller

• The eyes of the fruit fly Drosophila are red, thanks to the expression of the w locus that is usually in a region of euchromatin near the telomere of the X chromosome

• He used X-rays and isolated mutants in which a segment of the X chromosome had inverted thereby placing the w locus near the centromere

  • The result was that in some eye cells, the gene was not expressed due to the spread of heterochromatin to the centromere; while other cells it was expressed giving a variegated appearance to the eyes

Germ line cells

Sperm and ovum

Chromosome segregation

The process in eukaryotes by which two sister chromatids formed as a consequence of DNA replication (or paired homologous chromosomes) separate from each other and migrate to opposite poles of the nucleus

• Occurs during both mitosis and meiosis

• Also occurs in prokaryotes, in contrast to eukaryotic ___ ___, replication and segregation are not temporally seperated but is simultaneous instead of being sequential like in eukaryotes. It is progressive after following replication

The cell cycle

1) Interphase

2) M phase or Mitosis

Interphase

• Occurs between cell divisions, chromosome/DNA replication occurs

• The nucleus is granular looking

• DNA is replicated

Homologous chromosomes

• Very similar to each other and have the same size and shape

• They carry the same type of genetic information, so they have the same genes in the same locations

Prophase

• Chromosomes condense

• Nucleolus dissapears

• Centrioles move to poles

• spindles form

• Nuclear membrane breaks down

Prometaphase:

• Movement of chromosomes to centre of the cell

Metaphase

Chromosomes align at equatorial plane of the cell

• Mitotic spindle formation complete

Anaphase

Starts when sister chromatids split

• chromosomes move to poles of cell

• cell begins to elongate

• cleavage furrow starts

Telophase

Begins when chromosomes reach the poles

• Nuclear membrane reforms (shady colour)

• chromosomes de-condense

• nucleoli reform

• spindle fibers disappear

• cleavage furrow continues

Cytokinesis

• Not part of mitosis but normally follows closely after mitosis

• Equals completion of cleavage furrow and production of two cells

• Not always part of the M phase but this is not always true - Drosophila development

Meiosis

It is a specialized type of mitosis

• Occurs in the germ line/cells

• Necessary for the production of gametes in diploid, sexually reproducing organisms

• Involves two sequential cell divisions without DNA replication between divisions

  • 8 stages in total

  • Happens in germ cells

  • Purpose is sexual reproduction

  • Produces 4 haploid daughter cells

  • Chromosomes number is halved in each daughter cell

  • Genetic variation increased

Meiosis 1

Reduction division step

• Segregation of homologous chromosomes reduction, diploid to haploid

• Odd step

• More complex

• Subdivided into more steps

Leptotene

• 1st stage of prophase 1

• Chromosomes begin to condense and become visible

• Thickened regions (chromomeres) appear

Zygotene

• 2nd stage of prophase 1

• Homologous pair (bivalent) of chromosomes

Chromosomes continue to condense and there is active pairing of the chromosome threads between non-sister chromatids

Prophase 1 - Pachytene

• 3rd stage of prophase 1

Chromosomes become fully aligned

• Slow dissociation of nuclear envelope

• Allows for recombination

Diplotene

4th stage of prophase 1

• Aligned homologous pairs become less tightly aligned

• Chiasmata appear and mark the locations where crossing over has occured

Diakinesis

5th stage of prophase 1

• Compaction is completed and the chromosomes are ready to be segregated

Telophase 2

• 1 out of 4 recombinant from the mother

• 1 out of 4 recombinant from the father

• The 2 others are non-recombinant

How meiosis differs from mitosis

• Reduction in the number of chromosomes (from diploid to haploid)

• recombination between chromosomes

Importance of meiosis

Recombination increases diversity even more by reshuffling genetic information between the chromosomes

• The total is not the sum of the parts

• Production of haploid cells by meiosis is a critical component of sexual reproduction

• Independent assortment of chromosomes during meiosis 1 (Mendel’s fourth law) produces diversity in offspring (two offspring will almost never have exactly the same chromosome complement)

Mendel’s fourth law

Also known as Independent assortment

• Occurs during meiosis 1, and produces diversity in offspring which ensures that they will almost never have exactly the same chromosome component

• 8 possible combinations with only 3 chromosomes

Mitosis

• 4 stages in total + interphase

• Happens in somatic cells

• Purpose is cell proliferation

• Produces 2 diploid daughter cells

• Chromosome number remains the same

• Genetic variation doesn’t change