Lecture 15

Textbook Notes

12.1 How Do Cells Replicate?

Chromosome

• chromosomes consists of a single long DNA double helix wrapped around histone proteins

• gene — a region of DNA in a chromosome that codes for a particular protein or ribonucleic acid (RNA)

• chromatin — each of the double-stranded DNA copies in a replicated chromosome

• sister chromatids are chromatid copies that are only attached at their centromere

M Phase and Interphase

• M phase — occurs when cells are in the process of separating their chromosomes

• Interphase — when the cell is not in M phase

  • chromosomes uncoil into extremely long, thin structures

S Phase

• part of interphase

• DNA replication occurs

Gap Phases

• G1 phase — gap between the end of M phase and start of S phase

• G2 phase — between end of S phase and start of M phase

19.2 Chromatin Remodeling

Chromatin Structure/Organization

• nucleosomes — unit of chromatin

  • group of 8 histones with about 200 nucleotides wrapped around them

  • between nucleosomes there is “linker” DNA with no histones

• topologically associating domain (TAD) — first level of chromatin structure

  • formed by a loop of the 10 nm fiber anchored at its base by a ring made of the protein cohesin and by binding between proteins that associate with specific sequences of DNA

  • individual TADs come together in larger chromatin compartments

• all chromatin compartments of a single chromosome associate in one area of the interphase nucleus called a chromosome territory

Chromatin Structure Alteration in Active Genes

• chromatin must be decondensed to expose the promoter so RNA polymerase can bind to it

• eukaryotic genes in their standard state are turned off because its normally tightly wrapped in chromatin

• DNA methylation

  • DNA methyl transferases add methyl groups (—CH3) to cytosine residues in DNA

  • in mammals the sequence is a CpG(5’-C-phosphate-G-3’)

  • the methyl gets added to the C in a CG sequence

  • methylated CpG sequences are recognized by proteins that condense chromatin

    • actively transcribed genes have few methylated CpG sequences near their promoters

    • non-transcribed genes usually have many methylated CpG sequences

• histone modification

  • a large set of enzymes adds a variety of chemical groups to specific amino acids of histones

    • acetyl groups (—COCH3), methyls, phosphates, and short polypeptide chains

  • histone code hypothesis — particular combinations of histone modifications on specific amino acids set the state of chromatin condensation for a particular gene

  • histone acetylation usually promotes decondensed chromatin

  • HATs (histone acetyletransferase) are the on switch, HDACs (histone deactylases) are an off switch

  • chromatin-remodeling complexes — macromolecular machines made of proteins

    • harness ATP to reshape chromatin

    • either cause nucleosomes to slide along the DNA or to knock the nucleosomes completely off to open up stretches of chromatin for transcription

  • when cells divide, the patterns of chromatin modifications will be passed on to the daughter cells, called epigenetic inheritance

Lecture Slides

• the “end replication” workaround

  1. a strand of parental DNA remains unreplicated after the RNA primer is removed from the end of the lagging strand

  2. telomerase extends unreplicated end

     • telomerase binds to the 3’ end of the overhanging strand of parental DNA and, using its own internal RNA template, extends the strand

  3. telomerase shifts and repeats activity

     • telomerase extends the DNA strand by shifting down the newly synthesized DNA and adding additional repeats, multiple times

  4. extended single-stranded DNA acts as a template

     • standard DNA synthesis on this template creates double-stranded DNA to prevent chromosome shortening

• DNA organization

  • nucleotides make up nucleic acid chain

  • bases pair with each other to make a double helix

  • a very long double helix of DNA and associated proteins is a chromosome

  • chromosomes can be linear or circular

    • nuclear genome is typically a linear form for eukaryotes

• how do we pack millinois or billions of base pairs of DNA into such a small space

  • we need to compact all the chromatin into a space that’s only 1-10 um

• bacterial chromosomes are supercoiled:

  • done by topoisomerases, which nick DNA (cutting the backbone), wind or unwind (depending on what’s needed), then reseal DNA

• eukaryotes: need to get 2 meters of DNA into a nucleus that is 5-8 micrometers in diameter

• organization of chromatin in the nucleus

  • first described by R. Kornberg in 1974 “beads on a string”

  • these are the complexes of DNA and protein (chromatin)

• what are the proteins that are part of chromatin?

  • histones

• histones

  • small, basic (pH wise) proteins (positively charged)

  • five major types of histones: H1, H2A, H2B, H3, and H4

  • sequence is highly conserved among species that have them

    • histone organization does not change much between organisms

  • bacteria do not have histones, but they have histone-like proteins

  • archae do have histones

• the (core) nucleosome

  • two each of histones H2A, H2B, H3, and H4

  • 146/147 base pairs of DNA

  • nucleosome “bead”: 8 histone molecules and 146 base pairs of DNA

  • packing of DNA and histones into nucleosomes yields chromatin fiber of approximately 10 nm in diameter

• chromatosome structure:

  • a nucleosome plus a single molecule of H1

  • H1 “tapes” the nucleosome closed

  • non-histone proteins (like CTCF) connect to linker DNA

• 10-nm chromatin fibers only shorten the length of DNA by 6-fold

  • next level of organization is the 30-nm fiber

• possible conformations of the 30-nm chromatin fibers

  • solenoid model versus flexible zigzag model

• the eukaryotic cell cycle:

  • M phase (mitotic), Interphase

• interphase chromatin exists in loosely condensed form (euchromatin) and highly condensed form (heterochromatin)

  • euchromatin

    • distributed through the nucleus

    • most in the form of 30-nm fibers

    • actively being transcribed

  • heterochromatin

    • found at periphery of the nucleus and in pockets elsewhere in the nucleus

    • gets them out of the way because they’re not being actively transcribed, they “hang on the wall”

• as a cell enters mitosis, chromatin must condense further:

  • eventually condense into familiar “chromosomes”

  • heterochromatin becomes much more widespread

• typical mitotic chromosome structure

  • sister chromatids are joined at the centromere with chromosome arms

  • ends are telomeres

• summarizing DNA replication

  • semiconservative — each daughter double helix is one parental strand and one newly synthesized strand

  • initiates at ori

  • synthesis only happens in 5’ → 3’ direction

  • requires a single-stranded DNA template, free NTPs and dNTPs, and nucleic acid-synthesizing enzymes

  • also requires unwinding enzymes, stabilizing proteins, and glue

  • DNA polymerases require a free 3’-OH, which the primer provides

  • RNA synthesizing-enzymes require a free 3’-OH for synthesis, but they can hybridize a nucleotide to a nucleic acid strand