Mod 3- Genomes Flashcards
What is a genome?
The complete set of DNA molecules possessed by an organism
What is the size of the human genome?
3200 Mb DNA
How many linear DNA molecules are in the human genome?
24 linear DNA molecules
What is the range of sizes for the chromosomes in the human genome?
Shortest is 48 Mb, longest is 250 Mb
How many chromosomes are in a normal diploid human cell?
46 chromosomes
What is the size of the E. coli genome?
4.64 Mb DNA
Contained in a single DNA
What shape does the E. coli genome take?
Circular
Where does the replication of the E. coli genome begin?
At an origin of replication
Always the same position on the genome as it’s a small genome (IN E.Coli)
How many replication forks are formed during E. coli DNA replication?
Two replication forks - bidirectional
One will be the leading strand and one will be the lagging strand
What initiates E. coli DNA replication?
DnaA proteins bind close to the origin of replication
The DNA becomes wound round these proteins
What is the role of DnaA in E. coli DNA replication?
DNA becomes wound around these proteins
It forces the base pairs to break at the origin of replication due to the stress of DNA winding around the barrel
Original is A-T rich (easier to come apart as it only has 2 hydrogen bonds)
What is the prepriming complex in E. coli DNA replication?
The attachment of DnaB proteins to the origin
What forms the primosome in E. coli DNA replication?
Attachment of two primase enzymes
These make the RNA primers that initiate replication of the 2 leading strands (on each parent strand)
What is the function of DNA topoisomerase at the replication fork?
Unwinds the double helix, preventing DNA supercoiling
What do Single Strand Binding proteins (SSBs) do?
Protect the bare single strands of DNA
What is the role of DNA pol III during replication?
Synthesizes DNA
What is the function of FEN1 in DNA replication?
Removes primers and joins Okazaki fragments
What is the role of the ‘proliferating cell nuclear antigen’ (PCNA) in human DNA replication?
Sliding clamp that holds DNA Pol delta tightly onto the DNA
What are terminator sequences in the E. coli genome?
Binding sites for Tus proteins
These sequences are directional
What is the function of Tus proteins?
Allow the replication fork to pass in one direction but not the other
Permissive face (round side) = allows fork to pass through
Non-permissive face (flat side) = prevents fork from passing through
What results in replication fork arrest in E. coli?
DNA strand separation followed by specific interaction of the Ter C6 base with the Tus lock domain
There are 5 terminator sequences on each side = 5 chances to stop the fork (fork may stop at any of these)
What happens at the replication forks in human DNA?
Forks just merge, don’t need exact control like in circular DNA
Fill in the blank: The human genome comprises _______ Mb DNA.
3200
True or False: Each replication fork in human DNA copies about 150 kb of DNA.
True
What is the length of human chromosome 8?
About 3.5 µm or 0.0035 mm
How many linear DNA molecules comprise the human genome?
24 linear DNA molecules
What is the total DNA content in a normal diploid human cell?
6400 Mb DNA
What is chromatin?
DNA extracted from the nucleus
(as DNA is associated with lots of protein)
What structure does endonuclease digestion of chromatin reveal?
A DNA-protein complex with proteins spaced at regular intervals
What is the ‘beads-on-a-string’ structure in chromatin?
A structure observed through electron microscopy
The circles/ ‘beads’ are DNA associated with proteins (histones)
These beads are called nucleosomes
What are the proteins in chromatin called?
Histones
Associated with and package DNA
What is a nucleosome?
The basic unit of DNA packaging, consisting of an octamer of histones
Two of each histones, H2A, H2B, H3 and H4
What is the role of histone H1?
It is the linker histone that attaches outside the nucleosome
A linker histones keeps the linker onto the nucleosome
Fill in the blank: Nucleosome + DNA + linker histone = _______.
Chromatosome
What is the next level of DNA packaging after nucleosomes?
30 nm chromatin fibre
The 4.9cm molecule in chromosome 8 would now be about 1.2mm in length
How much do the beads on a string reduce the length of DNA?
By one-sixth as it wraps around the histones
There must be high levels of packaging
What stabilizes the 30 nm chromatin fibre?
Off-axis asymmetric binding of histone H1
What is the importance of histones and nucleosomes?
- Histones allow vast amounts of DNA to be packed into nucleosomes and other higher order DNA structures
- Chromatin structure can be changed (dynamic) through the reversible chemical modification of histones to allow things to happen to the DNA
- DNA packaging into nucleosomes is not permanent but dynamic
- Nucleosomes detach or shift to allow transcription of DNA
- Nucleosomes must detach for replication of DNA
- Nucleosome presence or modification can control gene expression
What characterizes euchromatin?
Contains active genes (probably as 30nm fibres)
Appears light under a microscope
Starts replicating first as it is open and accessible
What characterizes heterochromatin?
Contains inactive genes
Appears dark under a microscope (more densely packed)
What is constitutive heterochromatin?
DNA that is always tightly packed in all cells
Never needs to be accessed i.e. one of the X chromosomes in females
What is facultative heterochromatin?
DNA that is tightly packed only in some cells
Can be opened and used
What is the role of the nucleolus?
Contains ribosomal RNA genes and is the site of ribosome biogenesis (for translation)
What is the highest level of DNA packaging found in dividing cells?
Metaphase chromosome which is only found in dividing cells
What is the function of centromeres?
Hold daughter chromosomes together
Contains special histones = CENP-A instead of H3 (allows DNA to associate)
Serve as attachment points for microtubules that pull chromosomes apart
What do telomeres do?
Produces buffer sequences
Protect the ends
- From exonuclease attack (which digest ends of DNA)
- from being mistaken for chromosomes breaks and joined together by DNA repair mechanisms (identify ends of real chromosomes)
What is a karyogram?
A complete set of stained metaphase chromosomes used for mapping gene positions
True or False: All bands in a karyogram have names.
True
What is the significance of the nuclear matrix and nuclear lamina?
Nuclear matrix = inside nucleus
Nuclear lamina = around edge of nucleus
They maintain the shape of the nucleus
The 30 nm fibre is attached to these to prevent DNA tangling
What diseases are associated with nuclear matrix and nuclear lamina?
Progeria, Down’s Syndrome, Huntington disease
Where does replication of the human DNA begin?
Origins of replication
- Many on each chromosomal DNA molecule
- Not always at the same sites
- Each replication fork copies about 150kb of DNA
What is the role of DnaB in E. coli DNA replication?
- The pre priming complex is formed by attachment of DnaB proteins to the origin
- DnaB is a helicase (separates strands)
- DnaB breaks more base pairs so the replication forks move away from the origin (starts replication process)
What happens at the replication fork in E.coli?
- Helicase (DnaB) breaks base pairs
- Single strand binding proteins (SSBs) protect the bare single strands to prevent degrading
- DNA topoisomerase unwinds the double helix preventing DNA supercoiling (works ahead of the leading strand)
- Primase makes primers on the leading & lagging strands
- DNA pol III (two copies- on each strand) synthesises DNA
- DNA pol I and DNA ligase removes primers and joins Okazaki fragments (works on lagging strand)
- The gamma complex (clamp loader) attaches and detaches Pol III from the lagging strand
- Beta complex (sliding clamp) holds Pol III onto template allowing it to slide
Gamma and Beta complexes are accessory complexes
What happens at the replication fork in humans?
- Helicase breaks base pairs
- Single strand binding proteins (SSBs) protect the bare single strands (replication protein A)
- DNA topoisomerase unwinds the double helix preventing DNA supercoiling
- Primase/ DNA pol Alpha makes primers on leading & lagging strands
- DNA pol Delta (two copies) synthesises DNA = switches to Pol Delta as Pol Alpha doesn’t have proofreading activity
- FEN1 (acts on flap) and DNA ligase removes primers and joins Okazaki fragments
- The ‘proliferating cell nuclear antigen’ (PCNA) sliding clamp holds DNA pol Delta tightly onto the DNA
What is special about the replication of the E.coli genome?
Can replicate in both directions (as it’s circular)
Replication occurs at different rates = not identical speeds
The replication forks always meet around half way
What is special about the replication of the E.coli genome?
Can replicate in both directions (as it’s circular)
Replication occurs at different rates = not identical speeds
The replication forks always meet around half way
What are the different types of histones?
H1
- Molecular mass = 23,000 kD
- 30% basic amino acid content
- Different types of this exist
H2A
- Molecular mass = 14,000 kD
- 20% basic amino acid content
H2B
- Molecular mass = 13,750 kD
- 22% basic amino acid content
H3
- Molecular mass = 15,350 kD
- 23% basic amino acid content
H4
- Molecular mass - 11,300 kD
- 25% basic amino acid content
