Chromosomes and DNA Flashcards
When individual chromosomes be easily distinguished?
during metaphase of mitosis
Diploid eukaryotic cells
contain two copies of each chromosome
each chromosome pair differs in size and DNA sequence
What is the karyotype of the parent organism?
organisaed representation of all the chromosomes in a eukaryotic cell at metaphase
individual chromosomes occupy distinct subnuclear territories even in interphase
Chromosome
highly coiled fibre of chromatin
under electron microscope interphase chromatin resembles beads on a string
beads are nucleosomes
Nucleosomes
a protein core with DNA wound around it
protein subunits are core histones
Core histones structure
N-terminal tails of core histone subunits project out from the nucleosome core and are free to interact with otehr proteins
faciliate regulation of chromatin structure and function
Linker histones
strap DNA onto histone octamers and limit movement of DNA relative to the histone octamer
facilitates establishment of transcriptionally silent heterochromatin
How is DNA packed?
by histone octamers into a compact, flexible 30nm chromatin scaffold that can be remodelled to accomodate protein complexes involved in gene transcription and DNA replication
Chromatin is engineered to permit…
flexible responses to altered transcription factor activity caused by changes in cell differentiation status + signalling pathway activities
Interphase chromatin
comprises of a set of dynamic fractal globules that can reversible condense and decondense without becoming knotted
Nuclear periphery in interphase cells
composed of transcriptionally inactive DNA
RNA transcripts are excluded from the periphery
Chromosomes contain specialised DNA sequences that facilitate:
reliable and complete DAN replication
segregation of duplicated chromosomes during cell division
Telomeres
specialised repetitive DNA sequences at the ends of chromosomes
define chromosome ends and maintain their integrity
Telomerase
specialised DNA polymerse that replicated telomeres
synthesises single stranded 3’ overhanging TTAAGGG repeat arrays
ribonucleoprotein with an intrinsic RNA component that acts as a template
Centromeres
contain specialised proteins and DNA sequences that facilitate chromosome segregation during cell division
Alpha-satellite DNA repeats
in centromeres
readily form condensed chromatin with histone octamers containing unusual subunits
Kinetochore outer plate proteins
bind to protein components of the mitotic spindle
What is the function of kinetochore proteins?
part of the mechanism of ensuring faithful segregation of sister chromatids at cell division
Yeast kinetochore
basket that links a single nucleosome of centromeric chromatin to a single microtubule
Parasitic DNA
repeated DNA sequence elements that make up half of the human genome
copies of retrotransponons
Increasing biological complexity is accompanied by
increasing numbers of protein coding genes
increasing amounts of non-protein-coding DNA for regulating transcription and organising access to protein coding genes
Cis-regulatory information
encoded by non-protein-coding DNA sequences
determines where and when in the body adjacent protein-coding genes are transcribed
What are the 3 different types of transponons?
DNA transponons
retroviral trasnponons
non-retroviral polyA retrotransponons
Transponons
repeated DNA sequences
mobile genetic elements that jump around the genome
How do DNA transponons move?
by a cut and paste mechanism without self-duplication, requiring enzyme transposase
Non-retroviral polyA retrotransponsons
abundant in vertebrate genomes
replicates via an RNA intermediate using its own encoded reverse transcriptase
What do products of L1 reverse transcription do?
integrated directly into the genome at a new location without the need to be packaged into virus like particles
some are known to disrupt genes and cause diseases such as haemophilia
Evolution of transposing elements
non retroviral retrotransponsons have expanded hugely in numbers in evolution of higher mammals
most of the copies of transposing elements in the genome are defective and ancient relics of formerly functional elements
How is DNA synthesis initiated?
creating a replication fork where the DNA strands are seperated by DNA helicase
What direction does DNA replication happen?
5’ → 3’
Why is there a leading and lagging strand?
antiparallel orientation of parental strands
unidirectional orientation of new DNA synthesis
both new strands can’t be synthesised continuously
What strand has continuous synthesis?
leading strand
Why are primers needed?
DNA polymerases can’t start making a DNA chain from scratch
requires a pre-existing chain or short stretch of nucleotides
How is the short RNA primer synthesised?
using template and NTPs by DNA primase
once the primer is in place DNA polymerase extends it
What enzymes does lagging strand synthesis require?
DNA primase
DNA polymerase
ribonuclease H
DNA ligase
Lagging strand synthesis
DNA primase makes RNA primer → DNA polymerase- extends RNA primer which requires primer-template junction → ribonuclease H removes RNA primer → DNA polymerase extends across gap → DNA ligase seals the nick
Werner syndrome
caused by mutations in genes encoding DNA helicases + accessory 3’ exonuclease
causes premature ageing, genome instability + cancer in several sites
Sliding clamp
increases the processivity of DNA polymerase
once frist step of DNA synthesis has been accomplished interaction of enzyme with primer-template junction is maintained
makes addition of further nucleotides very rapid
ATP dependent
Single stranded DNA binding proteins
expose single stranded DNA in the replication fork
makes it available for templating synthesis of the new DNA strand and eases fork progression
DNA topoisomerases
prevent DNA from becoming tangled during DNA replication and enhance processivity of DNA polymerase
What is the function of topoisomerases?
helicase unwinding introduces superhelical tension into the DNA helix → tension is relaxed by DNA topoisomerases which nick and reseal the backbone of the helix
Origin of replication
single point where DNA replication starts
specific DNA sequences recruit replication initiator proteins
What are the 2 phases of DNA replication in eukaryotes?
- replicator selection in G1 phase- formation of pre-replicative complex
- origin activation in S phase- unwinding of DNA and recruitment of DNA polymerase
Eukaryotic replicator selection
origin recognition complex binds to replicator sequence (ARS in yeast) → helicase loading proteins Cdc6 and Cdt1 bind to ORC → the helicase Mnm2-7 binds to complete formation of pre-RC → inactive
Ribonuclease H
removes RNA primers which further shortens the newly synthesised DNA strands at 5’ ends of chromosomes
chromosome shortening risks loss of valuble coding info
What are the 2 types of things cells are under constant attack from?
endogenous sources
exogenous sources
What are endogenous sources?
reactions with other molecules within the cell like hydrolysis, oxygen species and by-products of metabolism
What are exogenous sources?
reactions with molecules from outside the cell like UV, x rays, carcinogens and chemotherapeutics
Types of endogenous DNA damage
depurination
deamination
methylation
Types of exogenous DNA damage
pyrimidine dimers
double strand breaks
interstrand crosslinks
What DNA damage effects both strands of the alpha helix?
double strand breaks
interstrand crosslinks
Deamination
removal of the amino group by hydrolysis
results in changes to the DNA bases
Transition mutations
more likely than transversions
less likely to result in amino acid substitutions
Depurination
the N-glycosidic bond is a common substrate for hydrolysis as it is an abasic site
more frequent at purine bases
What effect does UV light have on DNA?
induces the formation of pyrimidine dimers
distorts DNA and can also cause interstrand DNA crosslinks and DNA protein crosslinks
these block relpication and transcription so are highly toxic
Double backbone break inducers
x rays
ionising radiation
topoisomerase II inhibitors
Single backbone break inducers
reactive oxygen species
hydroxyurea
camptothecin
NER
nucleotide excision repair
repairs DNA damage when more than one base is involved such as pyrimidine dimers
involves excision of short patches of single stranded DNA to remove affected bases
Translesion synthesis
translesional DNA polymerases can replicate highly damaged DNA
lack precision in template recognition and substrate base choice
What does translesion synthesis cause?
base substitution and single nucleotide deletion mutations
What are the 2 mechanisms to repair double strand breaks?
non-homologous end joining NHEJ
homologous recombination
Non-homologous end joining NHEJ
error prone
restricted to G1 phase
usually results in the loss of nucleotides surrounding the break site and important genetic information
Homologous recombination
error free and accurate repair
occurs only in S phase by using intact sister chromatids as a template
What 3 places in the cell cycle can DNA damage be detected?
G1
entry to S phase
entry to mitosis
How is damage detected?
ATM/ATR get activated and associate with the site of DNA damage and this activates other kinases to block the cell cycle → p53 is stabilised and activates p21 → p21 renders the G1/S-CDK and its complexes inactive → preventing cycle progression → DNA is repaired and if this isn’t possible, apoptosis
Xeroderma pigmentosum
autosomal recessive disease associated with a defect in NER
skin cancer, UV sensitivity, neurological abnormalities
BRCA2 deficient cells
exhibit genomic stability+ sensitive to DNA damaging elements
defective in HR
breast, ovarian and prostate cancer
What are the two types of topoisomerase?
type I topoisomerases nick and reseal one of the 2 DNA strands so no ATP is required → type II topoisomerases nick and reseal both DNA strands which requires ATP
What enzymes are involved in base excision repair?
DNA glycosylase
AP endonuclease + phosphodiesterase
DNA polymerase
DNA ligase
DNA glycosylase
removes base that has been deaminated from DNA sequence
AP endonuclease + phosphodiesterase
remove sugar phosphate that incorrect base was attached to from backbone
DNA polymerase + ligase role in BER and NER
DNA polymerase adds in correct base(s) into gap created
DNA ligase seals the nick
What enzymes are involved in nucleotide excision repair?
excision nuclease
DNA helicase
DNA polymerase + ligase
Translesion synthesis process
covalent modifications to sliding clamp when it encounters damaged DNA
replicative DNA polymerase released
translesion DNA polymerase loaded by assembly factors
DNA synthesis continues
BRCA1 mutations
defective homologous recombination
breast ovarian cancer
Bloom syndrome
defective DNA helicase needed for recombination
cancer at several sites, stunted growth + genome instability`
