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Summary 'Advanced genetics'
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Summary of the course “Advanced genetics” at KU Leuven by Prof. Jan Michiels. Combined with Prof. Buys's part “Genetic analysis of higher organisms”. (32p.)
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Summary Advanced GeneticsPart 1: Genetic Variation
Chapter 1: Structure,organization and replication of genomes
A) Nuclear genomes of eukaryotes
Three Important elements for maintenance of eukaryotic chromosomes:
1) Centromere
Correct segregation of the chromosomes after DNA replication
- Centromere index = p/ (p+q) -> telocentric, acrocentric or metacentric
- Centromere in S. cerevisiae: CDE I-III (Cell-cycle Dependent Element), non-repetitive.
CDE II is wound around a core (of Cse4 and Mif2), CDE I and III are bound by Cbf1 and
Cbf3 (centromere binding factor).
- Centromere in animal cells: H3-nucleosomes in inner core + CENP-A nucleosomes on
the outer shell to which kinetochore is attached.
2) Origin of replication
Found at 30-40 kb apart throughout the length of the eukaryotic chromosome at
which the DNA replication machinery assembles for initiation of replication.
- For initiation of replication you need a replicator (genes) and a initiator (proteins).
Ori is part of the replicator. Sequence specific DNA binding proteins recognizes DNA
element in the replicator and activates replication.
- Replicators share 2 common features:
1) Binding site for the initiator protein
2) Stretch of AT-rich DNA for DNA unwinding
- Functions of the initiator:
Binding to replicator, interaction with additional factors and unwinding of DNA.
In eukaryotes: initiator is six-protein complex: Origin Recognition Complex (ORC).
No origin can be initiated after it has been replicated so unactive until the next round
of replication.
Not all potential origins need to be activated, some can passively replicate by which
the replication fork system pushes away the initiator system.
- Initiation of replication in eukaryotes includes two events that occur at distinct time:
1) Replicator selection: In G1, assembly of multiprotein complex to each replicator.
2) Origin activation: In S, replicator-associated protein complex initiates DNA
unwinding and recruits polymerase.
->Temporal separation ensures DNA is replicated only once.
- Formation of the pre-replicative complex:
o Helicase loading: binding of Mcm-complex next to ORC (by cdc6 and Cdt1).
o Helicase activation: activated by 2 protein kinases: Cdk and Ddk which are
activated in S-phase. Results in assembly of additional replication proteins.
Mcm becomes part of the replication fork and leaves the replicator.
- Cdk also have a second very important role in the regulation of transcription: It
inhibits the formation of new pre-RC’s by inhibition of helicase loading by inhibiting
the function of ORC, Cdc6 and Cdt1.
- Conclusion in eukaryotes: regulation of the replication by regulation of the initial
loading and activation of MCM complex.
, 3) Telomere
Resistant to recombination and DNA degradation; survival
- Functions:
o Replication
o Protection of the chromosome ends; maintenance of length
o Distinguish natural ends from sites of chromosome breakage
- 100-1000 copies of repeated sequences with short G-T rich overhang at 3’-end
TRF1 and TRF2 specifically bind to repeated sequences.
- Reason why Linear DNA molecules can shorten due to replication:
The final Okazaki fragment (lagging strand) cannot be primed or is at the extreme
end. After replication molecule has been shortened. Solution:
Telomerase binds with homologue telomerase RNA -> Extension of the end by DNA
polymerase and ribonucleoprotein until there is enough place for Okazaki fragment.
Three proteins prevent further elongation by telomerase: Rap1, Rif1 and Rif2. In
human cells: POT1. Telomere binding proteins binds to TRF1 or TRF2 and so protects
the chromosome ends of DNA repair enzymes. Another alternative for protection is
the forming of t-loop by the 3’ end with help of TRF2.
- Telomerase is absent in terminally differentiated cells.
Three-dimensional structure of the chromosome
- In human body 2*1010 km DNA -> efficient packaging is essential!
- Chromatin: DNA and protein that makes up a chromosome
- Nucleosoom: Histone core (8 proteins, 4 dimers: H2A, H2B, H3, H4) surrounded by
core DNA. Nucleosomes bound by linker DNA.
- DNA that is not packaged also binds proteins: Gene expression, replication…
- Higher order structures:
o Heterochromatine: little gene expression, condensed
o Euchromatine: higher level of gene expression, more extended
- H1 (fifth histon) binds two DNA regions: linker DNA and in the middle of core DNA.
- Stabilization through interactions between N-terminal tails of core histones with
adjacent nucleosomes: interactions between pos. charged N-terminus of H4 with
neg. charged region of H2A -> compactation into the 30 nm formation (hetero) !
- Further levels of genome organization: (ranked from small to large size)
o Nanodomains
Enhancer- promoter contact
o Topologically associating domains (TAD’s)
CTCF-cohesine dependent chromatin loops
o Gene-active and gene-inactive compartments
A and B compartiment
o Chromosome territories
lamina-associated domain, nucleus associated domain….
- Transcription occurs at clustered sites: transcription factories
,- Regulation of chromatin structure:
o Interaction of dna with the histone octamer is dynamic-> non-covalent bonds
Probability of release depends on the position of the binding site within the
nucleosome.
o In addition to intrinsic dynamics, stability of histone octamer-DNA also
influenced by large protein complexes: Nucleosome-remodeling complexes
(NRC), using energy of ATP-hydrolysis.
All NRC’s can catalyze sliding of DNA, some can also transfer/ejection a
histone octomer and some can facilitate the exchange of the H2A/H2B dimer
with variants with other properties.
o Nucleosome positioning: most nucleosomes are not fixed on one location
but sometimes positioning is beneficial (e.g. a DNA-binding site remains
accessible in the linker DNA -> regions upstream of active transcription start
sites often are nucleosome-free). Two mechanisms:
1) By DNA-binding proteins, 2 options:
a) Competition between TF (= DNA binding proteins) and nucleosome
-> Nucleosome requires more than 150 bp of DNA (without TF’s)
-> They inhibit the formation of nucleosomes
b) Some DNA binding proteins bind to nucleosomes
-> Once bound to DNA, it facilitates the assembly of nucleosomes
-> They promote the formation of nucleosomes
2) By particular DNA sequences
-> These have high affinity for nucleosomes
-> nucleosomes preferentially form on DNA that bends easily.
-> sequences that alternate between A/T and G/C rich sequences are
preferred as nucleosome-binding sites.
o Modifications of the histone amino-terminal tails alters the accessibility of
chromatin. Position and nature of modification is crucial to the effect.
1) They affect the charge of N-terminal tails of histones: reduction of pos.
charge by acetylation or phosphorylation(HAT:histone acetyl transferase)
2) Influence on the formation of repressive 30 nm fibers due to acetylation
of H4 N-terminal domain interfering with the binding to H2A histone.
3) Modifications recruit specific proteins to the chromatin
e.g.: bromodomain, chromodomain ….
Which proteins are recruited to modified histones?
-> Modified nucleosomes recruit enzymes that will further modify
adjacent nucleosomes; enzymes for histone modifications.
o Nucleosome modifications and remodeling work together to increase the
DNA accessibility.
, o Where do nucleosomes come from? Nucleosome assembly:
-> They are assembled immediately after DNA replication: nucleosome
disassembly and reassembly-> critical role in the genetic inheritance!
- disassembly: old histones include the modifications = memory
* H3-H4 tetramer remain bound to one of the two daughter
duplexes for maintenance and propagation of modifications.
* H2A-H2B dimers are released to a local pool -> available
- assembly:
* first binding of H3-H4 tetramer
* then association of two H2A-H2B dimers
* finally binding of H1
* not a spontaneous process; assembly requires histone chaperones:
these are negatively charged proteins that forms complexes with H3-
H4 tetramer (CAF-I , recognizes PCNA) and H2A-H2B dimers (NAP-I)
and escort them to sites of nucleosome assembly.
o Epigenetic gene regulation:
Histone modifications
DNA methylation: imprinting
-> maintenance methylase recognizes methylations on old strand.
Protein distribution: e.g. bacteriophage lambda lysogen
o Repeated sequences in genomes, pseudogenes and gene fragments
Pseudogenes: genes that are no longer active
-> could be conventional (mutations) or processed (mRNA copy
inverted into cDNA and reinserted into the genome.
Truncated genes: lack a stretch from one end of the gene
Gene fragments: short isolated regions of gene
Intergenic regions (62% of human genome)
Often contain repetitive DNA
* genome wide repeats or ‘interspersed repeats’
* tandemly repeated DNA or ‘satellite DNA’
Minisatellites (up to 20 kb) or microsatellites (up to 150 bp;
dinucleotide repeats or single nucleotide repeats)
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