DNA as a molecule is very long. It needs to fit into a cell / into the nucleus

Ploidy refers to how many sets of corresponding (the lecture says identical but on a base to base level there can be differences) chromosomes an organism has

No, but they have similarly positively charged proteins, like IHF (Integration host factor)

H2A, H2B, H3, H4

No there are tails extending from the core, these are often the target of chemical histone modifications

As an outer particle, additional packing

~146b

micrococal DNAase digestion, the nucleosme protects the DNA, then run the fragments on a gel

These ae likely due to incomplete digestion

The 10nm fiber is further packaged into a 30nm fiber requiring the linker histone H1

By nucleosome remodelling factors. These can change the localization of the nucloesomes with respect to the DNA

1) SWI/SNF: disrupts nucleosome positioning 2) ACF (ISWI family): positions nucleosomes during chromatin assembly

Euchromatin is found in less condensed regions, whereas heterochromatin is more condensed and thus stains darker. Generally there is less transcription in heterochromatin.

–Acetylation (Ac)

–Ubiquitination (Ub)

–Methylation (Me)

–Phosphorylation (P)

–Sumoylation (Su)

Histone acetylase.

Histone deacetylase.

Histone methyltransferase

Histone 3 Lysine 27 triple methylated and it is associated with genes.

Metyhlation of Cytosines =>5 methyl cytosine; Methylation of Adenine e.g. for the bacterial restriction system

Methylated cytosines are less chemically stable, so more likely to undergo deamination, which changes the base to thymine (as opposed to U)

-Bromodomain (binds acetylated lysines)

-Chromodomain (binds methylated lysines)

e.g. through modified histones or through RNA based mechanisms

See slide 65

When two alleles at the same locus interact, and where one allels causes a heritable change in the other allele.

Point centromers are very small, regional are several hundred kb long and provide the regions for the spindles to attach. In holocentric centromers no defined centromer exists, the spindels attach everywhere.

telomers are the ends of the chromosomes, they protect the chromosomes and can be partly single stranded and/or folded back. As DNA polymerase can't copy the ends, one way to maintain the ends is telomerase an enzyme using internal RNA as template.

it solved the "end" problem by prolonging special structures "telomeres" at chromosome ends.

Purine versus Purine exchange

Purine versus Pyrimidine exchange, or vice versa

Every 100,000 to 1,000,000 bp (One in each 100kb to 1Mb). This would result in many many mutations as plant genomes are easily in the range of 1,000,000,000 Bases (Gbases)

tautomeric structures of DNA bases

cell internal metabolites, e.g. reactive oxygen species

external chemicals e.g. alkylation agents

radiation e.g. UV or X-ray

deamination

base slippage

Direct reversal of the damage by photolyases

The protein transfers the alkyl group onto itself, as this is irreversible the protein is no enzymes and is thus "used up". However it then stimulates its own production.

Its role is in mismatch repair MutS recognizes a mismatch, MutH a hemimethylated strand and nicks the strand

damaged bases are removed by glycosylase (there are many different ones) AP site is recognized by an endonuclease, it cleaves the backbone and the hole is filled => 2/17

For bulky lesions

Translesion polymerases, they are able to polymerize in a non-templated fashion when (bulky) damage has occurred

The TLs polymerase are error prone and just ensure that DNA can be replicated, this often leads to mutations. It can safeguard replication though 2/22ff

Normally LexA is blocking the synthesis of SOS genes by binding to a DNA operator. If RecA bind ssDNA it is activated and cleaves LexA. Cleaved LexA can't bind to the operator anymore, SOS genes are read. =>2/28,29

They play a role in the eukaryotic damage reponse e.g. to double strand breaks.

Cell cylce control, replication fork stabilization, replication origin control

See slide 2/38

Non homologous end joining, joining DNA ends, it is predominant in non dividing cells. (G1 phase)

A double stranded break is repaired by simply joining the ends potentially after trimming a few bases by a nuclease.

DNA ends are resected, regions of micro"homology" are found, DNA pairs, gaps are filled and overhangs trimmed

Resection occurs to generate 3' overhangs, one of the 3' overhangs invades an intact duplex, generating a heteroduplex and a displacement loop,

new DNA is synthesized, until this can pair with the other 3' overhang. Then repair replication and synthesis (Probably helpful if you could draw a diagram here)

It plays a role in homology directed repair, where it has nuclease and helicase activity, and in the end it generates 3' tails after encountering Chi sites.

Special DNA sequences recognized by the recBCD complex.

It loads onto the single stranded DNA and helps in strand exchange it also plays a role in homologous recombination

DNA is changed and is afterwards a copy of the homologous chromosome. This can lead to a loss of heterozygosity

The switching uses homology directed repair. There are three loci: the active MAT locus and two silent loci one carrying alpha and one a information.

The endonuclease HO cuts in the MAT locus and either the information of alpha or a is copied there potentially changing the mating type.

No this depends on how the junction is cleaved

Movement of the holiday junctions

After a double strand break, DNA is resected and bound by proteins which help in invading the other duplex. DNA

is synthesized from the invading strand. The displaced strand of the D-loop is captured by the remaining 3' end.

DNA synthesis occurs and holiday junctions form

They are similar and the initial processes are almost the same

A single strand nick leads to a ds break after strand separation. One strand of the "broken off" end invades the

"whole" part a D-loop structure is formed, DNA synthesis occurs, the displaces strand is captured by the lagging strand

lagging strand synthesis is restarted and after cleavage the fork is restored

Resection occurs to generate 3' overhangs, one of the 3' overhangs invades an intact duplex, generating a heteroduplex and a displacement loop,

new DNA is synthesized. The displaced strand from the D-loop is not captured. Here lagging strand synthesis starts occuring => 3/50

Potentially loss of heterozygousity

Potenntially this Replication is not as precise as normal replication 4/44f

Recombination on the same chromosome

Unequal crossover between chromatids or between homologs can lead to deletions and duplications

Crossing over between different chromosomes can lead to DNA translocation

See 4/2

transposition / transposable elements (in the wider sense, for an inspiring read: look at her short bio it shows how far ahead she was)

Autonomous elements encode the enzymes needed to move. Non-autonomous elements lack these and rely on enzymes of Autonomous elements 5a/4

High

They are inactive e.g. due to mutation (also see section 2)

The gene function or regulation can be affected potentially resulting in disease

No in general not, but be aware of the special cases

Class I elements are retrotransposons that move via an RNA intermediate, Class II elements are DNA only transposons

It has terminal inverted repeats and a transposase gene.

LTR and non LTR

Class II elements can likely cross the species barrier

Cut and Paste

E.g. pathogenicity factors and antibiotics resistance

A bacterial cut and paste DNA element only encoding its own transposase

Two simple transpons (IS elements) flanking (another) gene(s)

Two

The transposase cuts in trans, i.e. they cut the other end that they don't bind

GCATxxxxTransposonxxxxGCAT

Upon insertion a piece of DNA is duplicated, when the transposon leaves, this duplicated DNA can remain -> a footprint

When the transposon leaves and the resulting gap can be repaired by homolgy directed repair from a transposon free site, the original state can be restored and no footprint remains

Antibodies joining of different pieces, more possible antibodies, several possible fragments for each V D J pieces

A DNA mobile element. It was only recently introduced into Drosophila by crossing the species barrier.

Using microinjection this is a way to make transgenic flies.

This is a fish transposon which was no longer active. Using the consensus sequence the ancestral working state could be restored

long terminal repeat

>LTR=== GAG, PR, IN, RT-RH ===LTR>

INtegrase, GAG, PRotease, RT-RnaseH reverse transcriptase+ RNAse activity

ENV for extracellular stage

It can get lost due to homologous recombination between the elements

very high

Reverse Transcription is error prone. reverse transcription is part of the life cycle.

ORF1/ORF2 and polyA

a stretch of Ts that pair with the polyA tail

DNA methylation both prevents transposase expression and transposon end activity

It is active in germ line but not somatic cells.

They block transcription of certain transposons.

If transposons preferably insert in a location close to the donor site.

Conservative site-specific recombination

They are topoisomerases

Serine and Tyrosine type

Tyrosine recombinases break DNA and form DNA-3P-tyrosine linkage, Serine recombinases break DNA and form a DNA 5P-serine linkage

=>4/47 f

=>4/51 ff

a (tyrosine) recombinase that recognizes lox sites.

To delete DNA between two lox sites, this is often used as a tool where Cre is expressed under a specific promoter. After Cre expression, a piece of DNA is removed and for example a tissue specific knockout is made.

attP, attB -> attL, attR

phage lambda recombination

RNA has U instead of T and ribose instead of deoxyribose.

Initiation, Elongation, Termination

RNA polymerase I (large ribosomal RNA), II (mRNA), III (tRNA and 5S rRNA)

They have an additional polymerases (apart form I, II , III) to transcribe regulatory RNAs

It contacts the promoter

TATA box

TATA binding protein TBP

RNA polymerase is not immediately working fully instead it enters cycles of abortive initiation resulting in short pieces of RNA being released. This is likely due to a loop of eukaryotic TFIIB and bacterial sigma factors extending into the polymerase

When the loop of TFIIB/sigma inserted into the RNA polymerase is displaced and the polymerase breaks away from the promoter

It becomes phosphorylated

Incubate RNA polymerase holoenzyme with radioactive nucleotides. Run products on gel and visualize with Xray film

locked nucleic acids (these are locked in a conformation more favourable for hybridization)

Short complementary regions in the nascent transcript forming hairpins or weak DNA-RNA hybrids

Elongation factors

RNA polymerases reverses direction, most recently made RNA separates off from DNA, this protruding end can be chopped off

This is often capping, happening while RNA is still being made

It helps in nuclease degradation, elongation termination of transcript, mRNA processing, export from the nucleus and directing translation

7-methylguanine is lined via a 5'-5' triphosphate to the 5' end of the RNA. (In more complex eurkaryotes 2' O of ribose in the second and third base gt methylated) 6a/37

Changes in supercoiling

A site where bacterial RNA polymerase terminates transcription without any additional factors

An inverted repeat that forms a stem loop in the RNA, and a polyA (DNA) region, that leads to less stable AU base pairs

An enzymatic terminator

The former make a hairpin and have a poly A region the latter (Rgho) don't

It is coupled to 3' end processing

They get polyadenylated

No, the nascent transcript is cleaved first and then the polyA tail is added.

It must specifically recognize its right regulatory sequence e.g. through a protein domain

In the major grove all bases can potentially be distinguished this is not necessarily the case for the minor grove

positively charged amino acids, as the DNA is negatively charged

It cleaves it into short pieces

siRNA -> silencing (Give some details about AGO, Dicer etc)

They can spread between cells.

When a construct is brought into a genome in normal sense orientation and both the introduced gene and the endogenous gene are silenced

It produces secondary siRNA

By combing them to achieve sequence specificity and then adding a FokI nuclease domain.

By combing them to achieve sequence specificity and then adding a FokI nuclease domain.

One Zinc finger => three nucleotides; One TAL repeat one nucleotide

see lecture

see lecture