Part I Packaging of DNA
Why does DNA need to be packaged? DNA as a molecule is very long. It needs to fit into a cell / into the nucleus
What is ploidy? 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
Do bacteria have histones? No but they have similarly positively charged proteins, like IHF (Integration host factor)
What are the four histones making up the core particle H2A, H2B, H3, H4
Are the four histones making up the core completely "hidden" by the DNA? No there are tails extneding from the core, these are often the target of chemical histon modifications
Where does H1 come into play? As an outer particle, additional packing
How long (in nt) is a strech of DNA around a nucleosme? ~146b
How can one determine the length of DNA around a nucleosome? micrococal DNAase digestion, the nucleosme protects the DNA, then run the fragments on a gel
If you digest chromosomal DNA with micrococal DNA and run them on a gel you get shortisch fragments of around 170-200bp but also longer and much longer bands, where are these coming from? These ae likely due to incomplete digestion
How is DNA that has been wrapped around the histone futher packaged? The 10nm fiber is further packaged into a 30nm fiber requiring the linker histone H1
Sometimes "packed" chromosomal DNA needs to be accessed, how is this problem solved? By nucleosome remodelling factors. These can change the localization of the nucloesomes with respect to the DNA
Give ONE example of nucleosome remodelling complexes. 1) SWI/SNF: disrupts nucleosome positioning 2) ACF (ISWI family): positions nucleosomes during chromatin assembly
What is hetero and euchromatin. What can you say as a rule of thumb about the transcriptional activity of these two? Euchromatin is less condensed regions, whereas heterochromatin is more condensed and thus stains darker. Generally there is less transcription in heterochromatin.(heterochromatin)
What histone modifications do you know? –Acetylation (Ac) –Ubiquitination (Ub) –Methylation (Me) –Phosphorylation (P) –Sumoylation (Su)
What does HAT stand for? Histone acetylase.
What does HDAC stand for? Histone deacetylase.
What does HMT stand for? Histone methyltransferase
What is H3K27me3 short for and with what is this histone modification associated? Histone 3 Lysine 27 triple metylated and it is associated with genes.
Which chemical modification of DNA do you know? Metyhlation of Cytosines =>5 methyl cytosine; Methylation of Adenine e.g. for the bacterial restriction system
Why is DNA methylation (in eukaryotes) risky? Methylated cytosines are less chemically stable, so more likely to undergo deamination, which changes the base to thymine
What does a bromo- and what does a chromodomain do, in the context of binding to certain structures? -Bromodomain (binds acetylated lysines) -Chromodomain (binds methylated lysines)
Methylation in CG can be propagted trhough DNA replication, as the site is symmetric how can the state be propagted in asymmetric sites ? e.g. through modified histones or through RNA based mechanisms
Explain position effect variegation ? See slide 64
What is para mutation? When two alleles at the same locus interact, and where one allels causes a heritable change in the other allele.
Explain what point, holocentric and regional centromers are? 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.
What are telomers and how are they maintained (pick one mechanism)? telomers are the ends of the chromosomes, they protect the chromsomes 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.
What is the role of telomerase? it solved the "end" problem by prolonging special structures "telomeres" at chromosome ends.
What is a transition? Purin versus Purin exchange
What is a transversion? Purin versus Pyrimidin exchange, or vice versa
Name some sources of mistakes which could eventually lead to mutations: 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
What is a simple mechanism to repair UV induced tymine dimerization? Direct reversal of the damage by photolyases
Ada is involved in alkylation repair, what is special about this repair? The protein transfers the alkyl group onto itself, as this is irreversible the protein is no enzymes and is thus "used up"
What is the role of Mut SLH? Its role is in mismatch repair MutS recognizes a mismatch, MutH a hemimethylated strand and nicks the strand
Explain the Mut SLH mechanism. See slide 2/16
Explain Base excision repair 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
When is nucleotide excision repair being used? For bulky lesions
What are TLS polymerase Translesion polymerases, they are able to polymerize in a non-templated fashion when damage has occurred
Why are TLS polymerase a last resort? The TLs polymerase are error prone and just enusure that DNa can be replicated, this often leads to mutations. It can sageguard replication though 2/21ff
Explain the LexA-RecA system. Normally LexA is blocking the syntehsis 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. =>2/27,28
Where do ATM and ATR play a role and what can activate them (in terms of which state not which protein)? They play a role in the eukaryotic damage reponse e.g. to double strand breaks.
What are effects of ATR activation? Cell cylce control, replication fork stabilization, replication origin control
Explain the regulation of p51. See slide 2/40
What is NHEJ Non homologous end joining, joining DNA ends, it is predominant in non dividing cells. (G1 phase)
Explain the simplest case of NHEJ A couble stranded break is repaired by simply joining the ends potentially after trimming a few bases by a nuclease.
When resection occurs during NHEJ, what happens? DNA ends are resected, regions of microhomology are found, DNA pairs, gaps are filled and overhangs trimmed
Briefly depict the process of homology directed repair with synthesis dependent strand annealing (no protein names are necessary, but give the neccessary terms for the DNA structures)? 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 diagrm here)
What is the role of RecBCD in E. coli? It plays a role in homology directed repair, where it has nuclease and helicase activity and in the end generates 3' tails after encountering Chi sites.
What are Chi sites? Special DNA sequences recognized by thr recBCD complex.
Genes involved in repair and recombination are often implicated in some diseases as well, name two example for such a disease Werner Syndrome Bloom Syndrome Rothmund Thomson Syndrome
What is the role of RecA (RAd51) in homology directed repair, where else does it occur? It loads onto the single stranded DNA and helps in strand exchange it also plays a role in homolgous recombination
What is the consequence of using homologous chromosomes as templates for homology-directed repair? DNA is changed and is afterwards a copy of the homologous chromosome. This can lead to a loss of heterozygousity
Explain how yeast switches its mating type and why this is interesting for Molecualr biology? The switching uses homology directed repair. There are thre loci the active MAT locus and two silent loci one carrrying 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.
Does the cleavage of a holiday junction always lead to large areas of recombinant product? No this depends on how the junction is cleaved
Where is RuvAB involved in the broad complex of recombination? Movement of the holiday junctions
Depict the first steps in homologous recombination After a double strand break, DNA is resected and bound by proteins which help in inavding the other duplex. DNA is synthesiszed 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 3/41
What is the relationship between homology directed repair and homologous recombination? They are similar and the initial processes are almost the same
How can damaged replication forks (specifically nicks) be repaired with a principle remeniscent of recombination? single strand nick leads to a ds break after strand separtion. 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 clevaged the fork is restored see
Describe Break Induced replication 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
What are some consequences of Break Induced replication? Potentially loss of heterozygousity Potenntially this Replication is not as precise as normal replication 4/58f
Name at least two problems that can occur with recombination due to sequence similarity regions? 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
Be ready to explain a C0t curve See 4/2
What did Barbara McClintock discover transposition / transposable elements
Explain autonmous and non-autonomous elements Autonomous elements encode the enzmes needed to move. Non-autonomous elements lack these and rely on enzymes of Autonomous elements 5a/4
Would you rate the number of transposable elements in the humann genome as high or low? High
What is the state of many transposons in the human genome? They are inactive e.g. due to mutation (also see sction 2)
If a Mobile Elementsjumps into a functional gene, what can be the consequence for gene function? The gene function or regulation can be affected potentially resulting in disease
Does transposition necessarily require homology? No in general not, but be aware of the special cases
What are Class I and Class II elements? Class I elements are retrotransposons that move via an RNA intermediate, Class II elements are DNA only transposons 5a/11
What is the general structure of a Class II element? It has terminal inverted repeats and a transposase gene.
What are the two major Class I element groups? LTR and non LTR
What is remarkable for Class II elements when it comes to the species barrier? Class II elements can likely cross the species barrier
What is the predominant movement mechanism for DNA elements? Cut and Paste
What is the less common movement mechanism for DNA elements? Nick and Paste
When it comes to bacterial transposons, which other genes apart from the transposase do they often carry? E.g. pathogenicity factors and antibiotica resistance
What is an IS Element? A bacterial cut and paste DNA element only encoding its own transposase
What is a compound transposon? Two simple transpons (IS elements) flanking (another) gene(s)
For a DNA cut and paste transposon, how many transposase proteins would you need in general to mobilize one transposon? Two
How is it ensured that transposon ends are brought together before cutting for cut and paste DNA elements? The transposase cuts in trans, i.e. they cut the other end that they don't bind
In the case of DNA cut and paste elements: When such an element inserts into the sequence CGAT. How will the sequence look after insertion. GCATxxxxTransposonxxxxGCAT
After a DNA cut and paste transposon leaves a certain site a footprint remains. Explain what this is. Upon insertion a piece of DNA is duplicated, when the transposon leaves, this duplicated DNA can remain -> a footprint
After a DNA cut and paste transposon leaves a certain site a precise excision can occur. Explain what this is and name conditions. 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
Explain in broad, superficial terms where VDJ recombination occurs? Antibodies joining of different pieces, more possible antibodies, several possible fragments for each V D J pieces
What is the relationship between VDJ recombination and transposons? They could be related as there are certain similarites both in the enzymes as well as in the signal DNA sequences
What is the P element and what is remarkable about it? A DNA mobile element. It was only recently introduced into Drosophila by crossing the species barrier.
Name an application in the lab for the Drosophila P element? Using microinjection this is a way to make transgenic flies.
What is sleeping beauty and how was "she" awakened from "her" sleep? This is a fish transpon which was no longer active. Using the consensus sequence the ancestral working state could be restored
What is sleeping beauty and how did "she" fall asleep in the first place? This is a fish transpon which was no longer active. Due to mutations the inserted transposons became "dormant"
Name the two clasess of RNA retrotransposons LTR and non-LTR elements
What does LTR stand for in LTR (RNA) elements? long terminal repeat
Draw an archetypical LTR element. >LTR=== GAG, PR, IN, RT-RH ===LTR>
Within a LTR Retroelement you usually find coding regions for 4 proteins, name these? INtegrase, GAG, PRotease, RT-RnaseH reverse transcriptase+ RNAse activity
You have a working LTR retroelement, when you compare that to a retrovirus which protein coding regions is missing? ENV for extracellular stage
Describe how a LTR element multiplies and integrates into new regions?
What can happen when you have two LTR elements spaced a bit apart from each other with the intervening DNA? It can get lost due to hmologous recombination bewteen the elements
Do you rate the non-LTR element occurrence in mamamalian genomes as absent/low/medium/high/very high? very high
Why are RNA elements very often mutated? Reverse Transcription is error prone. reverse transcription is part of the life cycle.
What are the typical elements for a LINE element ORF1/ORF2 and polyA
What is the sequence requirement for an insertion of some non-LTR elements (e.g. LINEs) and why? a stretch of Ts that pair with the polyA tail
How does DNA methylation affect IS10 transposition? DNA methylation both prevents transposase expresison and transposon end activity
In which cells is the Drosophila P element active and in which one is it not active? It is active in germ line but not somatic cells.
What is the role of piRNA? They block transcription of certain transposons.
Ty5 is a relatively "benign" element when it comes to gene mutations why? It preferabley inserts into heterochromatin
Explain what local hopping means for certain transposons? If transposons preferably insert in a location close to the donor site.
What does CSSR stand for? Conservaive site-specific recombination
When it comes to CSSR recombinases which enzyme class do they fall into? They are topoisomerases
Which two major classes of CSSR recombinases exist (hint name the AA)? Serine and Tyrosine type
What is the difference between Serine and Tyrosine Recombinases when it comes to the intermedeate form, describe both forms? Tyrosine recombinases break DNA and form DNA-3P-tyrosine linkage, Serine recombinases break DNA and form a DNA 5P-serine linkage
Draw and explain the recombination of a serine resolvase. =>4/99 ff
Draw and explain how the Cre recombination occurs (a tyrosine recombinase). =>4/103 ff
What is the Cre enzyme and what does it do? a (tyrosine) recombinase that recognizes lox sites.
For what can the cre lox system be used for? To delete DNA between two lox sites, this is often used as a tool where Cre is expressed under a specfic promoter. After Cre expression, a piece of DNA is removed and for example a tissue specific knockout is made.
The bacteriophage lambda system has four sites that are named on DNA (hint two sites get converted into two other sites), name the sites? attP, attB -> attL, attR
What is the Invitrogen Gateway system system building on (which molecular mechanism from which organisms)? phage lambda recombination
What are the major two differences between RNA and DNA? RNA has U instead of T and ribose instead of deoxyribose.
Name the three major phases in transcription Initiation, Elongation, Termination
Name the three major eukaryotic RNA polymerases and their primary role RNA polymerase I (large ribosomal RNA), II (mRNA), III (tRNA and 5S rRNA)
What is special for plant RNA polymerases? They have an additional polymerases (apart form I, II , III) to transcribe regulatory RNAs
What does the bacterial sigma factor do? It contacts the promoter
Eukaryotic promoters have a box very similar in sequence to the bacterial -10 element, how is this box called? TATA box
Which protein do all thee eukaryotic RNA polymerases need for establishing the pre-initiaion complex? TATA binding protein TBP
Interestingly, when RNA polymerase starts making RNA, a strange phenomena occurs resulting in short pieces of RNA, explain this? 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
What is promoter clearance? When the loop of TFIIB/sigma inserted into the RNA polymerase is displaced and the polymerase breaks away from the promoter
What happens to the CTD when eukaryotic RNA polymerase is converted into the elongating complex after promoter clearance? It becomes phosphorylated
Describe a simple! experiment to show that abortive initiation takes place in vitro Incubate RNA polymerase holoenzyme with radioactive nucleotides. Run products on gel and visualize with Xray film
To show that abortive initiation takes place in vivo special kinds of nucleic acids were used, name them and indicate why these were used? locked nucleic acids (these are locked in a conformation more favourable for hybridization)
What are potential reasons for transcriptional pausing? Short complementary regions in the nascent transcript forming hairpins or weak DNA-RNA hybrids
Which factors can relieve transcriptional pausing? Elongation factors
Explain how transcriptional pausing may be relieved? RNA polymerases reverses direction, most recently made RNA separates off from DNA, this protruding end can be chopped off
What is one of the first maturation steps that occurs to eukaryotic mRNA? This is often capping, happening while RNA is still being made
What is the role of the 5' cap for the mRNA? It helps in nuclease degradation, elongation termination of transcript, mRNA processing, export from the nucleus and directing translation
What is the minimal structure incorporated into a cap? 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
What topological problem can transcription cause? Changes in supercoiling
What is a (bacterial) intrinsic terminator? A site where bacterial RNA polymerase terminates transcription without any additional factors
What are the two main features of a bacterial terminator? An inverted repeat that forms a stem loop in the RNA, and a polyA (DNA) region, that leads to less stable AU base pairs
What is the bacterial Rho protein (in the field transcription)? An enzymatic terminator
What is the difference between intrinsic and Rho dependent genes in terms of termination? The former make a hairpin and have a poly A region the latter (Rgho) don't
What is eu. RNA polymerase II termination coupled to? It is coupled to 3' end processing
What happens to most eukaryotic 3' mRNAs? They get polyadenylated
Is the polyA tail of eukaryotic mRNA added to the last based made by RNA polymerase II? No, the nascent transcript is cleaved first and then the polyA tail is added.
What must a RNA transcription regulatory protein do? It must specifically recognize its right regulatory sequence e.g. through a protein domain
Why can it be favourable to contact the major grove of DNA in terms of specificity? In the major grove all bases can potentially be distinguished this is not necessarily the case for the minor grove
Which kind of amino acids would you expect to find in proteins that contact DNA and why? positively charged amino acids, as the DNA is negatively charged
What can Dicer (and Dicer Like) do to double stranded RNA? It cleaves it into short pieces
How can plants defend themselves against plant viruses? siRNA -> silencing (Give some details about AGO, Dicer etc)
What is remarkable about siRNA duplexes? They can spread between cells.
What is Co-suppression? When a construct is brought into a genome in normal sense orientation and both the introduced gene and the endogenous gene are silenced 7/29
What is Co-suppression? When a construct is brought into a genome in normal sense orientation and both the introduced gene and the endogenous gene are silenced 7/29