User:Loguej

From OpenWetWare

Jump to: navigation, search
BMCB625 Advanced Topics in Molecular Biology

Home        People        Materials        Schedule        Help        Discussion       

Contents

Education:

B.S. in molecular, cellular and developmental biology with minors in organic and biochemistry from the University of Oregon, Eugene, OR

Currently pursuing a Ph.D. in biochemistry and molecular biology from Oregon Health and Sciences University, Portland, OR

Thesis mentor Dr. John D. Scott

Thesis project:

Compartmentalized cAMP/PKA signalling via the A-kinase anchoring protein 220 (AKAP 220).

Publications:

Mooers, BH, Logue, JS, Berglund, JA "The structural basis of myotonic dystrophy from the crystal structure of CUG repeats" (2005) PNAS Nov 15;102(46):16626-31.

Contact:

loguej AT ohsu DOT edu


Homework and Questions:

DNA Replication - Week 2

Lopes et al


Despite the remarkable discovery that DNA polymerase can transcribe through UV lesions when yeast cells lack NER, list potential genotoxic consequences of transcribing through these lesions?

Answer:

Leaving behind DNA lesions and ssDNA, poses many problems to the cell. The lack of nucleotide excision repair is sufficient to introduce mutations into the genome by mis-pairing to DNA UV-adducts. Additionally, ssDNA gaps that are filled in post-replication, would be expected to have abberant chromatin structure, according to current models of DNA replication and chromatin structure. Current models suggests nucleosomes are added during replication, and subsequently modified. Filled in ssDNA post-replication, would likely produce hypersensitive sites, if in the right place, this could dramatically upregulate a gene. It would be interesting to know whether nucleosomes at these sites can be added post-replication, or if neighboring nucleosomes can slide and fill in the gaps to allow for proper chromatin stucture.


Heller and Marians


Why might the mechanism described by Heller and Marians be considered heretical?

Answer:

The mechanism proposed by Heller and Marians could certainly be considered heretical. The mechanism proposed, where DNAG primase fills in ssDNA gaps post replication suggests DNAG can act independently of the transcription machinery. DNAG is most often associated with the lagging strand during DNA replication, acting in concert with the replication fork and replication machinery. This mechanism suggests DNAG can be recruited to ssDNA independent of the replication machinery, and independent of an origin of replication, of which is tightly regulated. This mechanism in some ways shatters the current models of DNA replication and repair, and revives old ones. The work by Heller and Marians redefines the functionality of DNAG, and mechanisms of repair.


New Components of DNA Replication - Week 3

Moyer et al


1. If the purification strategy in figure 1A hadn't been so stringent, what other kinds of factors might of been isolated with cdc45?

Answer:

The stringent purification used in Moyer et al not suprisingly isolated a core helicase complex. A less stringent approach may have identified new components of the replication or pre-replication machinery like cdt-1. Mass spectrometry based approaches to identify new protein partners or complexes is a compromise between isolating tightly bound proteins or loose or transient partners, the latter can often produce false positives since this requires gentle conditions.


2. What does figure 5 prove?

Answer:

Figure 5 demonstrates that cdc45 is necessary for S-phase progression. The others suggest this shows that cdc45 is a necessary component of the replication machinery in vivo, but cdc45 may also trigger the S/M cell cycle check point. Further experiments in vivo are necessary to prove that cdc45 is a component of the replication machinery. Co-localization, for example, of cdc45 using replication specific antibodies towards polII on a replicating chromosome may provide further evidence.


Structure Function of DNA Helicases - Week 8

Q1. Does the fact that helicase subunits are stabely bound by ADP in the crystal hint at something mechanistically important?

A1. The fact the structure is stable when bound by ADP suggests a stable intermediate, mechanistically. In other words, an unloading step, where ADP is removed so that ATP may enter may be required for helicase function.


Q2. The structure reveals several polar contacts between the helicase and DNA, but highly non-polar residues are within VDW distance of the DNA. However counter-intuitive, why might this be important?

A2. Polar contacts are necessary to obtain a grip on DNA, the non-polar, or greasy contacts however, may provide speed for rapid helicase function. There may be a balance between polar and non-polar contacts to allow rapid unwinding, where the DNA slides across the greasy surfaces.


RNA Exon Junction Complex - Week 9

Q1. The authors down play the observation that the RNA is in a bent conformation. Why might this actually be important?

A1. The bent conformation observed for the bound polyU RNA may help explain why the EJC complex binds specifically at exon junctions. The spliceosome bends RNA through out the splicing cycle. The EJC complex may therefore bind at particular bends imposed by the spliceosome - this would lower the entropic cost of binding for the EJC complex.


Q2. Suggest some possible experiments to test the observed protein-protein interaction network and it's effect on assembly on RNA?

A2. As for all x-ray crystal structures, the crystallographers or collaborators should make an attempt to support their structure using experiments in solution. This is particularly important when characterizing protein-protein interactions, where point mutations should be made at sites thought to be important from the crystal structure and tested in a solution based assay. This helps to rule out crystal packing artifacts, a somewhat common artifact of shoving proteins together to form a crystal lattice. The authors unfortunately did not do this for the EJC crystal structure. GST pull down assay and native PAGE using radiolabeled RNA could be used to test important contacts, and investigate potential cooperativity between protomers in the EJC complex as it forms on RNA.


Mathematics in Biology

Q1. Thermodynamically speaking, why might it not be surprising that biological events exhibit hysteresis and bimodal behavior?

Part 1. Considering that the majority of protein-protein interactions or protein-ligand interactions take place at concentrations close to or near the Kd suggests that most processes exhibit bimodal like behavior. In other words, a small change in ligand concentration results in a large change in activity within the cell. The authors observe linear graded activity only when the concentration of protein (lacI) is reduced. The authors achieve this by introducing multiple lacI sites on a plasmid - titrating away protein and effectively reducing the concentration of free lacI.

Part 2. Kinetically speaking, if a protein ligand interaction has a slow off-rate.. the state of the complex depends on it's history and therefore exhibits hysteresis.

When comparing protein-ligand interactions it's often easy to forget the importance of comparing on and off rates. Two ligands may have similar affinities but have very different on and off rates. A ligand with a slow off rate would have a longer lasting effect.


DNA gyrases

Q1. Why and how might an antimicrobial target DNA gyrase?

Part 1. Fortunately, bacterial gyrases differ significantly from our gyrases, making them an attractive antimicrobial target.

Part 2. Potential targets: ATP binding or hydrolysis, DNA cutting, binding or wrapping.


Non-coding Y RNA

Q1. The authors propose that the Y RNAs guide the formation of specific RNP complexes by strand hybridization and that these complexes are necessary for DNA replication. How might you identify nucleic acids which hybridize to a Y RNA?

Q2. How could you test the physiological importance of hybridization between Y RNA and it's target nucleic acid?

Hint: The paper I will be presenting uses some of these techniques to study BC1 RNA.


A1. Zalfa et al. (2003) Cell identify target RNAs which hybridize specifically to BC1 RNA by mixing and pulling down synthetic biotin labeled BC1 with purified total brain mRNA, followed by gene specific RT-PCR (among other experiments). Nucleic acids which hybridize specifically with Y RNAs could be found in a similar manner, by mixing synthetic biotin labeled Y RNA with purified total mRNA from fibroblasts, for example. mRNAs which hybridize to the Y RNA could be identified by gene specific RT-PCR or by microarray analysis.

A2. Zalfa et al. (2003) Cell design oligos which compete for regions of complementarity between BC1 and target mRNAs and test whether hybridization is necessary for FMRP activity. Once target nucleic acids of Y RNAs are identified, a similar approach could be tested in vivo by assaying for DNA replication following the introduction of oligos that compete for specific sites of hybridization.


Topo

Q1. Propose an experiment that tests whether Stn1 and Ten1 do indeed bind DNA in vivo.

Q2. Also, what additional experiment would show that the substrates used in vitro are similar in structure to telomeres?


A1. Fluorescence anisotropy measurements using fluorescein labeled DNA and purified proteins is another useful method for assaying protein-nucleic acid interactions. Upon protein binding, the anisotropy of the DNA increases as reflected by differences in the amount of polarized light collected at left and right handed angles. This approach is often less cumbersome than EMSA, but can be less informative. For example, EMSA allows the direct observation of multiple binding sites and which nucleic acid population is bound by protein as indicated by it's gel mobility. For instance, double stranded and single stranded nucleic acid run differently on a gel - EMSA would indicate which population is shifted with protein.

A2. According to the authors model, Stn1 and Ten1 cap the ends of chromosomes where DNA is single stranded. A melting curve for the substrates used would confirm whether substrates are single or double stranded, where absorbance at 260 nm differs for single and double stranded DNA. Single stranded DNA absorb 1.37 units at 260 nm, where as double stranded DNA absorb 1.00 units at 260 nm. A melting curve showing an absorbance of 1.00 units at physiological temperature, and of the experiment, would indicate the DNA is in fact single stranded, for example. An old school, but nearly fool proof technique for detecting whether a nucleic acid is structured.

Personal tools