Hoatlin: Fundamentals

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Equipped with his five senses, man explores the universe around him and calls the adventure Science. ~Edwin Powell Hubble, The Nature of Science, 1954

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Links for Maureen Hoatlin's Fundamentals 2018 Class

Movies for Lecture. We will view several of these movies and discuss together.
Materials for the Skills Session on XP

Questions and Answers

  • Taken from emailed questions over the years about topics I cover including transcription, chromosome and gene structure, DNA replication and DNA repair. Thank you to all students who have contributed these questions--we all benefit:
Question
  • I had one question about the study guide. It says HATs acetylate and therefore neutralize a negative charge. I was taught that lysine is positive, and putting an acetyl group on it neutralized a positive charge. (note--this issue has been revised for clarification in the study guide as of 2014, but I left this q&A in because it may be informative).
Answer
  • I think I see the confusion. In the sentence you got confused by (II. A.4a), the charge referred to is the negative charge on the DNA phosphate backbone. This is a problem with wording because the study guide and slides are so condensed that they make a general case for both HATs and HDACs. Other students had the same question. I hope this clears it up:

Reversible modification of the ε-amino group of lysine by acetylation neutralizes a negative charge on DNA.

  • The longer version for the study guide that would be better is:
  • Histone tails contain amine groups on lysine and arginine. These positively charged residues interact with the negatively charged phosphate groups on the DNA phosphate backbone. Acetylation by HATs neutralizes the positive charges on the histone tails thus reducing histones binding to DNA, leading to chromatin expansion. HDACs remove acetyl groups resulting in a positive charge of histone tails increasing binding between the histones and DNA backbone, condensing chromatin.
Question
  • Are transcription factors, basal transcription factors, basal factors all the same thing?
Answer
  • Yes. (see pg 239 in your reading, Essential Cell Biology pg 239) and the lecture notes that describe initiation of transcription with the stepwise assembly of proteins at the promoter (the TFIIA, TFIIB etc plus RNA polymerase) and slide 24 of the last lecture where the first point is “Basal transcription factors: TATA-binding protein TFIIB, TFIIA, TFIIE, TFIIH etc.” The basal transcription factors are a subset of all transcription factors.
Question
  • It is not clear what the functional classes of transcription factors are.
Answer

Transcription factors can be constitutively-active – present in all cells at all times or can be conditionally-active – requires activation (e.g., cell specific or signal-dependent).

  • Lecture Slides have the overview of the list of proteins that regulate transcription (as follows):
    • Basal transcription factors
      • TATA-binding protein TFIIB, TFIIA, TFIIE, TFIIH etc.
    • DNA-Binding Factors
      • Signal-regulated proteins: posttranslational modifications (phosphorylation).
      • Nuclear Hormone Receptors: require ligand binding.
    • Co-regulatory protein complexes
      • Interact with DNA binding proteins, but not (necessarily) with DNA
      • TBP-Associated Factors (TAFs)
      • Histone modifying enzymes.
      • Chromatin remodeling factors
Question
  • I was a bit confused with: Using a steroid receptor as an example, explain how gene expression can be regulated by hormonal signals.
Answer
  • Just a general description from the standpoint of transcription can be found in the related text and in the figure in the lecture notes: "Reciprocal regulation of transcription. In the absence of ligand, nuclear hormone receptors repress transcription through the action of co-repressor complexes with associated HDAC activity. Ligand-induced conformational changes lead to dissociation of co-repressor complexes with recruitment of co-activator/HAT complexes. (from Glass and Rosenfeld, Genes Dev. 14:121-41, 2000)"
Question
  • Cis elements- these are regulatory sequences on DNA. Can they be both enhancers and silencers? Does their proximity to the gene matter to still be considered "cis"?
Answer
  • Yes, they can be both enhancers and silencers, and can be very far away from the promoter, but on the same piece of DNA. Enhancers can even be located on a different chromosome than the promoter. Here’s a recent paper that is nteresting to scan briefly because it describes the complexities in transcription control. de Laat, W., & Duboule, D. (2013). Topology of mammalian developmental enhancers and their regulatory landscapes. Nature, 502(7472), 499–506. doi:10.1038/nature12753
Question
  • Regarding constitutive and inducible enhancer elements, it was my understanding that enhancer refers to the DNA sequence, whereas activator refers to the DNA binding protein, so I’m not sure of what a constitutive and inducible enhancer is.
Answer
  • The simplest answer is that an enhancer is a short region of DNA that can be bound with proteins to enhance transcription levels of genes in a gene cluster. It is different than an promoter because it can act at great distances from the promoter and can act upstream or downstream of the promoter itself. A constitutive enhancer, is "on" all the time, versus an inducible enhancer that is conditionally able to influence transcription based on the presence of a particular protein (e.g., in a certain cell type or during a specific moment or location during development).
Question
  • What is the difference between "alternative RNA Processing" and "alternative protein processing"?
Answer
  • Alternative RNA processing is exhibited in the example of calcitonin and calcitonin gene related peptide. The same transcript is spliced in different ways to achieve different mRNAs and thus alternative proteins.
  • Alternative protein processing can refer to a number of things. Rather than confuse the issue with examples, Let me know where the statement was in the notes so I can clarify.
Question
  • Can you shed some light on what is important about SINEs and LINEs, other than being transposons?
Answer
  • LINEs and SINEs are important to know about because (1) they provide knowledge about a large part of the human genome (2) the role of SINEs and LINEs in genome evolution (e.g., via recombination) and (3) it is likely that a clearer picture of the activities of LINEs and SINEs in genomic stability and clinically-important influences will continue to emerge (see abstract for a recent review: http://www.ncbi.nlm.nih.gov/pubmed/20307669).
Question
  • Can you explain the difference between XP-V and a mutation in XPA-XPG?
Answer
  • XP patients have a defect in one of the many genes involved in NER (nucleotide excision repair) e.g., XPA, XPC, XPC, XPG etc. Thus, XP patients cannot repair DNA damage (e.g., photoproducts like cyclobutane pyrimidine dimers) caused by exposure to sunlight. The XP-V patients have a different defect but the same phenotype. They have a defective bypass polymerase, called Pol eta. Normally, Pol eta can synthesize (error-free) past a thymine dimer by inserting two A residues. In XP-V patients, other bypass Pols are substituted that are not error-free for this lesion. Also, in XP-V patients, the upstream XPA-XPG proteins are functional and NER repair is intact. Only the bypass polymerase is defective.
Question
  • I was reading over your notes and can't quite seem to understand why telomeres have high C-T content. Would it not necessarily be true that they would have high C-A content as well?
Answer
  • Telomeres usually contain some version of tandem copies of sequences like 5'-CCCCAA-3' on one strand and 5'-TTGGGG on the complementary strand. The GT-rich strand comprises the 3'-end and **sticks out** longer than the CA strand (and forms a loop, sealing the end). Specifically for human telomeres, there are 300-8,000 sets of repeats of the sequence CCCTAA /TTAGGG, then a 100-200 nucleotide extension of single-stranded TTAGGG repeats, hence the comment that telomeres have high GT content. But why? The best way to understand this beautiful system is to watch the very simple but very revealing short animation in the link above called telomere animation of how telomerase works at the telomere (see especially step 5 and later). Note that it is all about the requirements of polymerase for a free 3' -OH (and a ss DNA template strand)! It might also help to scan quickly through the very good article from Nature Network listed above as well.
Question
  • I am a bit confused about the DNA polymerases that you mention on slide 56 of your lecture. Are the leading and lagging strand synthesied by different polymerases? Secondly, you mention polymerase eta and its role in synthesis of the leading strand. I thought eta was only involved in the bypass mechanism? Thanks for your clarification and help.
Answer
  • I think I see the confusion. I believe it is partly confusion over greek letters used to name the pols. Pol eta (looks like an italics small n) is a translesion pol---so you are correct about that. However, slide 56 mentions pol epsilon (Greek letter looks like like an italics e) which is the replicative pol that has recently been shown to primarily replicate the leading strand in eukaryotes. The lagging strand is synthesized primarily by pol delta. This division of labor at the replication fork in eukaryotes is a recent discovery, so many texts will have it backwards. Note that in eukaryotes there are two replicative pols: polymerase delta and epsilon, whereas in prokayotes there is only one main replicative polymerase, Pol III. Primase is required in both systems. Hope this helps.
Question
  • I am still having trouble understanding PARPi's mechanism. Can you explain to me the normal interactions and mutated interactions of PARP and BRCA along with PARPi's function?
Answer
  • The essence of the idea in the PARP/BRCA example, very briefly, is that the BRCA-deficient tumor cell has a defect in repair of DNA double strand breaks by a repair process called homologous recombination (HR). PARP1 is in charge of repairing single strand DNA breaks, which become DNA double strand breaks during replication. Thus, if PARP1 is inhibited, the DNA ss breaks convert to DNA ds breaks and must be repaired by HR. If HR is defective, the cell accumulates so much damage that it cannot survive. HR is defective in the tumor cell...but not in the wild-type cell!

That's how PARP1 inhibitors are relatively selective in killing the HR deficient tumor cell but not the wild-type cell (which is competent for HR)

Question
  • 1) When you talk about the number of base pairs in a genome, the number 3 x 10^9 was mentioned and I wanted to double check, is this for the haploid genome? And just out of curiosity, why is it reported for the haploid genome?
  • 2) When you were talking about DNA replication a "licensing factor" was mentioned, and I was wondering, is this a protein? And what is it's function?
Answer
  • 1. Yes, for haploid. Diploid cells have two homologous copies of each chromosome (in humans, one from the mother and one from the father) so you would be "counting twice." You could do that, but it is important to indicate haploid/diploid. I think the haploid number is reported b/c it is the total number of unique sequences.
  • 2. Yes licensing factor is the old name for a set of proteins that bind to the origins. Licensing factor is now thought to include the proteins Cdc6 and Cdt1. These proteins bind to the origin recognition complex proteins, and are synthesized only in a specific phase of the cell cycle (G1). Once replication origins "fire" (or start) these proteins are degraded or exported and the origin can't be "licensed" for firing again until the proteins are synthesized and enter the nucleus in the next cell cycle. That's how the cell controls replication so that the DNA is replicated once and only once.
Comment
  • there was a typo in lecture notes. the haploid human genome is 3x10^9 (billion)

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