MCB 540: "Nucleic Acid Enzymes" Overview and Syllabus

Offered every other year, alternating with MCB 544 ("Protein Structure, regulation and modification")

Weeks 1 to 5 during winter quarter of odd years
Tuesdays and Thursdays - 3:15 to 4:45 pm 

In this class, we survey a wide variety of enzymatic process that control the structure and modification of DNA and RNA, with particular focus on structure, function and mechanism.  Unifying features of major reaction types (such as phosphoryl transfers and base modifications) will constitute core material.

The course begins with an overview of the structure and reactivity of DNA and RNA substrates that influence fundamental aspects of relevant enzymatic reaction mechanisms, particularly (1) phosphoryl transfer reactions leading to polymerization, ligation, hydrolysis and recombination; and (2) reactions at DNA bases leading to repair or modification of nucleic acid targets.  By the end of the course, students should be very comfortable describing the mechanisms of several major classes of nucleic acid modifying enzymes, the structural protein families involved in those processes, and in the modeling and study of enzyme-nucleic acid complexes using online and downloadable software tools.

The purpose of this class is to take students to a higher level of understanding and confidence in the details of biomolecular structure/function analyses, and to introduce them to a few key tools of visualization and analysis that might prove useful during their time in graduate school.  What you put into the class will obviously translate into what you take away from it.  If you come to all the lectures, do the readings, and put forth a good faith effort to deliver a high quality final assignment, you will be happy with the outcome of this class.

Course Schedule and readings

Session 1: DNA and RNA structure and recognition - January 8

General background:

1. Watson JD and Crick FHC (1953) "Molecular structure of nucleic acids" Nature April 25 p. 737.
2. Westheimer FH (1987) "Why nature chose phosphates" Science 235: 1173 - 1178.  PubMed ID 2434996
3. Rohs et al. (2010) "Origins of specificity in protein-DNA recognition" Ann. Rev. Biochem. 79: 233 - 269.

Research Article:

Chevalier et al. (2002) "Flexible DNA target site recognition by divergent homing endonuclease isoschizomers I-CreI and I-MsoI" J. Mol. Biol. 329: 253 – 269. 

Coordinates:
1M5X

Websites and tools:

• RCSB PDB Database  
• Pymol (Graphics Program)  
• Free educational copy of Pymol

Session 2: Phosphotransfer reactions and DNA polymerization - January 10

General background:

1. Yang W et al. (2006) "Making and breaking nucleic acids: two Mg ion catalysis and substrate specificity" Molecular Cell 22 (1): 5 - 13.
2. Rothwell and Waksman (2005) "Structure and mechanism of DNA polymerases" Advances in Protein Chemistry 71: 401 – 440.

Research Article:

1. Nakamura et al. (2012) "Watching DNA polymerase n make a phosphodiester bond" Nature 487: 196 – 202.

Coordinates: 
4ECQ
4ECZ
4ED0
4ED3
4ED6
4ED8

Websites and tools:

Homology searching and modeling website #1: Protein HomologY REcognition (PHYRE) 2       

Session 3: DNA ligation - January 15

General background:

1. Tomkinson, AE, et al. and Ellenberger T. (2006) "DNA ligases: structure, reaction mechanism and function" Chemical Reviews 106 (2): 687 - 699.
2. Pascal, JM (2008) "DNA and RNA ligases: structural variations and shared mechanisms" Current Opin. Struct. Biol. 18: 96 - 105.
3. Pascal JM, O'Brien PK, Tomkinson AE and Ellenberger T (2004) "Human DNA ligase I completely encircles and partially unwinds nicked DNA" Nature 423 (7016): 473 - 478.

Research Article:

1. Cotner-Gohara et al. (2010) "Human DNA ligase III recognizes DNA ends by dynamics switching between two DNA-bound states" Biochemistry 49 (29): 6165 – 6176.

Coordinates:
3L2P

Websites and tools:

Homology modeling website #2: SWISS-MODEL

Session 4: DNA hydrolysis - January 17

General background:

1. Yang W et al. (2011) "Nucleases: diversity of structure, function and mechanism" Quarterly Reviews of Biophysics  44 (1): 1 – 93.
2. Orlowski, J. and Bujnicki, JM (2008) "Structural and evolutionary classification of Type II restriction enzymes based on theoretical and experimental analyses" Nucleic Acids Research 36 (11): 3552 - 3569.

Research Articles:

1. Rusling, et al. (2012) "DNA looping by FokI: the impact of synapse geometry on loop topology at varied site orientations" Nucleic Acids Research 40 (11): 4977 – 4987.

Read this follow up if you wish (we will not be discussing in class):

Laurens et al. (2012) "DNA looping by FokI: the impact of twisting and bending rigidity on protein-induced looping dynamics" Nucleic Acids Research 40 (11): 4988 – 4997.

Coordinates:
1FOK
2FOK

Websites and tools:

Structure similarity search engines:
DALI
FATCAT

Session 5: Methylation/base modification - January 22

General background:

1. Jeltsch (2002) "Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA methyltransferases" ChemBioChem  3: 274 – 293.
2. Jurkowska, et al. (2011) "Structure and function of mammalian DNA methyltransferases" ChemBiochem 12: 2066 – 222.

Research Article:

1. Matje and Reich (2012)  "Molecular drivers of base flipping during sequence-specific DNA methylation" ChemBioChem 13: 1574 – 1577.

Coordinates:
2ZCJ

Nucleic Acid conformation analysis server:
Web3DNA
CURVES+

Session 6: RNA as an enzyme and/or substrate (1): tRNA topogenesis January 24

General background:

1. Esakova and Krasilnikov (2010) "Of proteins and RNA: The RNase P/MRP family" RNA 16: 1725 – 1747.
2. El Yacoubi et al. (2012) "Biosynthesis and function of posttranscriptional modifications of transfer RNAs" Annual Reviews in Genetics.  Epub ahead of print.

Research Articles:

1. Reiter, et al. (2010) "Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA"  Nature 468: 784 – 789.
2. Howard et al. (2012) "Mitochondrial ribonuclease P structure provides insight into the evolution of catalytic strategies for precursor-tRNA 5’ processing."  PNAS USA 109 (40): 16149 – 16154.

Coordinates:
3Q1R  (Bacterial RNA based RNAse P in complex with tRNA)
3Q1Q (Bacterial RNA-based RNAse P in complex with tRNA in the presence of 5' leader)
4G24 (Mitochondrial protein only RNAse P)

Websites and tools:

Protein fold classification servers:
SCOP
PFAM
CATH

Session 7: RNA as an enzyme and/or substrate (2): the ribosome January 29

(Dr. Clint Spiegel, Western Washington University, guest lecturer.)

General background:

1.  Leung EK et al. (2011) "The mechanism of peptidyl transfer catalysis by the ribosome" Ann. Rev. Biochem. 80: 527 - 555. 

2.  Clementi and Polacek (2010) "Ribosome-associated GTPases: the role of RNA for GTPase activation" RNA Biology 7(5): 521 - 527.

Research Article:

 1.  Walter J.D. et al. (2012) "Thiostrepton inhibits stable 70S ribosome binding and ribosome-dependent GTPase activation of elongation factor G and elongation factor 4" Nucleic Acids Research 40 (1): 360 - 370.

Session 8: Recombination and RecBCD - January 31

(Dr. Gerry Smith, FHCRC Basic Sciences, guest lecturer)

General background:

1. Singleton et al. (2004) "Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks" Nature 432:  187 – 193.

    2.   Smith G. (2012) "How RecBCD enzyme and Chi promote DNA break repair and recombination: a molecular biologist's view" MMBR  76 (2): 217 - 228.

Research Articles:

1. Amundsen, et al. (2007) "Intersubunit signaling in RecBCD enzyme, a complex protein machine regulated by Chi hot spots" Genes & Development 21: 3296 – 3307.

Coordinates:
1W36

Session 9: DNA repair - February 5

General background:

1. Kim, Y. J. and Wilson, D. M. (2012) "Overview of base excision repair biochemistry" Curr. Mol. Pharmacol. 5 (1): 3 - 13.
2. Berquist, B. R. and Wilson, D. M. (2012) "Pathways for repairing and tolerating the spectrum of oxidative DNA lesions" Cancer Letters epub Feb. 19.
3. Chapman et al. (2012) "Playing the end game: DNA double-strand break repair pathway choice" Molecular Cell 47: 497 – 510.

Research Article:

1. Orans et al. (2011) "Structures of human exonuclease 1 DNA complexes suggest a unified mechanism for nuclease family" Cell 145: 212 – 223.

Session 10: Engineering and gene targeting - February 7

General background:

1. Baker, M. (2012)  "Gene-editing nucleases" Nature Methods 9 (1): 23 – 26.

Research article:

1. Thyme et al. (2009) "Exploitation of binding energy for catalysis and design" Nature 461: 1300 – 1304.
2. Streubel et al. (2012) "TAL effector RVD specificities and efficiencies" Nature Biotechnology 30: 593 - 595.

Grading:

Overall philosphy:  The purpose of this class is to take students to a higher level of understanding and confidence in the details of biomolecular structure/function analyses, and to introduce them to a few key tools of visualization and analysis that might prove useful during their time in graduate school.  What you put into the class will obviously translate into what you take away from it.  If you come to all the lectures, do the readings, and put forth a good faith effort to deliver a high quality final assignment, you will be happy with the outcome of this class.

General participation and preparation (50%)

Each session will include a literature-review style discussion of at least one recent research papers. Individuals will routinely be asked to provide questions and/or to discuss answers and important about the work being discussed.  Please don't show up and demonstrate that you haven't prepared for the day's reading and topic.

A final take-home assignment and modeling exercise (50%).   (Due Friday, February 14 via email)

Assignment:
As you may know, the NIH and several other funding agencies around the world have for about ten years funded large consortiums of investigators (comprised of individual academic labs, national laboratory facilities, research centers and industrial groups) to determine structures of as many proteins as possible (from specific model organisms, putative biochemical pathways and large homlogous gene superfamilies).  Termed "Structural Genomics", this effort has thus far led to the determination of several thousand distinct protein crystal just by the consortiums funded by the NIH through their "Protein Structure Initiative".

Because the laboratories engaged in Structural Genomics will solve structures of pretty much anything that they can express, purify and crystallize without overdue concern for biological context or information, a large number of protein structures now exist for which there is no functional annotation--1682structures as of November 19, 2012 from the four separate NIH PSI consortium members alone.   Here is your chance to get involved in this area of investigation, using only your knowledge of protein structure, modification and regulation, your internet service provider and your imagination.

The gallery of these "nonannotated" protein structures, with links to their PDB entries, is provided at the somewhat stupidly named "Functional Sleuth" website: (Start by clicking on "View by PSI Center") ...with the invitation for really smart people such as yourself to conduct "further research for proteins in the Protein Data Bank archive whose functions are unknown or minimally characterized" ( i.e., the crystallographers are too busy collecting X-ray data to spend time actually investigating the structures they solve).

Your assignment:  Choose any one of the thousands of functionally nonannotated structures from the Functional Sleuth website (how will you choose?) and subject it to EVERY POSSIBLE method of analysis that you have learned in this class and any others that you can think of, to try to generate one or more hypotheses about potential function.  Don't limit yourself to only analyses of the coordinates--feel free to go find the reading frame in the NCBI database and look at its surrounding genetic context; also see if its popped up in any phenotypic screens.   Write up a report, with original figures, that summarizes your findings.  Limit the length of your write up to no more than 3000 words of text, plus citations and figures.

If you are able to identify a protein of unknown function that you can argue plays a role in nucleic acid enzymology (either structural or catalytic) that will be great, but it is not a requirement for this assignment.

This write-up must be of publication quality;  i.e. it needs to be well-written, with actual complete sentences and paragraphs, proper grammar, and original figures (NOT snapshots of desktop output!!!).  If you send me a lame excuse for an analysis, I will send it back to you ungraded and respectfully ask that you redo it.

Properly cite all webservers and on-line tools (they all provide a seminal paper that they want cited if you use their tool).  Also cite the PDB and the corresponding entry, and the structural genomic consortium from which the structure originated.
 
Things to address in your analysis MIGHT  include (not necessarily in this order)

1. The biological source of the protein, and multisequence alignment analyses:
   1. What fold family(s) are represented in the structure?
   2. What are the most closely related folds from SCOP, CATH or PFAM?
   3. Where are the most conserved residues on the surface of the protein? What are their chemical properties and abilities?
   4. What does the electrostatic surface potential of the protein look like? Are there obvious highly charged or uncharged surfaces that might be involved in molecular recognition?
2. The similarity of the protein structure to other protein structures in the database (DALI analysis). Are the critical residues conserved with those proteins that most closely resemble your candidate?
3. Single protein fold or domain per subunit or multiple protein folds or domains per subunit? If the latter, is there evidence for a cleft and/or 'hinge points' that might indicate a ligand binding site and/or comformational changes?
4. Dynamic information: b-factor plots, disordered regions. Disordered regions are often involved in binding interactions.
5. Sites of possible covalent modifications? Examine the sequence for known sites of glycosylation, lipidation, etc.
6. Back to the gene: is it part of an operon or gene cluster with annotated proteins that might provide clues to function?
7. Are there knockout or knockdown studies in the model organism source that might indicate importantce or function?
8. What happens if you submit the sequence of the protein to a structural threading algorithm? Other than the actual structure, what other structures does the sequence 'hit' on?
9. Oligomeric state of the protein (monomer, dimer, tetramer, etc)--any signs of cooperativity? allostery?
10. Higher symmetry--what types of biological functions have involved proteins with 6-fold, 7-fold, 8-fold or higher symmetry?
11. Secondary structure composition: what types of motions, functions etc are associated with all helical bundles, with extended beta sheets, etc? What types of functions can you comfortably rule out by looking at the structure?
12. Bound ligands: did anything 'come along for the ride' in the purification and crystallization experiment? If so, perhaps it gives a clue regarding high affinity binding by the protein?

These are just a few suggestions.  I encourage you to go wild, think up additional ways to look at the protein, and above all else HAVE FUN with this assignment.  I look forward to seeing your answers.