Dna binding proteins

DNA
Histones
Non histones
helix turn helix motif
zinc finger
leucine zipper

1. DNA Binding Proteins Hari S haran Makaju M.Sc. Clinical Biochemistry 1 st year 2076/4/12

2. DNA DNA is a polymer of deoxyribonucleoside monophosphates covalently linked by 3′→5′–phosphodiester bonds F ound in chromosomes, mitochondria and chloroplasts C arries the genetic information

3. DNA The Primary structure of DNA is Sequence

4. DNA

5. The secondary structure of DNA Two anti-parallel polynucleotide chains wound around the same axis. Sugar-phosphate chains wrap around the periphery. Bases (A, T, C and G) occupy the core , forming complementary A –T and G –C The double helical structure of DNA was proposed by lames Watson and FrancisCrick in 1953 The DNA double helix is held together mainly by- Hydrogen bonds

6. Types of DNA Property A-DNA B-DNA Z-DNA Helix Right Right Left Base pair per turn 11 10.4 12 Pitch (Each turn) 2.46 3.40 4.56 Rise per base pair along axis 0.23 0.34 1.84 Diameter 2.55 2.37 1.84 Major Groove Present Present Absent Minor Groove Present Present Deep cleft

7. DNA Tertiary Structure DNA double helical structure coils round Histones. DNA bound to histones forms NUCLEOSOMES (10nm FIBRES) Nucleosomes contain 146 nucleotides

8. DNA Binding Proteins Histone Non-Histones Regulatory Proteins Structural Proteins Motor proteins

9. Histones Histones are a special group of proteins found in the nuclei of eukaryotic cells responsible for DNA folding and chromatin formation Are Basic Proteins Molecular weights between 11,000 Da and 21,000 Da Histones are positively charged Due to abundance of positive amino-acids, arginine and lysine

10. Histones Histones have five major classes : H1, H2A, H2B, H3 and H4 Histones are characterized C entral nonpolar domain, forms a globular structure N-terminal and C-terminal regions that contain most of the basic amino acids The C-terminal end is primarily responsible for histone-DNA and histone-histone interactions . The N-terminal tails stand as targets of post- transational modifications (PTMs),

11. Histones 5 classes of Histones are classified : Core Histones : H2A ,H2B ,H3 & H4 . Linker Histones: H1 Core Histones: H2A and H2B are lysine rich. H3 and H4 are arginine rich histones The basic N terminal regions of H2A, H2B, H3, and H4 are the major sides of interaction with DNA Two of each of these core histone proteins assembles to form one octameric (H3/H4)2- (h2a-h2b) 2 nucleosome core particle, and 147 base pairs of DNA wrap around this core particle .

12. Histones Linker histone : Binds the nucleosome at the starting and ending sites of the DNA, thus locking the DNA into place and help in the formation of higher order structure

13. Classes of Histones H1 Not part of the nucleosome core particle. Binds to the linker DNA and is referred to as a linker histone . H1 is half as abundant as the other histones, which is consistent with the finding that only one molecule of H1 can associate with a nucleosome.

14. Classes of Histones H2A H2A packages DNA molecules into chromatin, the packaging process will effect gene expression. H2A plays a major role in determining the overall structure of chromatin. Inadvertently, H2A has been found to regulate gene expression . H2B Involved with the structure of the nucleosomes of the 'beads on a string' structure.

15. H3 Featuring a main globular domain and a long N-terminal tail. Its sequence variants and variable modification states are thought to play a role in the dynamic and long term regulation of Genes H4 Structural component of the nucleosome, Subject to covalent modification ,including acetylation and methylation, which may alter expression of genes .

16. Function of Histones in chromosomes The DNA is housed in chromosomes in the form of nucleosomes Positively charged histones are linked with negative charged phosphate groups of DNA Some histone proteins function as spools for the thread-like DNA to wrap around looks like beads on a string

17. Histones Modification Each type of histone has variant forms Because certain amino acid side chains are enzymatically modified by Acetylation Methylation , Phosphorylation ADP- Ribosylation , Unibiquitination Sumoylation

18. Histones Modification Such modifications affect: The net electric charge, shape, and other properties of histones The structural and functional properties of the chromatin They play a role in the regulation of transcription

19. Histones Acetylation Adds acetyl groups group to the Lysine amino acid of the histone tails Enzymes: Histone acetyl transferases (HATs) Reduces positive charge and weakens interaction of histones with DNA Facilitates transcription by making DNA more accessible to RNA polymerase II

20. Histone deacetylation Removes acetyl groups from histone tails Enzyme: Histone deacetylases (HDACs) Increases interaction of DNA and histones Represses transcription

21. Sites of histone acetylation with their function

22. Histones Methylation Addition of an Methyl functional group to Lysine or Arginine of the histone tail. Enzymes "histone methyltransferase”

23. Histones Methylation Methylation can result in activation or repression of expression . Activation (H3K4, H3K36, H3K79) Trimethylation of histone H3 at lysine 4 (H3K4) is an universal active mark for transcription. Repression (H3K9, H3K27, H4K20) Dimethylation of histone H3 at lysine 9 (H3K9) and at 27 (H3K27) are the universal signal for transcriptional silencing.

24. Sites of histone Methylation with their function

25. Histones Phosphorylation Addition of a phosphate group (PO 43−) to a molecule. Phosphorylation is catalyzed by various specific protein kinases, whereas phosphatases mediate removal of the phosphate group. Phosphorylation of histones, in particular phosphorylation of H2AX, has a role in DNA damage response and DNA repair . Most studied sites of histone phosphorylation are the serine 10 of histone H3 (H3S10) that is deposited by the Aurora-B kinase during mitosis.

26. Sites of histone Phosphorylation with their function

27. Ubiquitination Refers to the post-translational modification of the amino group of a lysine residue by the covalent attachment of one or more ubiquitin monomers. Ubiquitin is a 76 amino acid protein highly conserved in eukaryotes. Histone ubiquitination alters chromatin structure and allows the access of enzymes involved in transcription.

28. Sites of histone ubiquitination with their function

29. ADP- ribosylation Addition of an ADP-ribose moiety onto a protein using NAD+ as a substrate . Mono ADP- ribosylation is mediated by ADP ribosyl transferases (ART) and the enzymes responsible for the Poly-ADP- ribosylation are the poly ADP ribose polymerases (PARPs ). PARP1 prefers to linker histone H1 while PARP2 prefers core histones

30. Sumoylation Addition of a “Small Ubiquitin-related MOdifier protein ” ( SUMO) of ~100 amino acids. Histone sumoylation was first reported in 2003, Shiio et al. F ound that H4 can be modified by SUMO and They suggested that this modification leads to the repression of transcriptional activity The putative sumoylation sites were identified as K6/7 T o a lesser extent K16/17of H2B, K126 of H2A, A ll four lysine in the N-terminal tail of H4.

31. DNA Binding Proteins Apart from histones, there are many other special proteins which will interact at specific regions of DNA. The protein–DNA interactions are mainly mediated by 3 motifs :– Helix-turn-helix Z inc finger L eucine zipper motifs. Only small regions of the protein make direct contact with the DNA T he rest of the proteins are involved in other activities , like dimerization, ligand-binding, interaction with coactivators and corepressors, etc .

32. DNA Binding Proteins DNA sequence-specificity of DNA binding proteins Sequence-specific interactions Frequently involve DNA major groove The protein-DNA interactions are maintained by hydrogen bonds, ionic interactions and van der Waals forces . Non-specific interactions Interactions with DNA phosphate backbones

33. Helix-Turn-Helix Comprises about 20 amino acids in two short α -helical segments Each seven to nine amino acid residues long, Separated by a β- turn One of the two α -helical segments is called the recognition helix, Because it usually contains many of the amino acids that interact with the DNA in a sequence-specific way. When bound to DNA, the recognition helix is positioned in or nearly in the major groove.

34. Classification of helix-turn-helix B ased on their structure and the spatial arrangement of their helices . Di-helical Simplest helix-turn-helix motif. Example: Homeodomain Tri-helical Example:  Transcriptional activator Myb Tetra- helica Example TetR repressors. Multihelical versions with additional helices also occur . Winged helix-turn-helix F ormed by a 3-helical bundle and a 3- or 4-strand  beta-sheet(wing ). Example : transcription factor ETS   S caffold arranged in the order α1-β1-β2-α2-α3-β3-β4 where the third helix is the DNA recognition  helix

35. Examples of helix-turn-helix Organism Regulatory protein E coli lac repressor, Cap Phages λcI , cro , and 434 repressors Mammals homeobox proteins pit-1, Oct1, Oct2 DNA-binding domain of the Lac repressor Homeobox protein

36. Zinc Finger Very common in eukaryotes About 30 amino acid residues form an elongated loop held together at the base by a single Zn 2+ ion α-helix plus two antiparallel β-sheets And a Zn 2+ ion that is coordinated by cysteines or histidines α-helix makes sequence-specific contacts along the major groove . Zinc Finger Proteins may have more than one Zn finger per protein.

37. Zinc Finger A Zn 2+ ion coordinated by 4 Cys or 2 Cys and 2 His residues. Often occur as tandem repeats with two, three, or more fingers.

38. Zinc Finger Structure of the six-finger TFIIIA–DNA complex

39. Zinc Finger Some zinc fingers contain the amino acid residues that are important in sequence discrimination. Zinc fingers can also function as RNA binding motifs—for example, in certain proteins that bind eukaryotic mRNAs and act as translational repressor Zinc fingers designed to bind targeted DNA sequences with ultimate goal of therapeutics Fig. Three zinc fingers (gray) of the regulatory protein Zif268, complexed with DNA (blue and white)

40. Zinc Finger Zinc Fingers typically function as Interaction modules and bind to a wide variety of compounds , such as nucleic acids, proteins and small molecules. Functions are extraordinarily diverse Include DNA recognition, RNA packaging, transcriptional activation, regulation of apoptosis, protein folding and assembly, and lipid binding.

41. Nuclear receptors - DNA interaction DNA interaction First “finger” binds DNA Second “finger” involved in dimerisation Binds to neighboring “major grooves” on same side of DNA Extensive phosphate contact and recognition helix docked into the groove specificity determined by 3 aa in recognition helix

42. Types of Zinc finger Classical Zinc finger (C 2 H 2 ) Gag-knuckle Treble-clef Zinc ribbon Zn 2 /Cys 6 TAZ2 domain

43. Example of Zinc Finger Organism Regulatory protein E coli Gene 32 protein Yeast Gal 4 Xenopus TFIII A Mammals Steroid receptor family Sp1

44. Leucine Zipper Motif Contain leucine residues every 7 th position in an α-helix. Form homo- or heterodimers with coiled coil structure (blue region ) Although researchers initially thought the Leu residues interdigitated (hence the name “zipper ”) The basic region with arginine and lysine residues bind to the major groove of DNA The basic amino acids interact with the phosphate backbone of DNA through electrostatic interactions and also the DNA bases through hydrogen bonding .

45. Leucine Zipper Leucine zippers also function as dimers to regulate gene transcription Example Organism Regulatory Protein Yeast GCN4 Mammals C/EBP, Fos , Jun, Fra-1, cAMP response element-binding protein (CREB), c- myc , n- myc , I- myc proto-oncogene  JUN  (purple) binding as a homodimer to DNA.

46. Methods for Detecting DNA-Protein Interactions In vitro and In vivo techniques which are useful in detecting DNA-Protein Interactions. Electrophoretic mobility shift assay Widespread technique to identify protein–DNA interactions.   DNase footprinting assay to identify the specific site of binding of a protein to DNA.  Chromatin immunoprecipitation  : to identify the sequence of the DNA fragments which bind to a known transcription factor.  

47. Methods for Detecting DNA-Protein Interactions Yeast one-hybrid System (Y1H) to identify which protein binds to a particular DNA fragment.  Bacterial one-hybrid system (B1H) to identify which protein binds to a particular DNA fragment. Structure determination using X-ray crystallography has been used to give a highly detailed atomic view of protein–DNA interactions

48. References Robert k. Murray, D.K.Granner , P.A.Mayes & Victor W.Rodwell Harpers illustrated biochemistry 26th edition Lippincot - Marks' Basic Medical Biochemistry A Clinical Approach Thomas M.Devlin , textbook of Biochemistry with clinical correlation 5th edition Lehninger Principle of Biochemistry 4th edition https:// en.wikipedia.org/wiki/zinc finger https://en.wikipedia.org/wiki/Helix-turn-helix https ://www.mycancergenome.org/content/pathways/protein-degradation-ubiquitination / https://epigenie.com/key-epigenetic-players/chromatin-modifying-and-dna-binding-proteins/zinc-finger-proteins/ Gregory R.Dressler,Epigenetics , Development, and the Kidney, J Am Soc Nephrol 19: 2060 –2067, 2008. doi : 10.1681/ASN.2008010119