Biochemical and molecular mechanisms

AID/APOBECs are challenging to express and purify due to their genotoxicity to host cells and the proteins’ highly charged surface, which renders them prone to extensive non-specific interactions with DNA/RNA and with other proteins. We have developed unique expertise, methods and reagents that allow for expression and purification of AID/APOBECs and other DNA/RNA-altering enzymes, as well as high-throughput assays to measure every facet of their enzymatic activity. This allows us to tackle biochemical, structural, and molecular aspects of these key enzymes. We measure the enzyme activity parameters (e.g. Michaelis-Menten kinetics) and enzyme: substrate interactions. For substrates, we can use either DNA/RNA substrates ranging in size from simple to complex oligonucleotides, to several kb-long gene sequences and plasmid DNA. To date, we have characterized many of the biochemical features of the AID/APOBECs and discovered that the biochemical properties of these enzymes are a direct and important regulato r of their in vivo activities in immunity and cancer. We establish and optimize multiple expression and purification systems for each DNA/RNA-editing enzyme using different expression hosts (bacterial, yeast, mammalian) and purification strategies (e.g.various fusion tags). For each enzyme, we typically employ multiple expression/purification systems to ensure that our findings reflect a bona fide property. Further, for each enzyme, we construct, express and purify extensive libraries of variants (mutants ,chimeras, species orthologs) which enables in-depth structure: function studies.

Representative publications in this area:

  1. Branton SA, Ghorbani A, Bolt BN, Fifield H, Berghuis LM, Larijani M. Activation-induced cytidine deaminase can target multiple topologies of double-stranded DNA in a transcription-independent manner. FASEB J. 2020. Jul;34(7):9245-9268
  2. Diamond CP, Im J, Button EA, Huebert DNG, King JJ, Borzooee F, Abdouni H, Bacque L, McCarthy E, Fifield H, Berghuis LM, Larijani M. AID, APOBEC3A and APOBEC3B efficiently deaminate deoxycytidines neighboring DNA damage induced by oxidation or alkylation. Biochimica et Biophysica Acta. 2019. 1863(11):129415
  3. Borzooee F, Larijani M. Pichia Pastoris as a host for production and isolation of mutagenic human enzymes involved in cancer and immunity. New Biotechnology. 2019. Jul 25;51:67-79
  4. Methot SP, Ludivine LC, Poorani SG, Eranki AK, Fifield H, Patenaude AM, Gilmore J, Bagci H, Cote JF, Larijani M, Verdun RE, Di Noia JM. A checkpoint for AID at the chromatin promotes selective tethering to elongating RNAPII to license mutagenesis. Nature Comm. 2018. Mar 28;9(1):1248
  5. Abdouni H, King JJ, Ghorbani A, Fifield H, Berghuis L, Larijani M. DNA/RNA hybrids structures modulate the catalytic activity of purified AID. Mol. Immunology 2017. Nov 18;93:94-106
  6. Zahn A, Eranki AK, Patenaude AM, Methot SP, Fifield H, Cortizas EM, Foster P, Imai K, Durandy A, Larijani M, Verdun RE, Di Noia JM. Activation induced deaminase C-terminal domain links DNA breaks to end protection and repair in class switch recombination. PNAS.2014. Mar 18; 111(11):E988-97
  7. Dancyger A, King J, Quinlan M, Fifield H, Tucker S, Saunders H, Berru M, Magor B, Martin A, Larijani M. Differences in the enzymatic efficiency of bony fish and human AID are mediated by a single residue in the C-terminus that modulates single-stranded DNA binding. FASEB J. 2012. Apr; 26(4):1517-25.
  8. Frieder D, Larijani M, Collins C, Shulman M, Martin A. The concerted action of MSH2 and UNG stimulates error-prone repair at A:T basepairs in hyper mutating B cells. Mol Cell. Biol.2009. (18):5148-57
  9. Larijani M and Martin A. ssDNA structure and positional context of the target cytidine determine the enzymatic efficiency of AID. Mol Cell. Biol.2007. (23):8038-8048
  10. Larijani M, Petrov A, Kolenchenko O, Berru M, Krylov S, Martin A. AID associates with single-stranded DNA with high affinity and a long complex half-life in a sequence-independent manner. Mol Cell. Biol.2007. (27): 20-30
  11. Larijani M, Chen S, Cunningham LA, Volpe JM, Cowell LG, Lewis SM, Wu GE. The recombination difference between mouse kappa and lambda segments is mediated by a pair-wise regulation mechanism. Mol Immunol. 2006. 43(7):870-81
  12. Larijani M, Zaheen A, Frieder D, Wang Y, Wu GE, Edelmann W, Martin A. Lack of MSH2 involvement differentiates V(D)J recombination from other non-homologous end joining events. Nucleic Acids Res.2005. 33(21):6733-42
  13. Larijani M, Frieder D, Sonbuchner TM, Bransteitter R, Goodman MF, Bouhassira EE, Scharff MD, Martin A. Methylation protects cytidines from AID-mediated deamination. MolImmunol. 2005. 42(5):599-604
  14. Larijani M, Frieder D, Basit W, Martin A. The mutation spectrum of purified AID is similar to the mutability index in Ramos cells and in ung(-/-)msh2(-/-) mice. Immunogenetics.2005. 56(11):840-5

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