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USC College > Faculty > Departments > Department of Biological Sciences > Phuong Pham
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Phuong Pham
Assistant Professor (research) of Biological Sciences

Contact Information
Office: RRI 113
Phone: (213)740-5191
E-mail: ppham@usc.edu

 

Education
M.S. Biology, St. Peterburg State University, 6/1989
Ph.D. Genetics, St. Petersburg State University, 5/1993
 

Description of Research

Detailed Statement of Research Interests
My research interest currently is focused on biological functions and biochemical properties of “Apobec” protein family of DNA dependent cytidine deaminases. Members of this family include activation-induced cytidine deaminase (AID) and Apobec3G. By modifying DNA, AID and Apobec3G play an essential role in adaptive and innate immunity. AID is required for B cells to undergo somatic hypermutation (SHM) and class switch recombination (CSR), two processes that are needed to produce high-affinity antibodies. Apobec3G and other Apobec proteins are responsible for innate immunity against HIV infection by triggering the destruction of HIV-1 reverse transcribed DNA. Furthermore, several members of the Apobec family have been shown to play a role in genomic instability by inhibiting action of retrotransposons in human and mice. My current research aims to decipher the biochemical basis for SHM, in which AID plays an essential role. We have shown that purified human AID deaminates C on single stranded DNA, but not double stranded DNA, RNA or DNA/RNA hybrid molecules. Importantly, AID-catalyzed deamination simulates several hallmark properties of SHM. First, AID preferentially targets the dC in ssDNA at 5’ WRC (W=A or T, R = A or G) motifs similar to SHM bone fide hotspot motifs in vivo. Second, AID appears to act processively, generating broad clonal heterogeneity and cluster mutations. Third, AID activity on dsDNA is transcriptional dependent and the 5’WRC hotspot preference is preserved when AID acts on T7 RNA polymerase-driven transcribed dsDNA. These biochemical data, together with genetic data strongly suggest that generation of mutations at WRC (or GYW on the complementary strand) SHM hotspots are initiated by the direct action of AID on DNA. Advancing along with our long-term goal to reconstitute SHM in vitro, we plan to develop an in vitro model system to study SHM. Biochemical reconstitution of SHM will be performed in several stages. In the first stage, we will examine the biochemical interaction governing the SHM initiation step by AID-catalyzed C?U conversion during transcription. We will employ a bona fide human RNA polymerase II transcription system, containing purified pol II holoenzyme including transcriptional enhancer elements. Subsequent diversification, generating a variety of SHM mutations from G:U intermediates will be investigated using an arsenal of enzymes, including error-prone DNA polymerases ?, ?, and perhaps ? and ?, in conjunction with mismatch repair and base excision repair. The second area of my research plans will be focused on the enzymatic mechanism of processive cytosine deamination reactions and function-structure studies of AID and Apobec3G. We and others have shown that each member of Apobec family posseses a unique deamination specificity. For example, AID prefers WRC hotspots, but Apobec3G prefers CCC hot-spots. The determinants for deamination specificities and processivity of Apobec enzymes are not known. With the structural data for a few members of Apobec family become available (Apobec2, Apobec3G and AID), it will be possible to address these questions by, for example, studies of systematically mutated important residues of the enzymes. We plan to use both steady state and pre-steady state kinetic analyses of Apobec enzymes to decipher the enzymatic steps of the deamination reactions. Third area of my research is to investigate the negative consequences of expressing DNA deaminases in the wrong place at the wrong time to catalyze aberrant deamination in “at risk” DNA sequences. Inappropriate expression of DNA deaminases, is likely to result in excess numbers of mutations and genomic instability which could lead to the disease development. The impact of DNA deaminases expression in the cells is currently unexplored. Specifically, I would like to address following problems: 1. Role of DNA deaminases in spontaneous mutations? In human, the most common type of mutations in normal and tumor cells is C ? T transitions, with a significant fraction occurring in CpG dinucleotides. The C ? T mutations are traditionally thought to occur spontaneously in both normal and tumor cells. Recent discoveries of DNA deaminases in higher eukaryotes indicate that a non-negligible fraction of C ? T mutations might also be caused by the enzymatic deamination. Although the expression of AID seems to be restricted to lymphoid cells, substantial quantities of Apobec 3G and 3F mRNA have been observed in lung, liver, kidney and pancreas, in addition lymphoid tissue. The tissue distribution of the other Apobec homologs is not currently known. However, it is possible, perhaps even probable, that even a low expression of one or more of the DNA deaminases might be sufficient to compete with, or possibly even override the effects of spontaneous deamination of C, especially in “at risk” sequences corresponding to hot spots. From a mechanistic perspective, it is important to reexamine the origins of C ? T mutations, what fraction are caused by spontaneous deamination of C and what fraction arise from the inappropriate action of DNA deaminases. 2. Possible role of DNA deaminases in triggering the cancer development. The necessity to keep mutations in check mandates that the cellular expression of DNA deaminases be limited to the “right place” and the “right time”. For instance, the transcription of AID is tightly regulated during normal B-cell development. A significant elevation in mutation and genomic instability is likely to occur when AID is expressed gratuitously, and this can lead to cancer. Aberrant SHM has been observed in about 50% of diffuse large B-cell lymphomas in about 20% of AIDS-related non-Hodgkin’s lymphomas. High level of point mutations at deaminated C sites may represent just the tip of the iceberg, because the unregulated action of AID can cause Ig chromosomal translocations frequently associated with mature B-cell malignancies in humans. In mice, C-myc/Ig chromosomal translocations in IL6-induced plasmacytomas are known to depend on AID expression The inability to regulate AID expression is also responsible for tumorigenesis in mice. Transgenic mice with constitutive expression of AID tend to develop maglinant T cell lymphoma, micro-adenomas and lung adenocarcinomas. Here one observes an increase in point mutations, but not translocations, in T cell receptors and c-myc genes from T cell lymphomas. The distribution and specificity of mutations is similar to those observed in B-cell lymphomas and in cultured cells that overexpress AID, which suggests that AID is responsible for tumorigenesis. In principal, however, any or all of the DNA deaminases have the potential to create similar havoc. The expression of Apobec-1 in rabbit and mouse liver correlates with the appearance of hepacellular carcinomas in transgenic animals. Thus, it is important to investigate how Apobec enzymes might be responsible for mutations leading to activation of protooncogenes and inactivation of tumor suppressor genes. 3. Characterization of aberrant deamination “at risk” sequences. Exposed ssDNA regions, occurring for example in genes undergoing active transcription, are likely targets for DNA deaminases. DNA deaminases may attack the non-transcribed strand and may also gain access to regions of supercoiled dsDNA situated adjacent to a moving transcription bubble. Favored mutational targets include WRC for AID, YCC for Apobec3G and TC for Apobec1 and Apobec3F. At risk motifs are likely to be tissue specific, depending on which of the DNA deaminases are present. For instance, mutations in proto-oncogenes in several types of B-cell tumors are frequently at C•G base pairs and a majority of these (64%) occur in WRC motifs, consistent with the “inappropriate” action of AID in B-cell tumors. In contrast, elevated mutations in a tumor suppressor gene APC were found at 5’TC sites in colorectal cancer, which is consistent with the action of Apobec1. The classic example of at risk sequences are methylated C in CpG dinucleotides, where C ? T mutations represent a disproportionately large fraction of mutations in human disease. It has been estimated that these comprise about a quarter of p53 tumor suppressor mutations. Since it has been shown that AID deaminates 5MeC residues with an efficiency of roughly 30% compared to C in vitro, it is possible that AID and other Apobec homologs can attack MeCpG sequences in vivo, with possible serious detrimental biological consequences. Secondary structures occurring in double stranded nucleic acids may also be susceptible to deamination at C sites. Sequences prone to assume non-W-C structures that have been implicated in human genetic disease include triplexes, left-handed DNA, G4 DNA tetrads, and slipped and sticky DNA structures including trinucleotide repeats implicated in about a dozen neurodegenerative diseases. These types of secondary structures could in principal be acted on by DNA deaminases to initiate dsDNA breaks leading to DNA rearrangements. The Ig switch region offers a concrete example of a sequence that assumes a secondary structure to serve as a substrate for AID. The Ig switch sequence provides a biologically beneficial substrate allowing AID to initiate CSR, and is technically not “at risk”. However, similar types of substrates are at risk, suggesting that DNA deaminases might contribute substantially to the mutational load by attacking regions containing DNA secondary structure.
 

Publications

Journal Article
Pham, P., Chelico, L., Goodman, M. F. (2007). DNA deaminases AID and APOBEC3G act processively on single-stranded DNA. DNA Repair (Amst). Vol. 6 (6), pp. 689-92.
Bransteitter, R. R., J, S. L., Allen, S., Pham, P. T., Goodman, M. F. (2006). First AID (activation-induced cytidine deaminase) is needed to produce high affinity isotype-switched antibodies. Journal of Biological Chemistry. Vol. 281, pp. 16833-16836.
Chelico, L., Pham, P. T., Calabrese, P., Goodman, M. F. (2006). APOBEC3G DNA deaminase acts processively 3' --> 5' on single-stranded DNA. Nature Structural & Molecular Biology/Nature Publishing Group. Vol. 13, pp. 392-399.
Pham, P. T., Zhao, W., Schaaper, R. M. (2006). Mutator mutants of Escherichia coli carrying a defect in the DNA polymerase III tau subunit. Molecular Microbiology/Blackwell Publishing. Vol. 59, pp. 1149-1161.
Schlacher, K., Pham, P. T., Cox, M., Goodman, M. F. (2006). Roles of DNA polymerase V and RecA protein in SOS damage-induced mutation. Chemical Reviews/American Chemical Society Press. Vol. 106, pp. 406-419.
Michell, D. L., Pham, P. T., Goodman, M. F., Nancy, M. (2005). AID binds to transcription-induced structures in c-MYC that map to regions associated with translocation and hypermutation. Oncogen/Nature Publishing Group. Vol. 24, pp. 5791-5798.
Pham, P. T., Bransteitter, R. R., Goodman, M. F. (2005). Reward versus Risk: DNA Cytidine Deaminases Triggering Immunity and Disease. Biochemistry/American Chemical Society. Vol. 44, pp. 2703-2715.
Bransteitter, R. R., Pham, P. T., Calabrese, P., Goodman, M. F. (2004). Biochemical analysis of hypermutational targeting by wild type and mutant activation-induced cytidine deaminase. Journal of Biological Chemistry. Vol. 279, pp. 51612-51621.
Tippin, B., Pham, P. T., Goodman, M. F. (2004). Error-prone replication for better or worse. Trends in Microbiology/Elsevier. Vol. 12, pp. 288-295.
Tippin, B., Pham, P. T., Bransteitter, R. R., Goodman, M. F. (2004). Somatic hypermutation: a mutational panacea. Advances in Protein Chemistry/Elsevier. Vol. 69, pp. 307-335.
 
 
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