Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glycosylase

A functional immune system depends on the production of a wide range of immunoglobulin molecules. Immunoglobulin variable region (IgV) genes are diversified after gene rearrangement by hypermutation. In the DNA deamination model, we have proposed that deamination of dC residues to dU by activation-induced deaminase (AID) triggers this diversification. In hypermutating chicken DT40 B cells, most IgV mutations are dC --> dG/dA or dG --> dC/dT transversions, which are proposed to result from replication over sites of base loss produced by the excision activity of uracil-DNA glycosylase. Blocking the activity of uracil-DNA glycosylase should instead lead to replication over the dU lesion, resulting in dC --> dT (and dG --> dA) transitions. Here we show that expression in DT40 cells of a bacteriophage-encoded protein that inhibits uracil-DNA glycosylase shifts the pattern of IgV gene mutations from transversion dominance to transition dominance. This is good evidence that antibody diversification involves dC --> dU deamination within the immunoglobulin locus itself.     

Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into somatic hypermutation

After gene rearrangement, immunoglobulin V genes are further diversified by either somatic hypermutation or gene conversion. Hypermutation (in man and mouse) occurs by the fixation of individual, non-templated nucleotide substitutions. Gene conversion (in chicken) is templated by a set of upstream V pseudogenes. Here we show that if the RAD51 paralogues XRCC2, XRCC3 or RAD51B are ablated the pattern of diversification of the immunoglobulin V gene in the chicken DT40 B-cell lymphoma line exhibits a marked shift from one of gene conversion to one of somatic hypermutation. Non-templated, single-nucleotide substitutions are incorporated at high frequency specifically into the V domain, largely at G/C and with a marked hotspot preference. These mutant DT40 cell lines provide a tractable model for the genetic dissection of immunoglobulin hypermutation and the results support the idea that gene conversion and somatic hypermutation constitute distinct pathways for processing a common lesion in the immunoglobulin V gene. The marked induction of somatic hypermutation that is achieved by ablating the RAD51 paralogues is probably a consequence of modifying the recombination-mediated repair of such initiating lesions.     

Two levels of protection for the B cell genome during somatic hypermutation

Somatic hypermutation introduces point mutations into immunoglobulin genes in germinal centre B cells during an immune response. The reaction is initiated by cytosine deamination by the activation-induced deaminase (AID) and completed by error-prone processing of the resulting uracils by mismatch and base excision repair factors. Somatic hypermutation represents a threat to genome integrity and it is not known how the B cell genome is protected from the mutagenic effects of somatic hypermutation nor how often these protective mechanisms fail. Here we show, by extensive sequencing of murine B cell genes, that the genome is protected by two distinct mechanisms: selective targeting of AID and gene-specific, high-fidelity repair of AID-generated uracils. Numerous genes linked to B cell tumorigenesis, including Myc, Pim1, Pax5, Ocab (also called Pou2af1), H2afx, Rhoh and Ebf1, are deaminated by AID but escape acquisition of most mutations through the combined action of mismatch and base excision repair. However, approximately 25% of expressed genes analysed were not fully protected by either mechanism and accumulated mutations in germinal centre B cells. Our results demonstrate that AID acts broadly on the genome, with the ultimate distribution of mutations determined by a balance between high-fidelity and error-prone DNA repair.     

The AID antibody diversification enzyme is regulated by protein kinase A phosphorylation

Antibodies, which are produced by B-lineage cells, consist of immunoglobulin heavy (IgH) and light (IgL) chains that have amino-terminal variable regions and carboxy-terminal constant regions. In response to antigens, B cells undergo two types of genomic alterations to increase antibody diversity. Affinity for antigen can be increased by introduction of point mutations into IgH and IgL variable regions by somatic hypermutation. In addition, antibody effector functions can be altered by changing the expressed IgH constant region exons through IgH class switch recombination (CSR). Somatic hypermutation and CSR both require the B-cell-specific activation-induced cytidine deaminase protein (AID), which initiates these reactions through its single-stranded (ss)DNA-specific cytidine deaminase activity. In biochemical assays, replication protein A (RPA), a ssDNA-binding protein, associates with phosphorylated AID from activated B cells and enhances AID activity on transcribed double-stranded (ds)DNA containing somatic hypermutation or CSR target sequences. This AID-RPA association, which requires phosphorylation, may provide a mechanism for allowing AID to access dsDNA targets in activated B cells. Here we show that AID from B cells is phosphorylated on a consensus protein kinase A (PKA) site and that PKA is the physiological AID kinase. Thus, AID from non-lymphoid cells can be functionally phosphorylated by recombinant PKA to allow interaction with RPA and promote deamination of transcribed dsDNA substrates. Moreover, mutation of the major PKA phosphorylation site of AID preserves ssDNA deamination activity, but markedly reduces RPA-dependent dsDNA deamination activity and severely impairs the ability of AID to effect CSR in vivo. We conclude that PKA has a critical role in post-translational regulation of AID activity in B cells.     

AID is required to initiate Nbs1/gamma-H2AX focus formation and mutations at sites of class switching.

Class switch recombination (CSR) is a region-specific DNA recombination reaction that replaces one immunoglobulin heavy-chain constant region (Ch) gene with another. This enables a single variable (V) region gene to be used in conjunction with different downstream Ch genes, each having a unique biological activity. The molecular mechanisms that mediate CSR have not been defined, but activation-induced cytidine deaminase (AID), a putative RNA-editing enzyme, is required for this reaction. Here we report that the Nijmegen breakage syndrome protein (Nbs1) and phosphorylated H2A histone family member X (gamma-H2AX, also known as gamma-H2afx), which facilitate DNA double-strand break (DSB) repair, form nuclear foci at the Ch region in the G1 phase of the cell cycle in cells undergoing CSR, and that switching is impaired in H2AX-/- mice. Localization of Nbs1 and gamma-H2AX to the Igh locus during CSR is dependent on AID. In addition, AID is required for induction of switch region (S mu)-specific DNA lesions that precede CSR. These results place AID function upstream of the DNA modifications that initiate CSR.     

Induction of somatic hypermutation in immunoglobulin genes is dependent on DNA polymerase iota

Somatic hypermutation of immunoglobulin genes is a unique, targeted, adaptive process. While B cells are engaged in germinal centres in T-dependent responses, single base substitutions are introduced in the expressed Vh/Vl genes to allow the selection of mutants with a higher affinity for the immunizing antigen. Almost every possible DNA transaction has been proposed to explain this process, but each of these models includes an error-prone DNA synthesis step that introduces the mutations. The Y family of DNA polymerases--pol eta, pol iota, pol kappa and rev1--are specialized for copying DNA lesions and have high rates of error when copying a normal DNA template. By performing gene inactivation in a Burkitt's lymphoma cell line inducible for hypermutation, we show here that somatic hypermutation is dependent on DNA polymerase iota.     

A single amino acid governs enhanced activity of DinB DNA polymerases on damaged templates

Translesion synthesis (TLS) by Y-family DNA polymerases is a chief mechanism of DNA damage tolerance. Such TLS can be accurate or error-prone, as it is for bypass of a cyclobutane pyrimidine dimer by DNA polymerase eta (XP-V or Rad30) or bypass of a (6-4) TT photoproduct by DNA polymerase V (UmuD'2C), respectively. Although DinB is the only Y-family DNA polymerase conserved among all domains of life, the biological rationale for this striking conservation has remained enigmatic. Here we report that the Escherichia coli dinB gene is required for resistance to some DNA-damaging agents that form adducts at the N2-position of deoxyguanosine (dG). We show that DinB (DNA polymerase IV) catalyses accurate TLS over one such N2-dG adduct (N2-furfuryl-dG), and that DinB and its mammalian orthologue, DNA polymerase kappa, insert deoxycytidine (dC) opposite N2-furfuryl-dG with 10-15-fold greater catalytic proficiency than opposite undamaged dG. We also show that mutating a single amino acid, the 'steric gate' residue of DinB (Phe13 --> Val) and that of its archaeal homologue Dbh (Phe12 --> Ala), separates the abilities of these enzymes to perform TLS over N2-dG adducts from their abilities to replicate an undamaged template. We propose that DinB and its orthologues are specialized to catalyse relatively accurate TLS over some N2-dG adducts that are ubiquitous in nature, that lesion bypass occurs more efficiently than synthesis on undamaged DNA, and that this specificity may be achieved at least in part through a lesion-induced conformational change.     

Dependence of DNA helix flexibility on base composition

We have used triplet anisotropy decay techniques to study the flexibility of synthetic DNA fragments with different base pair compositions. We have found major differences in the torsional and bending stiffness of poly(dG) . poly(dC), poly(dA) . poly(dT) and poly(dA-dC) . poly(dT-dG). Poly(dG) . poly(dC) has a torsional modulus more than 40 times larger than poly(dA-dC) . poly(dT-dG), and approximately 20 times larger than poly(dA) . poly(dT). These differences imply that the torsional stiffness of DNA can vary greatly with base composition. The Young's modulus (bending stiffness) we have measured for poly(dG) . poly(dC) is at least twice that of poly(dA-dC) . poly(dT-dG) or random sequence DNA, and is at least threefold greater than that of poly(dA) . poly(dT). This implies that the bending stiffness of DNA is also strongly dependent on base composition. In light of this dramatic base composition dependence, we suggest here that such stiffness variation may lead to local variations in the stability of chromatin or other protein complexes that require bending or twisting of the DNA helix.     

Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas.

Genomic instability promotes tumorigenesis and can occur through various mechanisms, including defective segregation of chromosomes or inactivation of DNA mismatch repair. Although B-cell lymphomas are associated with chromosomal translocations that deregulate oncogene expression, a mechanism for genome-wide instability during lymphomagenesis has not been described. During B-cell development, the immunoglobulin variable (V) region genes are subject to somatic hypermutation in germinal-centre B cells. Here we report that an aberrant hypermutation activity targets multiple loci, including the proto-oncogenes PIM1, MYC, RhoH/TTF (ARHH) and PAX5, in more than 50% of diffuse large-cell lymphomas (DLCLs), which are tumours derived from germinal centres. Mutations are distributed in the 5' untranslated or coding sequences, are independent of chromosomal translocations, and share features typical of V-region-associated somatic hypermutation. In contrast to mutations in V regions, however, these mutations are not detectable in normal germinal-centre B cells or in other germinal-centre-derived lymphomas, suggesting a DLCL-associated malfunction of somatic hypermutation. Intriguingly, the four hypermutable genes are susceptible to chromosomal translocations in the same region, consistent with a role for hypermutation in generating translocations by DNA double-strand breaks. By mutating multiple genes, and possibly by favouring chromosomal translocations, aberrant hypermutation may represent the major contributor to lymphomagenesis.     

Activation-induced cytidine deaminase turns on somatic hypermutation in hybridomas

The production of high-affinity protective antibodies requires somatic hypermutation (SHM) of the antibody variable (V)-region genes. SHM is characterized by a high frequency of point mutations that occur only during the centroblast stage of B-cell differentiation. Activation-induced cytidine deaminase (AID), which is expressed specifically in germinal-centre centroblasts, is required for this process, but its exact role is unknown. Here we show that AID is required for SHM in the centroblast-like Ramos cells, and that expression of AID is sufficient to induce SHM in hybridoma cells, which represent a later stage of B-cell differentiation that does not normally undergo SHM. In one hybridoma, mutations were exclusively in G*C base pairs that were mostly within RGYW or WRCY motifs, suggesting that AID has primary responsibility for mutations at these nucleotides. The activation of SHM in hybridomas indicates that AID does not require other centroblast-specific cofactors to induce SHM, suggesting either that it functions alone or that the factors it requires are expressed at other stages of B-cell differentiation.     

DNase I hypersensitive sites in the chromatin of human mu immunoglobulin heavy-chain genes

An immunoglobulin polypeptide chain is encoded by multiple gene segments that lie far apart in germ-line DNA and must be brought together to allow expression of an immunoglobulin gene active in B lymphocytes. For the immunoglobulin heavy chain genes, one of many variable (V) region genes becomes joined to one of several diversity (D) segments which are fused to one of several joining (J) segments lying 5' of the constant region (C) genes. Here we show that the rearranged mu genes of an IgM-producing human B-lymphocyte cell line exhibit pancreatic deoxyribonuclease (DNase I) hypersensitive sites in the JH-C mu intron that are absent in naked DNA or the chromatin of other differentiated cell types. DNA sequence analysis reveals that the major hypersensitive site maps to a conserved region of the JH-C mu intron recently shown to function as a tissue-specific enhancer of heavy-chain gene expression. A similar association of an enhancer-like element with a DNase I hypersensitive site has been reported for the mouse immunoglobulin light-chain J kappa-C kappa intron. These results implicate disruption of local chromatin structure in the mechanism of immunoglobulin enhancer function.     

Somatic hypermutation of an immunoglobulin transgene in kappa transgenic mice

Initial studies of somatically acquired mutations in immunoglobulin V regions from hybridomas and myelomas that are not derived from joining aberrations, suggested a controlled and specific hypermutation process, because spontaneous mutation rates observed for other genes are extremely low. Some evidence for the idea that mutations are introduced during V-gene rearrangement came from the clustering of mutations at the joining sites, from the absence of mutations in unrearranged V genes and from the low level of mutations in only partially (D-J) rearranged nonproductive heavy-chain alleles. Another model in which mutations accumulate with each cell division, rather than being introduced all at once, was supported by the finding that immunoglobulin genes of hybridomas derived from a single mouse frequently had several mutations in common, and so might be derived from the same precursor cell whose daughters then accumulated additional mutations. But the common mutations in some cases could be due to as yet unidentified related germline genes, or could represent the effect of antigen selection for certain amino acids. To try to detect hypermutation in the absence of V-gene rearrangement, we isolated B lymphocytes with endogenous heavy-chain gene mutations from transgenic mice carrying pre-rearranged kappa-transgenes. We found that these kappa-transgenes were also somatically mutated. This and other observations indicated that: ongoing rearrangement is not required for mutation; there are signals for hypermutation in the transgenes; the mutations are found only in the variable region, so the constant region may not be a target; different transgene insertion sites are compatible with hypermutations and more than one transgene is expressed in the same cell.     

应用大肠杆菌的核苷磷酸化酶合成胸苷

通过大肠杆菌核苷磷酸化酶的转脱氧核糖基作用，从胸腺嘧啶和２‘－脱氧核苷合成胸苷。优化了以ｄＵ为底物合成胸苷的反应条件。以３０ｍｍｏｌ／Ｌ ｄＵ为底物其合成胸苷的转化率可达５．３％；以ｄＡ，ｄＣ，ｄＵ，ｄＧ的混合物为底物转化度为５２．６％；以ＤＮＡ降解后的２’－０脱氧核苷混合液作底物，反应液中胸苷浓度从９．２ｍｍｏｌ／Ｌ提高到１７．９ｍｍｏｌ／Ｌ。