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Dependence of nucleotide substitutions on Ung2, Msh2, and PCNA-Ub during somatic hypermutation.

Krijger PH, Langerak P, van den Berk PC, Jacobs H - J. Exp. Med. (2009)

Bottom Line: Our data indicate that PCNA-Ub is required for A/T mutagenesis downstream of both Msh2 and Ung2.Furthermore, we provide evidence that both pathways are noncompetitive to initiate mutagenesis and even collaborate to generate half of all G/C transversions.These findings significantly add to our understanding of SHM and necessitate an update of present SHM models.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunology, The Netherlands Cancer Institute, 1066 CX Amsterdam, Netherlands.

ABSTRACT
During somatic hypermutation (SHM), B cells introduce mutations into their immunoglobulin genes to generate high affinity antibodies. Current models suggest a separation in the generation of G/C transversions by the Ung2-dependent pathway and the generation of A/T mutations by the Msh2/ubiquitinated proliferating cell nuclear antigen (PCNA-Ub)-dependent pathway. It is currently unknown whether these pathways compete to initiate mutagenesis and whether PCNA-Ub functions downstream of Ung2. Furthermore, these models do not explain why mice lacking Msh2 have a more than twofold reduction in the total mutation frequency. Our data indicate that PCNA-Ub is required for A/T mutagenesis downstream of both Msh2 and Ung2. Furthermore, we provide evidence that both pathways are noncompetitive to initiate mutagenesis and even collaborate to generate half of all G/C transversions. These findings significantly add to our understanding of SHM and necessitate an update of present SHM models.

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Related in: MedlinePlus

Current model: pathways of SHM downstream of AID. The three pathways: (a) replication across U, (b) Ung2-dependent SHM, and (c) Msh2-dependent SHM. Known and unknown (?) polymerases involved in the generation of specific mutations are indicated. TS, transitions; TV, transversions.
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fig1: Current model: pathways of SHM downstream of AID. The three pathways: (a) replication across U, (b) Ung2-dependent SHM, and (c) Msh2-dependent SHM. Known and unknown (?) polymerases involved in the generation of specific mutations are indicated. TS, transitions; TV, transversions.

Mentions: To generate high affinity antibodies, germinal center (GC) B cells are enabled to introduce point mutations into the variable region of their rearranged Ig genes. This process of somatic hypermutation (SHM) occurs at an astonishing rate of one per thousand bases per generation, six orders of magnitude greater than spontaneous mutagenesis (Di Noia and Neuberger, 2007). SHM is initiated by the activation-induced cytidine deaminase (AID), an enzyme found to be differentially expressed in B cells of the GC (Muramatsu et al., 2000). AID deaminates C to U within single-stranded DNA, and targets both DNA strands in the variable and switch regions of Ig genes. To establish point mutations at and around the U, three alternative pathways can handle this initial lesion (Fig. 1). (a) Replication across a U instructs a template T to DNA polymerases and generates G/C to A/T transitions (Rada et al., 2004; Shen et al., 2006). (b) The U can be excised from the DNA backbone by the base excision repair (BER) protein Ung2, and an abasic or apyrimidinic (AP) site is generated, causing replicative DNA polymerases to stall. To continue replication, specialized translesion synthesis (TLS) polymerases can be recruited, enabling a direct replicative bypass of AP sites. As AP sites are noninstructive, these TLS polymerases generate G/C transversions and may contribute to G/C transitions (Ung2-dependent SHM). Accordingly, Ung mutant B cells lack most G/C transversions (Rada et al., 2002). (c) Alternatively, the U can be recognized as a U:G mismatch by the mismatch repair complex Msh2–Msh6, resulting in Exo-1 activation and the formation of a single-stranded gap around the initial mismatch. Interestingly, Msh2-, Msh6-, and Exo-1–deficient B cells lack 80–90% of all A/T mutations, suggesting that the gap-filling process is executed by TLS polymerases predominantly generating A/T mutations (Msh2-dependent SHM; Rada et al., 1998; Wiesendanger et al., 2000; Bardwell et al., 2004). As a significant fraction of A/T mutations (10–20%) are found in Msh2-deficient GC B cells but not in Ung2/Msh2 double-deficient GC B cells, Ung2-dependent mutagenesis generates the described fraction of A/T mutations (Rada et al., 2004). Whether Ung2-dependent A/T mutations are generated during long-patch BER (i.e., within the strand containing the AP site) or, alternatively, during the extension phase of TLS across the AP site is currently unknown. In summary, these data suggest a specific role of these pathways in recruiting and activating selective TLS polymerases to establish defined mutations. The combination of these pathways enables hypermutating B cells to generate the entire spectrum of nucleotide substitutions.


Dependence of nucleotide substitutions on Ung2, Msh2, and PCNA-Ub during somatic hypermutation.

Krijger PH, Langerak P, van den Berk PC, Jacobs H - J. Exp. Med. (2009)

Current model: pathways of SHM downstream of AID. The three pathways: (a) replication across U, (b) Ung2-dependent SHM, and (c) Msh2-dependent SHM. Known and unknown (?) polymerases involved in the generation of specific mutations are indicated. TS, transitions; TV, transversions.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2806606&req=5

fig1: Current model: pathways of SHM downstream of AID. The three pathways: (a) replication across U, (b) Ung2-dependent SHM, and (c) Msh2-dependent SHM. Known and unknown (?) polymerases involved in the generation of specific mutations are indicated. TS, transitions; TV, transversions.
Mentions: To generate high affinity antibodies, germinal center (GC) B cells are enabled to introduce point mutations into the variable region of their rearranged Ig genes. This process of somatic hypermutation (SHM) occurs at an astonishing rate of one per thousand bases per generation, six orders of magnitude greater than spontaneous mutagenesis (Di Noia and Neuberger, 2007). SHM is initiated by the activation-induced cytidine deaminase (AID), an enzyme found to be differentially expressed in B cells of the GC (Muramatsu et al., 2000). AID deaminates C to U within single-stranded DNA, and targets both DNA strands in the variable and switch regions of Ig genes. To establish point mutations at and around the U, three alternative pathways can handle this initial lesion (Fig. 1). (a) Replication across a U instructs a template T to DNA polymerases and generates G/C to A/T transitions (Rada et al., 2004; Shen et al., 2006). (b) The U can be excised from the DNA backbone by the base excision repair (BER) protein Ung2, and an abasic or apyrimidinic (AP) site is generated, causing replicative DNA polymerases to stall. To continue replication, specialized translesion synthesis (TLS) polymerases can be recruited, enabling a direct replicative bypass of AP sites. As AP sites are noninstructive, these TLS polymerases generate G/C transversions and may contribute to G/C transitions (Ung2-dependent SHM). Accordingly, Ung mutant B cells lack most G/C transversions (Rada et al., 2002). (c) Alternatively, the U can be recognized as a U:G mismatch by the mismatch repair complex Msh2–Msh6, resulting in Exo-1 activation and the formation of a single-stranded gap around the initial mismatch. Interestingly, Msh2-, Msh6-, and Exo-1–deficient B cells lack 80–90% of all A/T mutations, suggesting that the gap-filling process is executed by TLS polymerases predominantly generating A/T mutations (Msh2-dependent SHM; Rada et al., 1998; Wiesendanger et al., 2000; Bardwell et al., 2004). As a significant fraction of A/T mutations (10–20%) are found in Msh2-deficient GC B cells but not in Ung2/Msh2 double-deficient GC B cells, Ung2-dependent mutagenesis generates the described fraction of A/T mutations (Rada et al., 2004). Whether Ung2-dependent A/T mutations are generated during long-patch BER (i.e., within the strand containing the AP site) or, alternatively, during the extension phase of TLS across the AP site is currently unknown. In summary, these data suggest a specific role of these pathways in recruiting and activating selective TLS polymerases to establish defined mutations. The combination of these pathways enables hypermutating B cells to generate the entire spectrum of nucleotide substitutions.

Bottom Line: Our data indicate that PCNA-Ub is required for A/T mutagenesis downstream of both Msh2 and Ung2.Furthermore, we provide evidence that both pathways are noncompetitive to initiate mutagenesis and even collaborate to generate half of all G/C transversions.These findings significantly add to our understanding of SHM and necessitate an update of present SHM models.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunology, The Netherlands Cancer Institute, 1066 CX Amsterdam, Netherlands.

ABSTRACT
During somatic hypermutation (SHM), B cells introduce mutations into their immunoglobulin genes to generate high affinity antibodies. Current models suggest a separation in the generation of G/C transversions by the Ung2-dependent pathway and the generation of A/T mutations by the Msh2/ubiquitinated proliferating cell nuclear antigen (PCNA-Ub)-dependent pathway. It is currently unknown whether these pathways compete to initiate mutagenesis and whether PCNA-Ub functions downstream of Ung2. Furthermore, these models do not explain why mice lacking Msh2 have a more than twofold reduction in the total mutation frequency. Our data indicate that PCNA-Ub is required for A/T mutagenesis downstream of both Msh2 and Ung2. Furthermore, we provide evidence that both pathways are noncompetitive to initiate mutagenesis and even collaborate to generate half of all G/C transversions. These findings significantly add to our understanding of SHM and necessitate an update of present SHM models.

Show MeSH
Related in: MedlinePlus