scholarly journals Somatic hypermutation of immunoglobulin genes: lessons from proliferating cell nuclear antigen K164R mutant mice

2008 ◽  
Vol 364 (1517) ◽  
pp. 621-629 ◽  
Author(s):  
Petra Langerak ◽  
Peter H.L Krijger ◽  
Marinus R Heideman ◽  
Paul C.M van den Berk ◽  
Heinz Jacobs
Keyword(s):  
B Cells ◽  
Proliferating Cell Nuclear Antigen ◽  
Somatic Mutations ◽  
Dna Polymerases ◽  
Critical Role ◽  
Dna Lesions ◽  
Nuclear Antigen ◽  
Immunoglobulin Genes ◽  
Proliferating Cell

Proliferating cell nuclear antigen (PCNA) encircles DNA as a ring-shaped homotrimer and, by tethering DNA polymerases to their template, PCNA serves as a critical replication factor. In contrast to high-fidelity DNA polymerases, the activation of low-fidelity translesion synthesis (TLS) DNA polymerases seems to require damage-inducible monoubiquitylation (Ub) of PCNA at lysine residue 164 (PCNA-Ub). TLS polymerases can tolerate DNA damage, i.e. they can replicate across DNA lesions. The lack of proofreading activity, however, renders TLS highly mutagenic. The advantage is that B cells use mutagenic TLS to introduce somatic mutations in immunoglobulin (Ig) genes to generate high-affinity antibodies. Given the critical role of PCNA-Ub in activating TLS and the role of TLS in establishing somatic mutations in immunoglobulin genes, we analysed the mutation spectrum of somatically mutated immunoglobulin genes in B cells from PCNA K164R knock-in mice. A 10-fold reduction in A/T mutations is associated with a compensatory increase in G/C mutations—a phenotype similar to Polη and mismatch repair-deficient B cells. Mismatch recognition, PCNA-Ub and Polη probably act within one pathway to establish the majority of mutations at template A/T. Equally relevant, the G/C mutator(s) seems largely independent of PCNA K164 modification.

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

2009 ◽  
Vol 206 (12) ◽  
pp. 2603-2611 ◽  
Author(s):  
Peter H.L. Krijger ◽  
Petra Langerak ◽  
Paul C.M. van den Berk ◽  
Heinz Jacobs
Keyword(s):  
B Cells ◽  
Proliferating Cell Nuclear Antigen ◽  
Mutation Frequency ◽  
Somatic Hypermutation ◽  
Nuclear Antigen ◽  
Immunoglobulin Genes ◽  
Nucleotide Substitutions ◽  
High Affinity ◽  
Proliferating Cell ◽  
Dependent Pathway

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.

scholarly journals APIM-Mediated REV3L–PCNA Interaction Important for Error Free TLS Over UV-Induced DNA Lesions in Human Cells

2018 ◽  
Vol 20 (1) ◽  
pp. 100 ◽  
Author(s):  
Synnøve Ræder ◽  
Anala Nepal ◽  
Karine Bjørås ◽  
Mareike Seelinger ◽  
Rønnaug Kolve ◽  
...  
Keyword(s):  
Cell Lines ◽  
Proliferating Cell Nuclear Antigen ◽  
Dna Lesions ◽  
Full Length ◽  
Nuclear Antigen ◽  
Wild Type ◽  
Replication Foci ◽  
Multiple Cell ◽  
Mutation Spectra ◽  
Proliferating Cell

Proliferating cell nuclear antigen (PCNA) is essential for the organization of DNA replication and the bypass of DNA lesions via translesion synthesis (TLS). TLS is mediated by specialized DNA polymerases, which all interact, directly or indirectly, with PCNA. How interactions between the TLS polymerases and PCNA affects TLS specificity and/or coordination is not fully understood. Here we show that the catalytic subunit of the essential mammalian TLS polymerase POLζ, REV3L, contains a functional AlkB homolog 2 PCNA interacting motif, APIM. APIM from REV3L fused to YFP, and full-length REV3L-YFP colocalizes with PCNA in replication foci. Colocalization of REV3L-YFP with PCNA is strongly reduced when an APIM-CFP construct is overexpressed. We also found that overexpression of full-length REV3L with mutated APIM leads to significantly altered mutation frequencies and mutation spectra, when compared to overexpression of full-length REV3L wild-type (WT) protein in multiple cell lines. Altogether, these data suggest that APIM is a functional PCNA-interacting motif in REV3L, and that the APIM-mediated PCNA interaction is important for the function and specificity of POLζ in TLS. Finally, a PCNA-targeting cell-penetrating peptide, containing APIM, reduced the mutation frequencies and changed the mutation spectra in several cell lines, suggesting that efficient TLS requires coordination mediated by interactions with PCNA.

scholarly journals Effect of Proliferating Cell Nuclear Antigen Ubiquitination and Chromatin Structure on the Dynamic Properties of the Y-family DNA Polymerases

2008 ◽  
Vol 19 (12) ◽  
pp. 5193-5202 ◽  
Author(s):  
Simone Sabbioneda ◽  
Audrey M. Gourdin ◽  
Catherine M. Green ◽  
Angelika Zotter ◽  
Giuseppina Giglia-Mari ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Proliferating Cell Nuclear Antigen ◽  
Dynamic Properties ◽  
Human Fibroblasts ◽  
Dna Polymerases ◽  
S Phase ◽  
Nuclear Antigen ◽  
Replication Foci ◽  
Y Family ◽  
Proliferating Cell

Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) η and ι are usually uniformly distributed through the nucleus but accumulate in replication foci during S phase. DNA-damaging treatments result in an increase in S phase cells containing polymerase foci. Using photobleaching techniques, we show that polη is highly mobile in human fibroblasts. Even when localized in replication foci, it is only transiently immobilized. Although ubiquitination of proliferating cell nuclear antigen (PCNA) is not required for the localization of polη in foci, it results in an increased residence time in foci. polι is even more mobile than polη, both when uniformly distributed and when localized in foci. Kinetic modeling suggests that both polη and polι diffuse through the cell but that they are transiently immobilized for ∼150 ms, with a larger proportion of polη than polι immobilized at any time. Treatment of cells with DRAQ5, which results in temporary opening of the chromatin structure, causes a dramatic immobilization of polη but not polι. Our data are consistent with a model in which the polymerases are transiently probing the DNA/chromatin. When DNA is exposed at replication forks, the polymerase residence times increase, and this is further facilitated by the ubiquitination of PCNA.

Characterization of cytosolic proliferating cell nuclear antigen (PCNA) in neutrophils: antiapoptotic role of the monomer

2013 ◽  
Vol 94 (4) ◽  
pp. 723-731 ◽  
Author(s):  
Alessia De Chiara ◽  
Magali Pederzoli-Ribeil ◽  
Julie Mocek ◽  
Céline Candalh ◽  
Patrick Mayeux ◽  
...  
Keyword(s):  
Proliferating Cell Nuclear Antigen ◽  
Nuclear Antigen ◽  
Proliferating Cell

scholarly journals DNA Polymerases BI and D from the Hyperthermophilic Archaeon Pyrococcus furiosus Both Bind to Proliferating Cell Nuclear Antigen with Their C-Terminal PIP-Box Motifs

2007 ◽  
Vol 189 (15) ◽  
pp. 5652-5657 ◽  
Author(s):  
Kazuo Tori ◽  
Megumi Kimizu ◽  
Sonoko Ishino ◽  
Yoshizumi Ishino
Keyword(s):  
Dna Synthesis ◽  
Proliferating Cell Nuclear Antigen ◽  
Pyrococcus Furiosus ◽  
Dna Polymerases ◽  
Nuclear Antigen ◽  
Hyperthermophilic Archaeon ◽  
Sliding Clamp ◽  
Replication Fork Progression ◽  
Proliferating Cell

ABSTRACT Proliferating cell nuclear antigen (PCNA) is the sliding clamp that is essential for the high processivity of DNA synthesis during DNA replication. Pyrococcus furiosus, a hyperthermophilic archaeon, has at least two DNA polymerases, polymerase BI (PolBI) and PolD. Both of the two DNA polymerases interact with the archaeal P. furiosus PCNA (PfuPCNA) and perform processive DNA synthesis in vitro. This phenomenon, in addition to the fact that both enzymes display 3′-5′ exonuclease activity, suggests that both DNA polymerases work in replication fork progression. We demonstrated here that both PolBI and PolD functionally interact with PfuPCNA at their C-terminal PIP boxes. The mutant PolBI and PolD enzymes lacking the PIP-box sequence do not respond to the PfuPCNA at all in an in vitro primer extension reaction. This is the first experimental evidence that the PIP-box motif, located at the C termini of the archaeal DNA polymerases, is actually critical for PCNA binding to form a processive DNA-synthesizing complex.

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