Deubiquitinating PCNA: a downside to DNA damage tolerance

Ulrich, Helle D.

doi:10.1038/ncb0406-303

Cited by 22 publications

(20 citation statements)

References 13 publications

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“…PCNA is a DNA-encircling homotrimer that is central to all forms of DNA replication and serves as a sliding platform that recruits MMR and activates polymerases and other factors in a reversible and competitive fashion (13,14). The monoubiquitylation of PCNA is regulated by a complex mechanism that involves the E2 and E3 ubiquitin ligases, RAD6 and RAD18, and deubiquitylation primarily by USP1 (12,15,16). Recent studies in the DT40 chicken B cell line (16,17) suggest that the recruitment and activation of Pol to the Ig V regions is mediated by PCNA that is monoubiquitylated at residue K164.…”

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confidence: 99%

Ubiquitylated PCNA plays a role in somatic hypermutation and class-switch recombination and is required for meiotic progression

Roa¹,

Avdievich²,

Peled³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

104

100

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Somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes are dependent upon activation-induced cytidine deaminase (AID)-induced mutations. The scaffolding properties of proliferating cell nuclear antigen (PCNA) and ubiquitylation of its residue K164 have been suggested to play an important role organizing the error-prone repair events that contribute to the AID-induced diversification of the Ig locus. We generated knockout mice for PCNA (Pcna ؊/؊ ), which were embryonic lethal. Expression of PCNA with the K164R mutation rescued the lethal phenotype, but the mice (Pcna ؊/؊ tg K164R ) displayed a meiotic defect in early pachynema and were sterile. B cells proliferated normally in Pcna ؊/؊ tg K164R mice, but a PCNA-K164R mutation resulted in impaired ex vivo CSR to IgG1 and IgG3, which was associated with reduced mutation frequency at the switch regions and a bias toward blunt junctions. Analysis of the heavy chain V186.2 region after NP-immunization showed in Pcna ؊/؊ tg K164R mice a significant reduction in the mutation frequency of A:T residues in WA motifs preferred by polymerase-(Pol ), and a strand-biased increase in the mutation frequency of G residues, preferentially in the context of AID-targeted GYW motifs. The phenotype of Pcna ؊/؊ tg K164R mice supports the idea that ubiquitylation of PCNA participates directly in the meiotic process and the diversification of the Ig locus through class-switch recombination (CSR) and somatic hypermutation (SHM).T o mount an effective antibody response, mice and humans create a highly diverse repertoire of antigen binding sites through the rearrangement of the germ line variable (V), diversity (D), and joining (J) Ig locus. Following interaction with antigen, B cells in the germinal centers (GCs) of secondary lymphoid organs express activation-induced cytidine deaminase (AID). AID, together with other enzymes, causes a very high rate (10 Ϫ5 -10 Ϫ3 /base pair/generation) of point mutations in Ig V regions resulting in the affinity maturation and the changes in fine specificity required to produce protective antibodies (1, 2). AID also initiates class-switch recombination (CSR) by mutating the switch regions (SRs) that are located just 5Ј of the constant region genes (3, 4). CSR allows antibodies to be distributed throughout the body and to carry out a wide variety of effector functions. AID deaminates deoxycytidines (dC) in single-stranded DNA in the V and SRs to generate deoxyuridine (dU) (1, 2). However, more than half of the mutations in the V and SRs of mice and humans are in A:T bases and are not the result of the direct biochemical action of AID. Rather, these mutations arise during a second phase of SHM and result from the error-prone base excision repair (BER) and mismatch repair (MMR), both of which are recruited to the dU:dG mismatch generated by AID (1, 2, 4).When critical MMR genes are deleted from mice, most of the mutations in A:T in the V region no longer occur, suggesting that MMR is responsible for the majority of the mutations that ar...

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confidence: 99%

Ubiquitylated PCNA plays a role in somatic hypermutation and class-switch recombination and is required for meiotic progression

Roa¹,

Avdievich²,

Peled³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

104

100

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“…This flexibility is accompanied by low fidelity. The recruitment of TLS polymerases is promoted by ubiquitylation of the polymerase processivity clamp (PCNA) at lysine 164 (19,26,48,50). HR is also associated with DNA synthesis, which is facilitated by TLS polymerases such as Pol and Pol (20,41).…”

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confidence: 99%

The 9-1-1 DNA Clamp Is Required for Immunoglobulin Gene Conversion

Saberi

Nakahara

Sale

et al. 2008

Molecular and Cellular Biology

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Chicken DT40 cells deficient in the 9-1-1 checkpoint clamp exhibit hypersensitivity to a variety of DNAdamaging agents. Although recent work suggests that, in addition to its role in checkpoint activation, this complex may play a role in homologous recombination and translesion synthesis, the cause of this hypersensitivity has not been studied thoroughly. The immunoglobulin locus of DT40 cells allows monitoring of homologous recombination and translesion synthesis initiated by activation-induced deaminase (AID)-dependent abasic sites. We show that both the RAD9 ؊/؊ and RAD17 ؊/؊ mutants exhibit substantially reduced immunoglobulin gene conversion. However, the level of nontemplated immunoglobulin point mutation increased in these mutants, a finding that is reminiscent of the phenotype resulting from the loss of RAD51 paralogs or Brca2. This suggests that the 9-1-1 complex does not play a central role in translesion synthesis in this context. Despite reduced immunoglobulin gene conversion, the RAD9 ؊/؊ and RAD17 ؊/؊ cells do not exhibit a prominent defect in double-strand break-induced gene conversion or a sensitivity to camptothecin. This suggests that the roles of Rad9 and Rad17 may be confined to a subset of homologous recombination reactions initiated by replication-stalling lesions rather than those associated with double-strand break repair.DNA replication is a complex and fragile reaction that is frequently stalled by damaged template strands. To alleviate replication blocks, cells have evolved two basic pathways: homologous recombination (HR) and translesion synthesis (TLS). HR facilitates a template switch to the intact sister chromatid. TLS involves the direct bypass of the damage by specialized DNA polymerases whose catalytic sites are flexible enough to accommodate damaged bases in the template DNA. This flexibility is accompanied by low fidelity. The recruitment of TLS polymerases is promoted by ubiquitylation of the polymerase processivity clamp (PCNA) at lysine 164 (19, 26, 48, 50). HR is also associated with DNA synthesis, which is facilitated by TLS polymerases such as Pol and Pol (20,41). These DNA polymerases thus perform a function in both TLS and HR. The role of posttranslational modification of PCNA in vertebrate HR has not been extensively studied.The heterotrimeric checkpoint clamp, consisting of the Rad9, Hus1, and Rad1 subunits (corresponding to the Rad17, Mec3, and Ddc1 subunits in budding yeast), is structurally homologous to PCNA (39). It serves as a damage sensor in the S and G 2 /M phases, eventually facilitating cell cycle arrest in vertebrate cells (8,39). In vitro, the 9-1-1 complex can be loaded onto DNA by a specific clamp loader, Rad17-RFC, which is analogous to the PCNA-RFC clamp-clamp loader system (5, 10).Although these checkpoint factors do not appear to play a major role in mitotic HR in budding yeast, mammalian Rad9 and Rad17 seem to contribute to some HR reactions (e.g., repair of X-ray-induced double-strand breaks [DSBs] and gene targeting), though they are not requi...

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“…28 Moreover monoubiquitination of PCNA facilitates the replicative bypass of DNA lesions by recruiting damage-tolerant polymerases. 29 It will be interesting to explore the possibility that LigI activity is regulated by phosphorylation-dependent monoubiquitination.…”

Section: Discussionmentioning

confidence: 99%

DNA ligase I and Nbs1 proteins associate in a complex and colocalize at replication factories

Vago¹,

Leva²,

Biamonti³

et al. 2009

Cell Cycle

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DNA ligase I is the main DNA ligase activity involved in eukaryotic DNA replication acting in the joining of Okazaki fragments. This enzyme is also implicated in nucleotide excision repair and in the long-patch base excision repair while its role in the recombinational repair pathways is poorly understood. DNA ligase I is phosphorylated during cell cycle at several serine and threonine residues that regulate its participation in different DNA transactions by modulating the interaction with different protein partners. Here we use an antibody-based array method to identify novel DNA ligase-interacting partners. We show that DNA ligase I participates in several multiprotein complexes with proteins involved in DNA replication and repair, cell cycle control, and protein modification. In particular we demonstrate that DNA ligase I complexes with Nbs1, a core component of the MRN complex critical for detection, processing and repair of double-stranded DNA breaks. The analysis of epitope tagged DNA ligase I mutants demonstrates that the association is mediated by the catalytic fragment of the enzyme. DNA ligase I and Nbs1 colocalize at replication factories during unperturbed replication and after treatment with DNA damaging agents. Since MRN complex is involved in the repair of double-stranded DNA breaks by homologous recombination at stalled replication forks our data support the notion that DNA ligase I participates in homology dependent pathways that deal with replicationassociated lesions generated when replication fork encounters DNA damage.

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Deubiquitinating PCNA: a downside to DNA damage tolerance

Cited by 22 publications

References 13 publications

Ubiquitylated PCNA plays a role in somatic hypermutation and class-switch recombination and is required for meiotic progression

Ubiquitylated PCNA plays a role in somatic hypermutation and class-switch recombination and is required for meiotic progression

The 9-1-1 DNA Clamp Is Required for Immunoglobulin Gene Conversion

DNA ligase I and Nbs1 proteins associate in a complex and colocalize at replication factories

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