Functional sites of human PCNA which interact with p21 (Cip1/Waf1), DNA polymerase δ and replication factor C

Oku, Takeo; Ikeda, Soichiro; Sasaki, Haruyo; Fukuda, Kotaro; Morioka, Hiroshi; Ohtsuka, Eiko; Yoshikawa, Hiroshi; Tsurimoto, Toshiki

doi:10.1046/j.1365-2443.1998.00199.x

Cited by 71 publications

(66 citation statements)

References 28 publications

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“…In addition, p21 is highly expressed in most cells and is known to have a strong affinity for PCNA. 12 Therefore, it is possible that under physiological conditions, PIDD upregulation amplifies rather than induces dissociation of p21 from PCNA upon UV-C irradiation. The dissociation of p21 from PCNA would suggest that PIDD and p21 interact with PCNA in close proximity and compete for nearby binding sites.…”

Section: Resultsmentioning

confidence: 99%

“…PIDD was still able to interact with these mutants (Figure 3c) indicating that the trimeric form of PCNA is not required for the interaction with PIDD. Another PCNA mutant (the KIE/A substitution mutant), 12 capable of forming trimers, but unable to bind p21 (Supplementary Figure S3B), still interacts with PIDD (Supplementary Figure S3C), indicating that the p21 and PIDD binding sites are distinct. In addition, this competition is not because of direct binding of PIDD to p21 as no interaction was found between these two proteins (Supplementary Figure S2A).…”

Section: Resultsmentioning

confidence: 99%

“…9 P21 binds to PCNA and inhibits its activity, 10,11 mainly because p21 obstructs the interaction of PCNA with DNA replication and repair factors. [12][13][14][15] In vitro studies have shown that p21 also inhibits the loading of PCNA onto DNA, 7 thereby compromising DNA repair. 9 UV is one of the major exogenous sources of DNA damage.…”

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confidence: 99%

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PIDD orchestrates translesion DNA synthesis in response to UV irradiation

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Schuepbach‐Mallepell²,

Eckert

et al. 2011

Cell Death Differ

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PIDD has been implicated in survival and apoptotic pathways in response to DNA damage, and a role for PIDD was recently identified in non-homologous end-joining (NHEJ) repair induced by c-irradiation. Here, we present an interaction of PIDD with PCNA, first identified in a proteomics screen. PCNA has essential functions in DNA replication and repair following UV irradiation. Translesion synthesis (TLS) is a process that prevents UV irradiation-induced replication blockage and is characterized by PCNA monoubiquitination and interaction with the TLS polymerase eta (polg). Both of these processes are inhibited by p21. We report that PIDD modulates p21-PCNA dissociation, and promotes PCNA monoubiquitination and interaction with polg in response to UV irradiation. Furthermore, PIDD deficiency leads to a defect in TLS that is associated, both in vitro and in vivo, with cellular sensitization to UV-induced apoptosis. Thus, PIDD performs key functions upon UV irradiation, including TLS, NHEJ, NF-jB activation and cell death. Cell Death and Differentiation (2011) 18, 1036-1045 doi:10.1038/cdd.2011; published online 18 March 2011 DNA lesions can result from endogenous metabolic processes as well as from exogenous DNA damaging agents. In both cases, different cellular responses are engaged (cell cycle arrest, repair pathways or apoptosis) to maintain genetic integrity. PCNA (proliferating cell nuclear antigen) acts as a DNA sliding clamp, which helps loading of replicative DNA polymerases. PCNA is involved in several forms of DNA repair, such as NER (nucleotide excision repair), BER (base excision repair) and MMR (mismatch repair), and also in other aspects of DNA metabolism, such as replication, chromatin assembly and cohesion. 1 PCNA mediates these different functions through interactions with proteins specific to each process. 2 Many of these PCNA interacting proteins (PIPs) contain a so-called PIP-box. 1,3 Competition between different partners for the same binding site on PCNA is one of the mechanisms that coordinate the functions of PCNA in DNA replication and repair. PCNA is loaded around the DNA by the conserved chaperone-like clamp loader complex RFC (replication factor C), consisting of five subunits (RFC1 to RFC5). 4,5 The RFC subunits all contain a PIP-box and thereby physically interact with PCNA. 6 Once PCNA is linked to the DNA, the RFC complex is ejected from the clamp to allow DNA polymerase access to the clamp. 7 The protein with the strongest affinity for PCNA is the PIPbox containing p21 (Cip1/Waf1), 8 a potent cyclin-dependent kinase (CDK) inhibitor involved in cell cycle regulation, except in response to UV, where cell cycle arrest is independent of p21. 9 P21 binds to PCNA and inhibits its activity, 10,11 mainly because p21 obstructs the interaction of PCNA with DNA replication and repair factors. [12][13][14][15] In vitro studies have shown that p21 also inhibits the loading of PCNA onto DNA, 7 thereby compromising DNA repair. 9 UV is one of the major exogenous sources of DNA damage. The most common...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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PIDD orchestrates translesion DNA synthesis in response to UV irradiation

Logette

Schuepbach‐Mallepell²,

Eckert

et al. 2011

Cell Death Differ

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“…In vitro, p21 blocks PCNAdependent DNA replication by interfering with its interaction with RFC (replication factor C) (Oku et al, 1998;Waga and Stillman, 1998), DNA polymerase d (Podust et al, 1995), and FEN1 (Chen et al, 1996). P21 also interferes with PCNA interaction with DNA repair factors required for NER (Gary et al, 1997).…”

Section: Introductionmentioning

confidence: 99%

P21Cip1/WAF1 downregulation is required for efficient PCNA ubiquitination after UV irradiation

et al. 2006

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p21Cip1/WAF1 is a known inhibitor of the short-gap filling activity of proliferating cell nuclear antigen (PCNA) during DNA repair. In agreement, p21 degradation after UV irradiation promotes PCNA-dependent repair. Recent reports have identified ubiquitination of PCNA as a relevant feature for PCNA-dependent DNA repair. Here, we show that PCNA ubiquitination in human cells is notably augmented after UV irradiation and other genotoxic treatments such as hydroxyurea, aphidicolin and methylmethane sulfonate. Intriguingly, those DNA damaging agents also promoted downregulation of p21. While ubiquitination of PCNA was not affected by deficient nucleotide excision repair (NER) and was observed in both proliferating and arrested cells, stable p21 expression caused a significant reduction in UVinduced ubiquitinated PCNA. Surprisingly, the negative regulation of PCNA ubiquitination by p21 does not depend on the direct interaction with PCNA but requires the cyclin dependent kinase binding domain of p21. Taken together, our data suggest that p21 downregulation plays a role in efficient PCNA ubiquitination after UV irradiation.

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“…Completion of a functioning holoenzyme requires addition of polδ to the RFC · PCNA · DNA complex. RFC and polδ bind the same face of PCNA (22,23); the evidence on the timing of RFC departure or whether it remains as a member of a RFC · PCNA · DNA · polδ complex is variable (24)(25)(26).…”

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confidence: 99%

Stepwise loading of yeast clamp revealed by ensemble and single-molecule studies

Kumar

Nashine

Mishra

et al. 2010

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

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In ensemble and single-molecule experiments using the yeast proliferating cell nuclear antigen (PCNA, clamp) and replication factor C (RFC, clamp loader), we have examined the assembly of the RFC · PCNA · DNA complex and its progression to holoenzyme upon addition of polymerase δ (polδ). We obtained data that indicate (i) PCNA loading on DNA proceeds through multiple conformational intermediates and is successful after several failed attempts; (ii) RFC does not act catalytically on a primed 45-mer templated fork; (iii) the RFC · PCNA · DNA complex formed in the presence of ATP is derived from at least two kinetically distinguishable species; (iv) these species disassemble through either unloading of RFC · PCNA from DNA or dissociation of PCNA into its component subunits; and (v) in the presence of polδ only one species converts to the RFC · PCNA · DNA · polδ holoenzyme. These findings redefine and deepen our understanding of the clamp-loading process and reveal that it is surprisingly one of trial and error to arrive at a heuristic solution.DNA replication | replication factor C | single-molecule FRET D NA replication is carried out by effective coordination of the activities of many proteins at the replication fork. Given the complex nature of DNA synthesis, a structural and functional conservation of the accessory proteins apparently has evolved across diverse organisms (1-3). However, eukaryotic DNA replication has diverged and become more complicated than its prokaryotic counterpart due in part to the multisubunit composition of several proteins. In yeast, lagging strand DNA synthesis requires the coordinated actions of polymerase δ (polδ), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) (4). Eukaryotic PCNA, a ring-shaped trimer of identical subunits, plays a pivotal role in both DNA replication and repair by tethering the DNA polymerase to significantly enhance the processivity of DNA synthesis (5-10). The productive assembly of a polymerase-PCNA complex bound to DNA requires loading of the PCNA clamp by RFC (11). RFC, an ATP-driven motor, is composed of a large subunit (RFC-A, 95 kDa) and four small subunits (RFC-B-RFC-E, 36-40 kDa) and forms a RFC · PCNA · ATP complex that binds specifically to primed DNA (12).The clamp-loading process occurs via multiple steps (13-16). X-ray crystallographic and electron microscopic studies of the clamp loader complexed with the clamp revealed that PCNA was either in a closed or slightly open conformation that perhaps represents the structure of an intermediate step in PCNA loading (13,17). PCNA, in solution, has a closed conformation and must be opened for loading on DNA and further reclosed to be retained on the DNA possibly through a specific single interface (18).The structure of the yeast RFC · PCNA complex (13) provided the coordinates for introducing a fluorescent donor-acceptor pair within the PCNA to probe the FRET efficiencies distance information on the conformation of the PCNA subunit interface (19). In the presence of ATP, the PCNA...

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Functional sites of human PCNA which interact with p21 (Cip1/Waf1), DNA polymerase δ and replication factor C

Cited by 71 publications

References 28 publications

PIDD orchestrates translesion DNA synthesis in response to UV irradiation

PIDD orchestrates translesion DNA synthesis in response to UV irradiation

P21Cip1/WAF1 downregulation is required for efficient PCNA ubiquitination after UV irradiation

Stepwise loading of yeast clamp revealed by ensemble and single-molecule studies

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