Purification of PCNA as a nucleotide excision repair protein

Nichols, Anne F.; Sancar, Aziz

doi:10.1093/nar/20.10.2441

Cited by 213 publications

(111 citation statements)

References 33 publications

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“…Recent studies have shown PCNA to be involved in numerous cellular functions associated with cell proliferation and to directly bind to or interact with various proteins, as listed in Table 1 (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)24). Our previous study (24) revealed that the PCNA multiprotein complexes purified by mAb to PCNA contained components of multiprotein machinery such as the DNA replication fork (Figure 1) (12,13,21,22) and a quaternary PCNA/ p21/CDK/cyclin complex reported to play a key role in the regulation of the cell cycle (14,17).…”

Section: Discussionmentioning

confidence: 99%

Autoimmune responses to proliferating cell nuclear antigen multiprotein complexes involved in cell proliferation are strongly associated with their structure and biologic function in patients with systemic lupus erythematosus

Kogure

Takasaki

Takeuchi

et al. 2002

Arthritis & Rheumatism

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Objective. To analyze the reaction of lupus sera with proliferating cell nuclear antigen (PCNA) multiprotein complexes (PCNA complexes), which are part of the protein machinery involved in cell proliferation.Methods. PCNA complexes were purified from rabbit thymus extract by affinity chromatography using anti-PCNA monoclonal antibodies (TOB7, TO17, and TO30); monomeric and trimeric PCNA forms (AK-PCNA) were purified using anti-PCNA serum AK. The reactions to these antigens of 10 anti-PCNA-positive and 40 anti-PCNA-negative sera selected from 560 lupus patients were tested by immunoblotting, immunoprecipitation, and enzyme-linked immunosorbent assays (ELISAs).Results. With one exception (serum OK), anti-PCNA-positive sera reacted exclusively with only the 34-kd polypeptide. In contrast, 14 of 40 anti-PCNAnegative sera reacted with multiple proteins within PCNA complexes. Most anti-PCNA-positive sera probably recognize as epitopes the binding sites for other proteins on PCNA, which are likely hidden when PCNA is complexed with other proteins. As a consequence, only serum OK reacted with the PCNA complex in a series of ELISAs. Using AK-PCNA as a competitive inhibitor, it was determined that serum OK reacts with both the 58-kd polypeptide and the 34-kd PCNA within complexes. Together with the results of a longitudinal analysis, these results suggest that the immune system of patient OK likely recognized the complexed PCNA protein, after which the autoimmune response spread to other elements of the complexes.Conclusion. Intermolecular-intrastructural help, leading to the spread of autoimmune response from PCNA to other proteins associated with its biologic function, plays a crucial role in the induction of the autoimmune response seen in lupus patients.Autoantibodies against proliferating cell nuclear antigen (PCNA) were first observed in a patient with systemic lupus erythematosus (SLE) (1). Using anti-PCNA antibodies from SLE patients as a probe, our group subsequently determined that PCNA is a 34-kd intranuclear polypeptide that exists mainly as a homotrimer (2,3), and that expression of PCNA is increased in the late G1 and early S phases of the cell cycle, immediately before DNA synthesis (4). These findings suggested an involvement of PCNA in DNA replication, and, indeed, PCNA was later identified as an auxiliary protein of DNA polymerase ␦ (Pol-␦), playing an essential role in DNA replication and repair (5-9).

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Section: Discussionmentioning

confidence: 99%

Autoimmune responses to proliferating cell nuclear antigen multiprotein complexes involved in cell proliferation are strongly associated with their structure and biologic function in patients with systemic lupus erythematosus

Kogure

Takasaki

Takeuchi

et al. 2002

Arthritis & Rheumatism

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“…In addition, PCNA is required for nucleotide excision repair (Shivji et al, 1992;Nichols and Sancar, 1992) and is involved in one pathway of base excision repair (Matsumoto et al, 1994). PCNA may be recruited to these di erent roles in replication and repair by interaction with proteins speci®c to each process.…”

Section: Discussionmentioning

confidence: 99%

“…PCNA is highly conserved and has been identi®ed as an essential gene required for DNA replication in the yeasts Schizosaccharomyces pombe (Waseem et al, 1992) and Saccharomyces cerevisiae (Bauer and Burger, 1990). In addition to its role in replication, however, PCNA is also required for nucleotide excision repair in cell-free systems (Shivji et al, 1992;Nichols and Sancar, 1992), and plays a role in one pathway of base excision repair (Matsumoto et al, 1994). The way in which PCNA activity is regulated to carry out these two separate roles is as yet unclear, though recently discovered interactions of PCNA with p21 Cip1 (Xiong et al, 1992;Waga et al, 1994b;Flores et al, 1994;Warbrick et al, 1995) and Gadd45 (Smith et al, 1994;Hall et al, 1995) may provide some insight into these regulatory phenomena.…”

Section: Introductionmentioning

confidence: 99%

Homologous regions of Fen1 and p21Cip1 compete for binding to the same site on PCNA: a potential mechanism to co-ordinate DNA replication and repair

et al. 1997

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Following genomic damage, the cessation of DNA replication is co-ordinated with onset of DNA repair; this co-ordination is essential to avoid mutation and genomic instability. To investigate these phenomena, we have analysed proteins that interact with PCNA, which is required for both DNA replication and repair. One such protein is p21 Cip1 , which inhibits DNA replication through its interaction with PCNA, while allowing repair to continue. We have identi®ed an interaction between PCNA and the structure speci®c nuclease, Fen1, which is involved in DNA replication. Deletion analysis suggests that p21 Cip1 and Fen1 bind to the same region of PCNA. Within Fen1 and its homologues a small region (10 amino acids) is su cient for PCNA binding, which contains an 8 amino acid conserved PCNA-binding motif. This motif shares critical residues with the PCNA-binding region of p21 Cip1 . A PCNA binding peptide from p21 Cip1 competes with Fen1 peptides for binding to PCNA, disrupts the Fen1-PCNA complex in replicating cell extracts, and concomitantly inhibits DNA synthesis. Competition between homologous regions of Fen1 and p21 Cip1 for binding to the same site on PCNA may provide a mechanism to co-ordinate the functions of PCNA in DNA replication and repair.

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“…This interpretation is supported by the observation that PCNA-positive cells were found within the shell in most of the specimens examined, a region where mitotic figures were never seen. PCNA is also involved in the repair of DNA (Nichols and Sancar, 1992;Shivji et al, 1992) and this function may explain the presence of PCNA not only in the shell but the generally higher numbers of PCNA-positive cells in other regions. Ki-67 labeled cells were more restricted in distribution.…”

Section: Discussionmentioning

confidence: 99%

Developmental expression of Ki‐67 antigen and proliferating cell nuclear antigen in macaque placentas

Blankenship

King

1994

Developmental Dynamics

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Placental growth is largely determined by the proliferation of cytotrophoblast cells. However, the distribution of cytotrophoblast cells engaged in the cell cycle during placental development is poorly understood. Recently, antibodies have been developed that identify two proteins directly involved with DNA synthesis: Ki-67 protein and proliferating cell nuclear antigen (PCNA). Immunolocalization of Ki-67 and PCNA provides a measure of the proliferating cells in tissues. We examined, in macaque placentas, the spatiotemporal pattern of expression of these proteins during gestation. Tissues from 24 macaque placentas collected from 22-153 days of pregnancy were prepared for paraffin sections. Standard immunoperoxidase techniques were used to identify Ki-67 and PCNA. The proteins generally co-localized, although PCNA was usually represented in more cells than Ki-67. Early in gestation the cell columns contained many labeled cells. The cytotrophoblastic shell was occupied by numerous cells with PCNA positive nuclei, but few were reactive for Ki-67. By 45 days of pregnancy the immunolabeled cells in the cell columns were concentrated in the proximal regions, adjacent to the anchoring villus tips. The number of positive cells decreased by 100 days when the cell columns were diminished, leaving the anchoring villus tips buried in the shell. Labeled cells were rarely present in the shell at late pregnancy. The single layer of cytotrophoblast cells in the chorionic plate contained numerous reactive cells throughout early and mid-gestation. After approximately 100 days the cytotrophoblast layer of the chorionic plate was stratified over large areas. Soon thereafter few cells of the chorionic plate were labeled. The chorionic villi contained reactive cytotrophoblastic cells throughout gestation. Extravillous cytotrophoblast cells invading spiral arteries were sometimes labeled for PCNA but not Ki-67. We conclude that compartments of the placenta are distinguished by specific patterns of cytotrophoblast cell proliferation. Moreover, these patterns correspond to macroscopic growth parameters of the placenta. Evidence suggests that the macaque placenta slows its rate of diametrical growth at approximately 100 days of gestation. It is at about this time that the cell columns are absorbed into the trophoblastic shell and this pool ofproliferating cells is diminished. The growth in diameter of the chorionic plate matches that of the shell. In this compartment also the architecture changes at about 100 days as the cytotrophoblast layer stratifies. This stratification may result from continued proliferation of cytotrophoblast cells when the diametrical rate of growth is decreasing. Soon thereafter, proliferation decreases in this compartment also. By contrast, labeled cells were found in chorionic villi throughout gestation. Continued villous growth and branching probably accounts for most of the increases in placental thickness and weight that occurs during late gestation. 0 1994 Wiley-Liss, Inc.

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Purification of PCNA as a nucleotide excision repair protein

Cited by 213 publications

References 33 publications

Autoimmune responses to proliferating cell nuclear antigen multiprotein complexes involved in cell proliferation are strongly associated with their structure and biologic function in patients with systemic lupus erythematosus

Autoimmune responses to proliferating cell nuclear antigen multiprotein complexes involved in cell proliferation are strongly associated with their structure and biologic function in patients with systemic lupus erythematosus

Homologous regions of Fen1 and p21Cip1 compete for binding to the same site on PCNA: a potential mechanism to co-ordinate DNA replication and repair

Developmental expression of Ki‐67 antigen and proliferating cell nuclear antigen in macaque placentas

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