Identification of Regions within the Four Small Subunits of Human Replication Factor C Required for Complex Formation and DNA Replication

Uhlmann, Frank; Gibbs, E. Michael; Cai, Jinsong; O’Donnell, Michael; Hurwitz, Jerard

doi:10.1074/jbc.272.15.10065

Cited by 59 publications

(36 citation statements)

References 32 publications

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“…In addition, the N-terminal mutant of ELG1 would probably also be defective in forming the functional RFC complex. Previous reports showed that the C-terminal regions of the yeast Elg1p protein and the large subunit of the human RFC complex are essential for complex formation with the RFC2 to -5 core subunits (31)(32)(33). These results indicate that intact alternative RFC complex formation is not necessary for ELG1 to down-regulate PCNA monoubiquitination.…”

Section: Discussionmentioning

confidence: 80%

Human ELG1 Regulates the Level of Ubiquitinated Proliferating Cell Nuclear Antigen (PCNA) through Its Interactions with PCNA and USP1

Lee

Yang

Cohn

et al. 2010

Journal of Biological Chemistry

116

157

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The level of monoubiquitinated proliferating cell nuclear antigen (PCNA) is closely linked with DNA damage bypass to protect cells from a high level of mutagenesis. However, it remains unclear how the level of monoubiquitinated PCNA is regulated. Here, we demonstrate that human ELG1 protein, which comprises an alternative replication factor C (RFC) complex and plays an important role in preserving genomic stability, as an interacting partner for the USP1 (ubiquitin-specific protease 1)-UAF1 (USP1-associated factor 1) complex, a deubiquitinating enzyme complex for PCNA and FANCD2. ELG1 protein interacts with PCNAs that are localized at stalled replication forks. ELG1 knockdown specifically resulted in an increase in the level of PCNA monoubiquitination without affecting the level of FANCD2 ubiquitination. It is a novel function of ELG1 distinct from its role as an alternative RFC complex because knockdowns of any other RFC subunits or other alternative RFCs did not affect PCNA monoubiquitination. Lastly, we identified a highly conserved N-terminal domain in ELG1 that was responsible for the USP1-UAF1 interaction as well as the activity to down-regulate PCNA monoubiquitination. Taken together, ELG1 specifically directs USP1-UAF1 complex for PCNA deubiquitination. Proliferating cell nuclear antigen (PCNA)4 functions in DNA replication, repair, and recombination as a sliding clamp for various DNA replication polymerases and a scaffold for many DNA repair and recombination enzymes (1). Various posttranslational modifications of PCNA, including ubiquitination, sumoylation, or phosphorylation, determine its specific functions (2, 3). PCNA ubiquitination directs different branches of the postreplication repair (PRR) pathway (4). When a DNA replication fork stalls at damaged DNA lesions, the ubiquitin conjugation enzyme, RAD6, and the ubiquitin ligase, RAD18, monoubiquitinate lysine 164 of PCNA (5). Monoubiquitinated PCNA recruits translesion synthesis (TLS) polymerases, which can insert a base opposite the damaged lesion to bypass the damaged DNA. Alternatively, PCNA can be polyubiquitinated to promote DNA damage bypass by a homologous recombination mechanism (6 -8).The process and consequence of PCNA deubiquitination after DNA damage bypass are not clearly understood. Recently, USP1 (ubiquitin-specific protease 1) was identified as a deubiquitinating enzyme for PCNA after DNA damage bypass (9, 10). USP1 reduces the accumulation of monoubiquitinated PCNA during normal cell division, which prevents mutagenesis by error-prone TLS polymerases. In response to damage that stalls DNA replication, USP1 is degraded, and consequently monoubiquitinated PCNA accumulates (9). However, the observation that levels of monoubiquitinated PCNA remain high, even in the presence of persistent levels of USP1 (11,12) suggests that there might be additional mechanisms to regulate USP1 activity. The identification of UAF1 (USP1-associated factor 1) indicates that interacting proteins may regulate the activity of USP1 in vivo (13).The loading a...

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Section: Discussionmentioning

confidence: 80%

Human ELG1 Regulates the Level of Ubiquitinated Proliferating Cell Nuclear Antigen (PCNA) through Its Interactions with PCNA and USP1

Lee

Yang

Cohn

et al. 2010

Journal of Biological Chemistry

116

157

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“…The N-termini of p37 and p40, although highly similar, are not interchangeable, supporting the idea of a unique function uitously, although the expression is highest in strongly proliferating tissue [49]. Overexpression of p140 in stably transfected for every subunit [40].…”

Section: Rf-c As a Clamp Loader And Unloadermentioning

confidence: 71%

“…Homologs of RF-C have also been found in Schizosaccharothe complex [40]. Thus, the unique sequences in this region might be important for the interaction between the subunits, ex-myces pombe : rad17 is one of six members of the 'checkpoint rad' class of genes and is absolutely required for G 2 arrest plaining why all five subunits are required, despite their sequence redundancy.…”

Section: Rf-c As a Clamp Loader And Unloadermentioning

confidence: 99%

Clamping down on clamps and clamp loaders

Mossi

Hübscher

1998

European Journal of Biochemistry

124

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DNA transactions such as DNA replication and DNA repair require the concerted action of many enzymes, together with other proteins and non-protein cofactors. Among them three main accessory proteins, replication factor C (RF-C), proliferating-cell nuclear antigen (PCNA) and replication protein A (RP-A), are essential for accurate and processive DNA synthesis by DNA polymerases. RF-C is a complex consisting of five polypeptides with distinct functions. RF-C can bind to a template-primer junction and, in the presence of ATP, load the PCNA clamp onto DNA, thereby recruiting DNA polymerases to the site of DNA synthesis. RF-C not only acts as a clamp loader in DNA replication and DNA repair, but there is some evidence that it could be involved in several other processes such as transcription, S-phase checkpoint regulation, apoptosis, differentiation and telomere-length regulation.Keywords : eukaryotic DNA replication; replication factor C; proliferating-cell nuclear antigen; clamp; clamp loader ; DNA polymerase δ ; S-phase checkpoint regulation ; apoptosis; differentiation; telomere. Of clamps and clamp loadersrespective clamps onto DNA. High sequence similarity between the clamp loaders suggest that their mechanisms of action may DNA replication, one of the most remarkable and challeng-be very similar [4]. The eukaryotic RF-C clamp loader consists ing cellular events, is the result of the collaboration of a formida-of five distinct subunits (Table 1), as does the E. coli γ complex. ble amount of proteins. The mechanisms at the basis of this By loading the homotrimer proliferating-cell nuclear antigen complicated event are similar in prokaryotes and eukaryotes, de-(PCNA) clamp onto DNA, it confers high processivity to polyspite the evolutionary distance. In each of these cell types, the merases δ and ε. Despite there being virtually no similarity in replicative polymerases are chaperoned by several accessory sequence between β and PCNA, the three-dimensional structures proteins that confer speed and high processivity.are almost identical [5]. The amino acid sequence of PCNA is Responsible for DNA replication in Escherichia coli is the only approximately 70% the length of that of β, so that three DNA polymerase III holoenzyme, a ten-subunit complex (Ta-monomers of PCNA are required to form the typical doughnutble 1) [1]. The DNA polymerase (polymerase III core) consists like structure, in contrast to β where only two monomers are of A (the polymerase), ε (the proofreading exonuclease) and θ enough. The T4 gene 44/62 protein complex contains five subsubunits. The clamp loader, called γ complex, contains five subunits, four protomers of the gene 44 protein and one copy of the units, γ, δ, δ′, ψ and χ, and is required for the ATP-dependent gene 62 protein [6]. It is required for the loading of the product assembly of the β clamp onto DNA. Contrary to all eukaryotes of gene 45, the T4 bacteriophage counterpart of PCNA and E. and the T4 phage, E. coli contains an additional connector procoli β. Despite the apparent similar...

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“…Purified replication factor C (RFC), in which the N-terminal 555 amino acids of the p140 subunit were deleted [28], was provided by Dr. J. Hurwitz (Memorial Sloan-Kettering Cancer Institute). BL21(DE3) was purchased from Stratagene Corporation, as was pRIL.…”

Section: Methodsmentioning

confidence: 99%

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

Bennett

2005

DNA Repair

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Uracil residues arise in DNA by the misincorporation of dUMP in place of dTMP during DNA replication or by the deamination of cytosine in DNA. Uracil-DNA glycosylase initiates DNA base excision repair of uracil residues by catalyzing the hydrolysis of the N-glycosylic bond linking the uracil base to deoxyribose. In human cells, the nuclear form of uracil-DNA glycosylase (UNG2) contains a conserved PCNA-binding motif located at the N-terminus that has been implicated experimentally in binding PCNA. Here we use purified preparations of UNG2 and PCNA to demonstrate that UNG2 physically associates with PCNA. UNG2 co-eluted with PCNA during size exclusion chromatography and bound to a PCNA affinity column. Association of UNG2 with PCNA was abolished by the addition of 100 mM NaCl, and significantly decreased in the presence of 10 mM MgCl 2 . The functional significance of the UNG2·PCNA association was demonstrated by UNG2 activity assays. Addition of PCNA (30-810 pmol) to standard uracil-DNA glycosylase reactions containing linear [uracil-3 H]DNA stimulated UNG2 catalytic activity up to 2.6-fold. UNG2 activity was also stimulated by 7.5 mM MgCl 2 . The stimulatory effect of PCNA was increased by the addition of MgCl 2 ; however, the dependence on PCNA concentration was the same, indicating that the effects of MgCl 2 and PCNA on UNG2 activity occurred by independent mechanisms. Loading of PCNA onto the DNA substrate was required for stimulation, as the activity of UNG2 on circular DNA substrates was not affected by the addition of PCNA. Addition of replication factor C and ATP to reactions containing 90 pmol of PCNA resulted in two-fold stimulation of UNG2 activity on circular DNA.

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Identification of Regions within the Four Small Subunits of Human Replication Factor C Required for Complex Formation and DNA Replication

Cited by 59 publications

References 32 publications

Human ELG1 Regulates the Level of Ubiquitinated Proliferating Cell Nuclear Antigen (PCNA) through Its Interactions with PCNA and USP1

Human ELG1 Regulates the Level of Ubiquitinated Proliferating Cell Nuclear Antigen (PCNA) through Its Interactions with PCNA and USP1

Clamping down on clamps and clamp loaders

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

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