Mutations in Yeast Proliferating Cell Nuclear Antigen Define Distinct Sites for Interaction with DNA Polymerase δ and DNA Polymerase ε

Eissenberg, Joel C.; Ayyagari, Rao; Gomes, Xavier V.; Burgers, Peter M.

doi:10.1128/mcb.17.11.6367

Cited by 156 publications

(187 citation statements)

References 57 publications

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“…Three different mutants were used: the interdomain connector loop (IDCL) mutant pcna-79 (I126A,L128A), which is particularly deficient in stimulation of Pol δ activity (12), the C-terminal mutant pcna-90 (P252A,K253A), which is particularly deficient in stimulation of FEN1 activity (12,13), and the double mutant, which we designate pcna-7990 (I126A,L128A, P252A,K253A), which we expect to be deficient in stimulation of both Pol δ and FEN1 activities. We denote, for example, a PCNA trimer containing two WT monomers and one monomer of pcna-7990 as WT 2 :7990 1 .…”

Section: Resultsmentioning

confidence: 99%

Sequential switching of binding partners on PCNA during in vitro Okazaki fragment maturation

Dovrat

Stodola

Burgers

et al. 2014

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

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The homotrimeric sliding clamp proliferating cell nuclear antigen (PCNA) mediates Okazaki fragment maturation through tight coordination of the activities of DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1) and DNA ligase I (Lig1). Little is known regarding the mechanism of partner switching on PCNA and the involvement of PCNA's three binding sites in coordinating such processes. To shed new light on PCNA-mediated Okazaki fragment maturation, we developed a novel approach for the generation of PCNA heterotrimers containing one or two mutant monomers that are unable to bind and stimulate partners. These heterotrimers maintain the native oligomeric structure of PCNA and exhibit high stability under various conditions. Unexpectedly, we found that PCNA heterotrimers containing only one functional binding site enable Okazaki fragment maturation by efficiently coordinating the activities of Pol δ, FEN1, and Lig1. The efficiency of switching between partners on PCNA was not significantly impaired by limiting the number of available binding sites on the PCNA ring. Our results provide the first direct evidence, to our knowledge, that simultaneous binding of multiple partners to PCNA is unnecessary, and if it occurs, does not provide significant functional advantages for PCNA-mediated Okazaki fragment maturation in vitro. In contrast to the "toolbelt" model, which was demonstrated for bacterial and archaeal sliding clamps, our results suggest a mechanism of sequential switching of partners on the eukaryotic PCNA trimer during DNA replication and repair.

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Section: Resultsmentioning

confidence: 99%

Sequential switching of binding partners on PCNA during in vitro Okazaki fragment maturation

Dovrat

Stodola

Burgers

et al. 2014

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

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“…PCNA is a highly conserved eukaryotic homotrimeric protein that assembles around DNA to form a sliding clamp and acts as a processivity factor for the replicative polymerase (reviewed in [16]). PCNA interacts with a large variety of proteins involved in DNA replication, lesion bypass, DNA repair, and cell cycle control, such as DNA polymerases δ and ε [17], DNA polymerase η [18], LIGI [19], MSH3 and MSH6 [20], FEN1 [21], GADD45 [22], and p21 [23]. An eight amino acid conserved motif (Q-x-x-[I/L/M]-x-x-F-[F/ Y]) in these proteins modulates their binding to PCNA [24].…”

Section: Introductionmentioning

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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“…Bluescript ssDNA primed with a single RNA-DNA primer was used in the Okazaki fragment maturation assays (28). mp18 ssDNA primed with a single primer was used in the processivity assays (29). All oligonucleotide substrates were prepared as described (27).…”

mentioning

confidence: 99%

“…The 20-l standard assay contained 25 mM Hepes 7.6, 50 mM NaCl, 0.1 mg͞ml BSA, 8 mM MgAc 2 , 1 mM ATP, 50 fmol of DECAprimed-SKII ssDNA, 2 pmol of RPA, 1 pmol of 32 P-labeled PCNA (29), 600 fmol of RFC, 4 pmol of Rad6͞Rad18, 1 pmol of Uba1, and 20 pmol of His 6 -ubiquitin at 30°C for 30 min. Samples were run on a 11% SDS͞ polyacrylamide gel, followed by STORM PhosphorImager (Molecular Dynamics) analysis.…”

mentioning

confidence: 99%

Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases η and REV1

Garg

Burgers

2005

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

223

254

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In response to DNA damage, the Rad6͞Rad18 ubiquitin-conjugating complex monoubiquitinates the replication clamp proliferating cell nuclear antigen (PCNA) at Lys-164. Although ubiquitination of PCNA is recognized as an essential step in initiating postreplication repair, the mechanistic relevance of this modification has remained elusive. Here, we describe a robust in vitro system that ubiquitinates yeast PCNA specifically on Lys-164. Significantly, only those PCNA clamps that are appropriately loaded around effector DNA by its loader, replication factor C, are ubiquitinated. This observation suggests that, in vitro, only PCNA present at stalled replication forks is ubiquitinated. Ubiquitinated PCNA displays the same replicative functions as unmodified PCNA. These functions include loading onto DNA by replication factor C, as well as Okazaki fragment synthesis and maturation by the PCNA-coordinated actions of DNA polymerase ␦, the flap endonuclease FEN1, and DNA ligase I. However, whereas the activity of DNA polymerase remains unaffected by ubiquitination of PCNA, ubiquitinated PCNA specifically activates two key enzymes in translesion synthesis: DNA polymerase , the yeast Xeroderma pigmentosum ortholog, and Rev1, a deoxycytidyl transferase that functions in organizing the mutagenic DNA replication machinery. We propose that ubiquitination of PCNA increases its functionality as a sliding clamp to promote mutagenic DNA replication.DNA replication ͉ postreplication repair ͉ translesion synthesis ͉ ubiquitination ͉ yeast M odification of the replication clamp proliferating cell nuclear antigen (PCNA) has recently emerged as an important regulatory switch during DNA replication and DNA damage response. The critical site of modification on PCNA is Lys-164, and either sumoylation or ubiquitination at this residue can occur (1, 2). Although sumoylation at Lys-164 is proposed to be associated with normal replicative functions, monoubiquitination of this residue by the Rad6͞Rad18 ubiquitin E2͞E3 complex channels DNA damage into the postreplication DNA repair (PRR) pathway (1,3,4). The PRR pathway is comprised of two error-prone branches involving translesion synthesis (TLS) by error-prone DNA polymerases, and an error-free damage avoidance branch that proceeds by fork regression and template switching (5, 6). The latter branch depends on further modification of monoubiquitinated PCNA involving Rad5 and Mms2͞Ubc13 E2͞E3 catalyzed formation of polyubiquitin chains via an unusual Lys-63 ubiquitin linkage (1).Replicative DNA polymerases of the B-family possess a very restrictive active site, which promotes high-fidelity DNA replication but inhibits bypass synthesis of many DNA lesions (5,7,8). On the other hand, Y-family DNA polymerases are particularly adapted to the bypass of DNA lesions because of a more open active site. For instance, DNA polymerase (Pol ), the ortholog of the human Xeroderma pigmentosum protein, can accommodate a cis-syn pyrimidine dimer in its active site allowing facile damage bypass (9). TLS in yeast ...

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Mutations in Yeast Proliferating Cell Nuclear Antigen Define Distinct Sites for Interaction with DNA Polymerase δ and DNA Polymerase ε

Cited by 156 publications

References 57 publications

Sequential switching of binding partners on PCNA during in vitro Okazaki fragment maturation

Sequential switching of binding partners on PCNA during in vitro Okazaki fragment maturation

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

Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases η and REV1

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