Genetic Instability Induced by Overexpression of DNA Ligase I in Budding Yeast

Subramanian, Jaichandar; Vijayakumar, Sangeetha; Tomkinson, Alan E.; Arnheim, Norman

doi:10.1534/genetics.105.042861

Cited by 38 publications

(58 citation statements)

References 82 publications

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“…In vivo and in vitro data suggest that Pol δ may bind PCNA together with FEN1, whereas Lig1 binding is exclusive (9,20,21). In contrast, another in vivo study in mammalian cells has demonstrated that PCNA is stably associated with DNA, whereas its partners are transiently associated (22).…”

Section: Discussionmentioning

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: Discussionmentioning

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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“…It is unclear how PCNA effects hand-off of DNA from polymerase and Fen1 to DNA ligase, thereby coordinating Okazaki fragments maturation. Biochemical studies have put forward a molecular tool-belt model, although this remains controversial [3][4][5]. However, recent biochemical studies [2] support a model where a single PCNA ring assembles an Okazaki fragment maturation complex composed of PolB1, Fen1 and Lig1.…”

Section: Introductionmentioning

confidence: 99%

The architecture of an Okazaki fragment-processing holoenzyme from the archaeon Sulfolobus solfataricus

Cannone

Beattie

et al. 2015

Biochemical Journal

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DNA replication on the lagging strand occurs via the synthesis and maturation of Okazaki fragments. In archaea and eukaryotes, the enzymatic activities required for this process are supplied by a replicative DNA polymerase, Flap endonuclease 1 (Fen1) and DNA ligase 1 (Lig1). These factors interact with the sliding clamp PCNA (proliferating cell nuclear antigen) providing a potential means of co-ordinating their sequential actions within a higher order assembly. In hyperthermophilic archaea of the Sulfolobus genus, PCNA is a defined heterotrimeric assembly and each subunit interacts preferentially with specific client proteins. We have exploited this inherent asymmetry to assemble a PCNApolymerase-Fen1-ligase complex on DNA and have visualized it by electron microscopy. Our studies reveal the structural basis of co-occupancy of a single PCNA ring by the three distinct client proteins.

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“…This suggests that secondary structures can form in vivo and impair replication/repair enzymes. Studies in model organisms, such as S. cerevisiae, have shown that mutations in DNA replication enzymes, particularly Rad27 (yeast FEN-1 homolog), lead to TNR instability (6,(22)(23)(24)(25)(26)(27)(28). Rad27/FEN-1 is a multifunctional nuclease that plays a critical role in maintaining genome stability through RNA primer removal and long patch base excision repair.…”

mentioning

confidence: 99%

Concerted Action of Exonuclease and Gap-dependent Endonuclease Activities of FEN-1 Contributes to the Resolution of Triplet Repeat Sequences (CTG) - and (GAA) -derived Secondary Structures Formed during Maturation of Okazaki Fragments

Singh

Zheng

Chavez

et al. 2007

Journal of Biological Chemistry

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There is much evidence to indicate that FEN-1 efficiently cleaves single-stranded DNA flaps but is unable to process double-stranded flaps or flaps adopting secondary structures. However, the absence of Fen1 in yeast results in a significant increase in trinucleotide repeat (TNR) expansion. There are then two possibilities. One is that TNRs do not always form stable secondary structures or that FEN-1 has an alternative approach to resolve the secondary structures. In the present study, we test the hypothesis that concerted action of exonuclease and gap-dependent endonuclease activities of FEN-1 play a role in the resolution of secondary structures formed by (CTG) n and (GAA) n repeats. Employing a yeast FEN-1 mutant, E176A, which is deficient in exonuclease (EXO) and gap endonuclease (GEN) activities but retains almost all of its flap endonuclease (FEN) activity, we show severe defects in the cleavage of various TNR intermediate substrates. Precise knock-in of this point mutation causes an increase in both the expansion and fragility of a (CTG) n tract in vivo. Taken together, our biochemical and genetic analyses suggest that although FEN activity is important for singlestranded flap processing, EXO and GEN activities may contribute to the resolution of structured flaps. A model is presented to explain how the concerted action of EXO and GEN activities may contribute to resolving structured flaps, thereby preventing their expansion in the genome.

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Genetic Instability Induced by Overexpression of DNA Ligase I in Budding Yeast

Cited by 38 publications

References 82 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

The architecture of an Okazaki fragment-processing holoenzyme from the archaeon Sulfolobus solfataricus

Concerted Action of Exonuclease and Gap-dependent Endonuclease Activities of FEN-1 Contributes to the Resolution of Triplet Repeat Sequences (CTG) - and (GAA) -derived Secondary Structures Formed during Maturation of Okazaki Fragments

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