Crystal structure of the catalytic core of Rad2: insights into the mechanism of substrate binding

Mietus, M.; Nowak, Elżbieta; Jaciuk, Marcin; Kustosz, P.; Studnicka, Justyna; Nowotny, Marcin

doi:10.1093/nar/gku729

Cited by 24 publications

(34 citation statements)

References 56 publications

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“…Multiple studies have demonstrated that overexpression of exonuclease 1 ( EXO1 ) rescues the DNA damage sensitivity of rad27Δ mutants [ 56 – 59 ]. Exo1 and Rad27 are both Rad2 family nucleases and crystal structures of their human homologs, FEN1 and EXO1, reveal highly conserved mechanisms of substrate binding and cleavage [ 60 – 62 ]. Thus, we hypothesized that Exo1, like Rad27, may cleave flaps before RPA can bind to them.…”

Section: Resultsmentioning

confidence: 99%

Genetic Interactions Implicating Postreplicative Repair in Okazaki Fragment Processing

et al. 2015

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Ubiquitination of the replication clamp proliferating cell nuclear antigen (PCNA) at the conserved residue lysine (K)164 triggers postreplicative repair (PRR) to fill single-stranded gaps that result from stalled DNA polymerases. However, it has remained elusive as to whether cells engage PRR in response to replication defects that do not directly impair DNA synthesis. To experimentally address this question, we performed synthetic genetic array (SGA) analysis with a ubiquitination-deficient K164 to arginine (K164R) mutant of PCNA against a library of S. cerevisiae temperature-sensitive alleles. The SGA signature of the K164R allele showed a striking correlation with profiles of mutants deficient in various aspects of lagging strand replication, including rad27Δ and elg1Δ. Rad27 is the primary flap endonuclease that processes 5’ flaps generated during lagging strand replication, whereas Elg1 has been implicated in unloading PCNA from chromatin. We observed chronic ubiquitination of PCNA at K164 in both rad27Δ and elg1Δ mutants. Notably, only rad27Δ cells exhibited a decline in cell viability upon elimination of PRR pathways, whereas elg1Δ mutants were not affected. We further provide evidence that K164 ubiquitination suppresses replication stress resulting from defective flap processing during Okazaki fragment maturation. Accordingly, ablation of PCNA ubiquitination increased S phase checkpoint activation, indicated by hyperphosphorylation of the Rad53 kinase. Furthermore, we demonstrate that alternative flap processing by overexpression of catalytically active exonuclease 1 eliminates PCNA ubiquitination. This suggests a model in which unprocessed flaps may directly participate in PRR signaling. Our findings demonstrate that PCNA ubiquitination at K164 in response to replication stress is not limited to DNA synthesis defects but extends to DNA processing during lagging strand replication.

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Section: Resultsmentioning

confidence: 99%

Genetic Interactions Implicating Postreplicative Repair in Okazaki Fragment Processing

et al. 2015

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“…SAXS data for the RPA DNA binding core bound to 30-nts of ssDNA was used to generate the model for RPA bound to the undamaged strand in the NER bubble 97 . While not incorporated in Figure 8 for clarity, further modeling can incorporate the structure of XPF-ERCC1 in complex with the XPA ERCC1-binding region and the structurally characterized portions of TFIIH and XPG 56-67,98 . XPA interactions with XPC and DDB1-XPE complexes are also relevant to modeling the early stages of assembling the NER incision complex.…”

Section: Xpa Interaction With Other Proteinsmentioning

confidence: 99%

XPA: A key scaffold for human nucleotide excision repair

et al. 2016

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“…The main section of the protein centers around a central seven‐strand mixed β sheet that is flanked on both sides by α helices. The connectivity of helices and sheet is generally very similar to that observed in the FEN1 family members . However, CtGEN1 lacks the helical arch that selects the single‐stranded flap of the DNA substrate in FEN1.…”

Section: The Structure Of Ctgen1mentioning

confidence: 60%

Holliday junction‐resolving enzymes—structures and mechanisms

Lilley

2017

FEBS Letters

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Holliday junction-resolving enzymes are nucleases that are highly specific for the structure of the junction, to which they bind in dimeric form. Two symmetrically disposed cleavages are made. These are not simultaneous, but the second cleavage is accelerated relative to the first, so ensuring that bilateral cleavage occurs during the lifetime of the DNA-protein complex. In eukaryotic cells there are two known junction-resolving activities. GEN1 is similar to enzymes from lower organisms. A crystallographic structure of a fungal GEN1 bound to the product of resolution has been determined. These complexes are dimerized within the crystal lattice such that the strands of the products may be simply reconnected to form a junction. These structures suggest a trajectory for the resolution process.

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Crystal structure of the catalytic core of Rad2: insights into the mechanism of substrate binding

Cited by 24 publications

References 56 publications

Genetic Interactions Implicating Postreplicative Repair in Okazaki Fragment Processing

Genetic Interactions Implicating Postreplicative Repair in Okazaki Fragment Processing

XPA: A key scaffold for human nucleotide excision repair

Holliday junction‐resolving enzymes—structures and mechanisms

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