Mechanism of Holliday junction resolution by the human GEN1 protein

Rass, Ulrich; Compton, Sarah A.; Matos, Joao; Singleton, Martin R.; Ip, Stephen C.Y.; Blanco, Miguel; Griffith, Jack D.; West, Stephen C.

doi:10.1101/gad.585310

Cited by 135 publications

(188 citation statements)

References 77 publications

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“…The evidence for diagonal digestion of cruciform DNA suggested that the resolution activity for Hollidayjunction structures in mammalian cells 28 could participate in this reaction. MUS81 nuclease, GEN1 and a complex of SLX4-SLX1 have been proposed as candidates for such a resolvase in mammalian cells [29][30][31][32][33][34][35][36][37] . To identify the cruciform resolvase working in this system, we performed siRNA screening coupled with monitoring the level of diagonal cleavage of the …”

Section: Resultsmentioning

confidence: 99%

Two sequential cleavage reactions on cruciform DNA structures cause palindrome-mediated chromosomal translocations

et al. 2013

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Gross chromosomal rearrangements (GCRs), such as translocations, deletions or inversions, are often generated by illegitimate repair between two DNA breakages at regions with nucleotide sequences that might potentially adopt a non-B DNA conformation. We previously established a plasmid-based model system that recapitulates palindrome-mediated recurrent chromosomal translocations in humans, and demonstrated that cruciform DNA conformation is required for the translocation-like rearrangements. Here we show that two sequential reactions that cleave the cruciform structures give rise to the translocation: GEN1-mediated resolution that cleaves diagonally at the four-way junction of the cruciform and Artemismediated opening of the subsequently formed hairpin ends. Indeed, translocation products in human sperm reveal the remnants of this two-step mechanism. These two intrinsic pathways that normally fulfil vital functions independently, Holliday-junction resolution in homologous recombination and coding joint formation in rearrangement of antigen-receptor genes, act upon the unusual DNA conformation in concert and lead to a subset of recurrent GCRs in humans.

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Section: Resultsmentioning

confidence: 99%

Two sequential cleavage reactions on cruciform DNA structures cause palindrome-mediated chromosomal translocations

et al. 2013

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“…These include enzymes that catalyze structure-specific reactions of an apparently disparate group of nucleic acid substrates including DNA bubbles (xeroderma pigmentosum complementation group G (XPG)) (35), four-way junctions (GEN-1 (xeroderma pigmentosum complementation group G-like gap endonuclease, a putative human Holliday junction resolvase)) (36), as well as flapped, nicked, and 3Ј-overhang DNAs (FEN and EXO1) (2,20,37,38). Because all of these enzymes carry out the same chemical transformation, like T5FEN, they too will require two ions to effect phosphate diester hydrolysis.…”

Section: Discussionmentioning

confidence: 99%

Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions

Tomlinson

Syson

Sengerová

et al. 2011

Journal of Biological Chemistry

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Flap endonucleases (FENs) are divalent metal ion-dependent phosphodiesterases. Metallonucleases are often assigned a "two-metal ion mechanism" where both metals contact the scissile phosphate diester. The spacing of the two metal ions observed in T5FEN structures appears to preclude this mechanism. However, the overall reaction catalyzed by wild type (WT) T5FEN requires three Mg 2؉ ions, implying that a third ion is needed during catalysis, and so a two-metal ion mechanism remains possible. , essential in all life forms, are members of the 5Ј-nuclease superfamily of structure-specific phosphate diesterases that carry out essential divalent metal ion-dependent nucleic acid hydrolysis (1). FENs act upon 5Ј-bifurcated structures known as flaps formed during lagging strand DNA replication and long patch base excision repair. Hydrolysis predominantly occurs one nucleotide into the 5Ј-duplex adjoining the site of bifurcation. An unusual feature of these enzymes is their high density of active site carboxylates that coordinate essential divalent metal ion cofactors. Bacterial and bacteriophage FENs possess eight active site acidic residues, seven of which are conserved in FENs from higher organisms and also in other members of the 5Ј-nuclease superfamily (1-4). In contrast, a typical metallonuclease active site consists of three or four carboxylates (5). However, a subclass of FEN paralogues, typified by Escherichia coli ExoIX, exists in eubacteria and appear to lack three of the metal-binding carboxylates (6).Although the most common mechanism suggested for metal-dependent phosphoryl transferases involves two metal ions in contact with the scissile phosphate diester, this is not universally accepted (5, 7-10). Debate about the validity or otherwise of two-metal ion catalysis has focused on several enzymes including flap endonucleases (11-16). Crystallographic studies of substrate-free FENs have demonstrated two active site-bound metal ions (14,(17)(18)(19). Metal ion 1 (M1) occupies a similar position in all structures. However, there is some variation in the position of the site coordinating a second metal ion (M2), often observed in FEN crystal structures. The distance between the M1 and M2 sites varies, and the separations observed are generally much greater than the 4 Å required for both metals to contact the same phosphate diester. For example, in bacteriophage T5FEN, 5 the M1-M2 separation of two Mn 2ϩ ions is 8 Å (Fig. 1A) (17). However, in a substrate-free structure of hFEN1, two magnesium ions are bound with a separation of 3.4 Å in one of three proteins bound to proliferating cell nuclear antigen (Fig. 1B) , 3-(N-morpholino)propane-sulfonic acid. 5 In early literature, T5FEN was referred to as T5 5Ј-3Ј-exonuclease and T5 5Ј-nuclease. In early literature, T4FEN was referred to as referred to also as T4 RNase H.

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“…It will be interesting to further dissect how GEN-1 affects DNA damage signalling. The biochemical activity of the mammalian GEN1 resolvase has been extensively characterized (Rass et al 2010 ) . At present no checkpoint function of mammalian GEN1 or yeast YEN1 was reported, and its Fig.…”

Section: Upstream Dna Damage Checkpoint Signalling In the C Elegans mentioning

confidence: 99%

Germ Cell Apoptosis and DNA Damage Responses

Bailly

Gartner

2012

Advances in Experimental Medicine and Biology

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In the past 12 years, since the fi rst description of C. elegans germ cell apoptosis, this area of research rapidly expanded. It became evident that multiple genetic pathways lead to the apoptotic demise of germ cells. We are only beginning to understand how these pathways that all require the CED-9/Bcl-2, Apaf-1/CED-4 and CED-3 caspase core apoptosis components are regulated. Physiological apoptosis, which likely accounts for the elimination of more than 50% of all germ cells, even in unperturbed conditions, is likely to be required to maintain tissue homeostasis. The best-studied pathways lead to DNA damage-induced germ cell apoptosis in response to a variety of genotoxic stimuli. This apoptosis appears to be regulated similar to DNA damage-induced apoptosis in the mouse germ line and converges on p53 family transcription factors. DNA damage response pathways not only lead to apoptosis induction, but also directly affect DNA repair, and a transient cell cycle arrest of mitotic germ cells. Finally, distinct pathways activate germ cell apoptosis in response to defects in meiotic recombination and meiotic chromosome pairing.

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Mechanism of Holliday junction resolution by the human GEN1 protein

Cited by 135 publications

References 77 publications

Two sequential cleavage reactions on cruciform DNA structures cause palindrome-mediated chromosomal translocations

Two sequential cleavage reactions on cruciform DNA structures cause palindrome-mediated chromosomal translocations

Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions

Germ Cell Apoptosis and DNA Damage Responses

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