Dataset Information

The XPF-ERCC1 Complex Is Essential for Genome Stability and Is Involved in the Mechanism of Gene Targeting in Physcomitrella patens.

ABSTRACT: The XPF-ERCC1 complex, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair, and homologous recombination. XPF-ERCC1 incises double-stranded DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here, we have examined the role of the XPF-ERCC1 complex in the model bryophyte Physcomitrella patens which exhibits uniquely high gene targeting frequencies. We undertook targeted knockout of the Physcomitrella ERCC1 and XPF genes. Mutant analysis shows that the endonuclease complex is essential for resistance to UV-B and to the alkylating agent MMS, and contributes to the maintenance of genome integrity but is also involved in gene targeting in this model plant. Using different constructs we determine whether the function of the XPF-ERCC1 endonuclease complex in gene targeting was removal of 3' non-homologous termini, similar to SSA, or processing of looped-out heteroduplex intermediates. Interestingly, our data suggest a role of the endonuclease in both pathways and have implications for the mechanism of targeted gene replacement in plants and its specificities compared to yeast and mammalian cells.

SUBMITTER: Guyon-Debast A

PROVIDER: S-EPMC6521618 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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The XPF-ERCC1 Complex Is Essential for Genome Stability and Is Involved in the Mechanism of Gene Targeting in <i>Physcomitrella patens</i>.

Guyon-Debast Anouchka A Rossetti Patricia P Charlot Florence F Epert Aline A Neuhaus Jean-Marc JM Schaefer Didier G DG Nogué Fabien F

Frontiers in plant science 20190509

The XPF-ERCC1 complex, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair, and homologous recombination. XPF-ERCC1 incises double-stranded DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here, we have examined the role of the XPF-ERCC1 complex i ...[more]

PMID: 31143199

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Project description:The ERCC1-XPF structure-specific endonuclease is necessary for correct processing of homologous recombination intermediates requiring the removal of end-blocking nonhomologies. We previously showed that targeting the endogenous CHO APRT locus with plasmids designed to generate such intermediates revealed defective recombination phenotypes in ERCC1 deficient cells, including suppression of targeted insertion and vector correction recombinants and the generation of a novel class of aberrant recombinants through a deletogenic mechanism. In the present study, we examined some of the mechanistic features of ERCC1-XPF in processing recombination intermediates by varying gene targeting parameters. These included altering the distance between the double-strand break (DSB) in the targeting vector and the inactivating mutation in the APRT target gene, and changing the position of the target gene mutation relative to the DSB to result in target mutations that were either upstream or downstream from the DSB. Increasing the distance from the DSB in the targeting vector to the chromosomal target gene mutation resulted in an ERCC1 dependent decrease in the efficiency of gene targeting from intermediates presenting lengthy end-blocking nonhomologies. This decrease was accompanied by a shift in the distribution of recombinant classes away from target gene conversions to targeted insertions in both wild-type and ERCC1 deficient cells, and a dramatic increase in the proportion of aberrant recombinants in ERCC1 deficient cells. Changing the position of the target gene mutation relative to the DSB in the plasmid also altered the distribution of targeted insertion subclasses recovered in wild-type cells, consistent with two-ended strand invasion followed by resolution into crossover-type products and vector integration. Our results confirm expectations from studies of Rad10-Rad1 in budding yeast that ERCC1-XPF activity affects conversion tract length, and provide evidence for the mechanism of generation of the novel, aberrant recombinant class first described in our previous study.

Dataset Information

The XPF-ERCC1 Complex Is Essential for Genome Stability and Is Involved in the Mechanism of Gene Targeting in Physcomitrella patens.

Publications

The XPF-ERCC1 Complex Is Essential for Genome Stability and Is Involved in the Mechanism of Gene Targeting in <i>Physcomitrella patens</i>.

Similar Datasets

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