GRB2 enforces homology-directed repair initiation by MRE11.
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ABSTRACT:
SUBMITTER: Ye Z
PROVIDER: S-EPMC8336959 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%-4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, approximately 50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2'-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.
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2018-06-04 | E-MTAB-6808 | biostudies-arrayexpress
Project description:RAD52 mediates homologous recombination by annealing cDNA strands. However, the detailed mechanism of DNA annealing promoted by RAD52 has remained elusive. Here we report two crystal structures of human RAD52 single-stranded DNA (ssDNA) complexes that probably represent key reaction intermediates of RAD52-mediated DNA annealing. The first structure revealed a "wrapped" conformation of ssDNA around the homo-oligomeric RAD52 ring, in which the edges of the bases involved in base pairing are exposed to the solvent. The ssDNA conformation is close to B-form and appears capable of engaging in Watson-Crick base pairing with the cDNA strand. The second structure revealed a "trapped" conformation of ssDNA between two RAD52 rings. This conformation is stabilized by a different RAD52 DNA binding site, which promotes the accumulation of multiple RAD52 rings on ssDNA and the aggregation of ssDNA. These structures provide a structural framework for understanding the mechanism of RAD52-mediated DNA annealing.
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Project description:The promyelocytic leukemia protein (PML) is a tumor suppressor critical for formation of nuclear bodies (NBs) performing important functions in transcription, apoptosis, DNA repair and antiviral responses. Earlier studies demonstrated that simian virus 40 (SV40) initiates replication near PML NBs. Here we show that PML knockdown inhibits viral replication in vivo, thus indicating a positive role of PML early in infection. SV40 large T antigen (LT) induces DNA damage and, consequently, nuclear foci of the key homologous recombination repair protein Rad51 that colocalize with PML. PML depletion abrogates LT-induced Rad51 foci. LT may target PML NBs to gain access to DNA repair factors like Rad51 that are required for viral replication. We have used the SV40 model to gain insight to DNA repair events involving PML. Strikingly, even in normal cells devoid of viral oncoproteins, PML is found to be instrumental for foci of Rad51, Mre11 and BRCA1, as well as homology-directed repair after double-strand break (DSB) induction. Following LT expression or external DNA damage, PML associates with Rad51. PML depletion also causes a loss of RPA foci following γ-irradiation, suggesting that PML is required for processing of DSBs. Immunofluorescent detection of incorporated BrdU without prior denaturation indicates a failure to generate ssDNA foci in PML knockdown cells upon γ-irradiation. Consistent with the lack of RPA and BrdU foci, γ-irradiation fails to induce Chk1 activation, when PML is depleted. Taken together, we have discovered a novel functional connection between PML and the homologous recombination-mediated repair machinery, which might contribute to PML tumor suppressor activity.
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