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. 2021 Jan 5;34(1):108565. doi: 10.1016/j.celrep.2020.108565

Figure 3.

Figure 3

Integrity of Active Site of Mre11, Irrespective of Its Nuclease Activity, Is Essential for RNAPII-Mediated Transcription

(A) Transcription assay with MRN, WT, or resection-deficient pCtIP (T847A) and RNAPII as indicated. m∗ indicates radioactive end-labeled single-stranded RNA ladder. Both WT pCtIP and its resection-deficient mutant stimulate MRN to support transcription by RNAPII to a similar extent. Bar plot shows the quantification of discrete band intensities of each lane (a.u.). Error bars indicate means ± SEMs from 2 independent experiments.

(B) Nuclease assay with MRN, pCtIP, and RNAPII. Biotinylated DNA labeled at the 3′ end was incubated with MRN, pCtIP, and RNAPII in the presence of ATP or rNTPs, and the products were resolved on 15% denaturing PAGE. In lane 10, streptavidin was added as a positive control to block DNA ends. m∗ indicates radioactively labeled DNA ladder. Exonuclease products are observed when MRN and pCtIP are both present with or without rNTPs, while endonuclease products are observed only when DNA is blocked with streptavidin. Bar plot shows quantification from each lane as a percentage of the degraded substrate. Error bars indicate means ± SEMs from 2 independent experiments.

(C) Generation of dilncRNA upon DSB induction by I-PpoI-empty vector (EV) is used as negative control—in the presence of WT or mutant MRN alleles as detected by strand-specific qRT-PCR with primers flanking DAB1 locus. MRN mutations abrogate dilncRNA generation from DSBs. Bar plot shows fold change of dilncRNA levels relative to control treated with I-PpoI. Error bars indicate means ± SEMs from 3 independent experiments (∗p < 0.05 and ∗∗∗p < 0.001). Lower panel: western blot analysis of the expression levels of MRE11 mutants. HeLa cells knocked down for endogenous MRE11 with siRNA against 3′ UTR of MRE11, were transfected with plasmids expressing hemagglutinin (HA)-tagged MRE11 (WT) or MRE11 (H129N, H63D, and H63S), along with either I-PpoI-expressing plasmid or EV as control. Expression levels of WT MRE11 and its mutants are comparable.

(D) Transcription assay with WT MRN or nuclease mutant (H129LD130V) MRN and RNAPII using blunt-ended DNA DSB. Nuclease mutant MRN does not support transcription by RNAPII from DNA ends. m∗ indicates radioactively labeled single-stranded RNA ladder. Bar plot shows the quantification of discrete band intensities of each lane (a.u.) of the autoradiograph. Error bars indicate means ± SEMs from 4 independent experiments (∗p < 0.05).