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

Figure 4.

Figure 4

Nuclease-Deficient Mutants of MRN Lack DNA Melting Activity

(A) Schematic of single-molecule FRET (smFRET) assay for probing DNA melting activity of MRN. Cy3 and Cy5 dyes are located on the opposite strands of duplex DNA, with a 5-bp distance between them.

(B) Representative smFRET trajectory of duplex DNA alone showing persistent high FRET. Top panel: donor (green) and acceptor (red) intensities; bottom panel: corresponding FRET efficiency (EFRET).

(C) Representative smFRET trajectory of duplex DNA in the presence of MRN and ATP showing transitions from higher to lower FRET state.

(D) Normalized frequency of smFRET distributions of duplex DNA alone, DNA in the presence of MRN WT + ATP, and M(H129LD130V)RN + ATP, respectively. More than 200 trajectories from at least 2 independent experiments were analyzed to generate the distributions.

(E) Quantification of population fraction (percentage) of trajectories displaying low FRET states for DNA substrate in the presence of MRN WT or M(H129LD130V)RN corresponding to the histograms shown in (D). Error bars indicate means ± SEMs from 2 independent experiments from >200 trajectories.

(F) Transcription assay with MRN and RNAPII in the presence of mirin, PFM39, and PFM01. PFM01 but not PFM39 abrogates transcription, while mirin has an intermediate effect. Extra irrelevant lanes have been removed from the gel. Bar plots show the quantification of discrete band intensities of each lane (a.u.) of the autoradiograph. Error bars indicate means ± SEMs from 2 independent experiments.

(G) Transcription assays using blunt linear DNA with MRE11(M), MRE11-RAD50 (MR), or MRE11-RAD50-NBS1(MRN) and RNAPII. Extra lane has been removed from the right end of the gel. RAD50 and NBS1 improve the efficiency of transcription mediated by MRE11 and RNAPII from DNA ends. Bar plot shows the quantification of discrete band intensities of each lane (a.u.) of the autoradiograph. Error bars indicate means ± SEMs from 3 independent experiments (*p < 0.05, **p < 0.01, ****p < 0.0001).

(H) Normalized frequency of smFRET distributions of duplex DNA alone and DNA in the presence of MR + ATP and MRN WT + ATP, respectively. More than 200 trajectories from 2 independent experiments were analyzed to generate the distributions. The data for duplex DNA and WT MRN are the same as in (D) and are replotted here for reference.