1. Nbs1 Converts the Human Mre11/Rad50 Nuclease Complex into an Endo/Exonuclease Machine Specific for Protein-DNA Adducts 
Rajashree A. Deshpande, Ji-Hoon Lee, Sucheta Arora, Tanya T. Paull 
Molecular Cell 
Volume 64, Issue 3, Pages (November 2016)
DOI: /j.molcel
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2. Molecular Cell 2016 64, 593-606DOI: (10.1016/j.molcel.2016.10.010)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
hMRN Complex Exhibits Endonuclease Activity on Protein-Blocked Ends
(A) hMR wild-type complex (50 nM) was assayed using a 50 bp dsDNA substrate containing a 5′ biotin-streptavidin block (B-Strep) and 3′ [32P] label as indicated. “sss” on the top strand denotes 5 phosphorothioate bonds at the 3′ end to prevent exonucleolytic degradation. Reactions contained Nbs1 (50 and 100 nM), 5 mM MgCl2, 1 mM MnCl2, and 1 mM ATP as indicated and were incubated at 37°C for 30 min. A total of 0.5 mM nucleotide was used for reactions in lanes 13–15. Reaction products were separated on a denaturing polyacrylamide gel and analyzed by phosphorimager.
(B) hMR wild-type complex (50 nM) was assayed as in (A) with 50 nM Nbs1. Mre11 inhibitors were added to reactions at 20, 100, and 500 μM, with equivalent volume of DMSO added to reactions without inhibitor.
(C) hMR (50 nM) was assayed as in (A) except with 197 bp dsDNA with Nbs1 added as indicated.
(D) hMR wild-type and H129N (50 nM) with equimolar Nbs1 was assayed as in (A) except with 197 bp dsDNA with ATP and streptavidin added as indicated.
Red arrows indicate sites of nuclease digestion on substrate, and arrows and brackets on gels indicate degradation products.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 Nbs1 Modulates Mre11 Nuclease Activities
(A) hMR wild-type complex (50 nM) was assayed using a 50 bp dsDNA substrate containing a 5′ biotin-streptavidin block (B-Strep) and a 5′ [32P] label as indicated. “sss” on the top strand denotes 5 phosphorothioate bonds at the 3′ end to prevent exonucleolytic degradation. A version of the same substrate containing a nick on bottom strand was used in the reactions in lanes 7–12 as indicated. Reactions contained 12.5, 25, and 50 nM Nbs1 and were performed as in Figure 1A.
(B) hMR wild-type complex (50 nM) was assayed with substrates containing 5′ biotin (B) and 50 nM Nbs1 and streptavidin as indicated.
(C) yMR wild-type complex was incubated with substrates as described in (A). Reactions contained 50 nM yMR, 50 nM Xrs2, and 100 nM wild-type Sae2 as indicated, except for lanes 11–13 (3.125 nM yMR and Xrs2), and were incubated at 30°C for 30 min.
(D) yMR wild-type complex (50 nM) was assayed as described in (C) in reactions containing 50 nM Xrs2 and streptavidin as indicated.
Red arrows indicate sites and direction of nuclease digestion.
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5. Figure 3
ATP Enhances 3′-5′ Exonuclease Activity at a Nick with 5′ Blocked End but Restricts 3′-5′ Exonuclease Activity on a 3′ Recessed End
(A) hMR wild-type complex (50 nM) was assayed using the 50 bp dsDNA substrate containing 5′ biotin (B), a 5′ [32P] label, and a nick or a 3′ recessed end on the bottom strand, as shown, with 50 nM Nbs1, and assays were performed as in Figure 1A. A total of 1 mM ATP and streptavidin were added to the reactions as indicated.
(B) 3′-5′ exonuclease activity of hMR complexes (50 nM) with equimolar Nbs1 was assayed in 1 mM MnCl2 using a 5′ [32P]-labeled substrate with a 4 nt 3′ recessed end and ATP as indicated.
(C) 3′-5′ exonuclease activity of yMR complexes (50 nM) with equimolar Xrs2 was assayed as in (B).
(D) hMR wild-type complex (50 nM) was assayed using a 50 bp dsDNA substrate with a nick in the bottom strand as indicated. Reactions were performed in presence of Mre11 inhibitors as in Figure 1B.
(E) hMR wild-type complex (50 nM) was assayed using a DNA substrate containing a 3′ recessed bottom strand. Reactions were performed in triplicate with 50 nM Nbs1, 1 mM MnCl2, and 100 μM inhibitors.
Red arrows indicate sites and direction of nuclease digestion.
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6. Figure 4
Nbs1 Enhances Mre11 Nuclease Activity on a 3′ Strand Opposite a Blocked End
(A) Human MR wild-type complex (50 nM) was assayed using the 50 bp substrate with 5′ radiolabel on top strand as shown, and assays were performed as in Figure 1A. A total of 50 nM Nbs1 was added as indicated.
(B) Human MR wild-type complex (50 nM) was assayed as in (A) with 50 nM Nbs1, and streptavidin, ATP, and Mn2+ were varied as indicated.
(C) Human MRN wild-type and H129N complexes (50 nM) were assayed using the 50 bp substrate with 5′ radiolabel on the top strand as shown, and assays were performed as in Figure 1A.
(D) yMR (50 nM) in presence of 50 nM Xrs2 and 100 nM Sae2 was assayed using the 50 bp substrate with 5′ radiolabel on top strand as shown.
(E) hMR wild-type complex (50 nM) was assayed using a 50 bp dsDNA substrate with a nick and a 5′ [32P] label on the top strand as shown.
(F) yMR (50 nM) with equimolar Xrs2 was assayed using the substrate in (E).
Red arrows indicate sites and direction of nuclease digestion on substrate, and arrows and brackets on gels indicate degradation products.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5 hMRN Endonucleolytic Activity Is Stimulated by a Nick
(A) hMR wild-type complex (50 nM) with 50 nM Nbs1 was assayed using 50 bp dsDNA substrates containing a 5′ biotin-streptavidin block (B-Strep) and a nick (substrate II, V, and VI) as in Figure 1A.
(B) hMR wild-type complex (50 nM) was assayed using a 50 bp dsDNA substrate containing a 3′ or 5′ [32P] label on the bottom strand and a nick in the top strand with Nbs1 (50 nM) as indicated.
(C) hMR wild-type complex (50 nM) was assayed as in (B) with ATP and streptavidin added as indicated.
(D) hMR wild-type complex (50 nM) with equimolar Nbs1 was assayed using a DNA substrate containing a 3′ biotin (B), a nick on one strand, and a 3′ or 5′ [32P] label as indicated.
Red arrows indicate sites and direction of nuclease digestion on substrate, and arrows and brackets on gels indicate degradation products.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6 CtIP Promotes hMRN Endonuclease Activity
(A) hMR (50 nM) and equimolar Nbs1 were incubated with 5′-radiolabeled 197 bp dsDNA as in Figure 1A. CtIP wild-type or T847/T859 mutants (50, 100, and 200 nM) were added as indicated.
(B) hMR (50 nM) was assayed as in (A) with CtIP-T847E/T859E (100 and 200 nM), with equimolar Nbs1 and other components varied as indicated.
(C) hMR (50 nM) was assayed as in (A) in reactions containing Nbs1 (50 nM) and CtIP-T847E/T859E (200 nM) as indicated. Reactions were digested with Proteinase K at 37°C for 1 hr, separated on 8% native polyacrylamide gel, and analyzed by phosphorimager.
Red arrows indicate sites of nuclease digestion on substrate, and arrows and brackets on gels indicate degradation products.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
MRN Removes Protein-DNA Adducts through Sequential Processing Events Regulated by Nbs1
(A) The current model for MRN nuclease activity (scenario I, dashed arrow) at 5′ protein adducts involves an initial endonucleolytic cleavage event followed by exonucleolytic degradation toward the blocked end. Based on our observations, hMRN mediates endonucleolytic cutting adjacent to the protein block, although this cut can occur on either the strand with the block (scenario I) or on the opposite strand (scenario II). The nicked strand is then further processed by hMRN 3′-5′ exonuclease activity, either toward the block (I) or away from it (II), creating ssDNA gaps. Another endonucleolytic event occurs at the ss-dsDNA transition, removing the block. This event releases a free DNA end that can be utilized by downstream processing machinery. A similar sequence of events is observed with a 3′ protein adduct (scenario III), if a nick close to the adduct is available for hMRN. All of the MR processing events shown in (A) are ATP and Nbs1 dependent, dependent on the protein block, and inhibited by PFM01 and PFM03.
(B) hMR-catalyzed 3′-5′ exonuclease activity at a 3′ open end is inhibited by Nbs1 in the presence of ATP and also by PFM39.
Molecular Cell  , DOI: ( /j.molcel )
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