1. The 3′ to 5′ Exonuclease Activity of Mre11 Facilitates Repair of DNA Double-Strand Breaks 
Tanya T. Paull, Martin Gellert 
Molecular Cell 
Volume 1, Issue 7, Pages (June 1998)
DOI: /S (00)

2. Figure 1 Expression of Recombinant hMre11 and hRad50
(A) The sequence of MRE11 cDNA isolated by PCR from human testis and HeLa cell cDNA libraries contained an extra 84 nucleotides in the C terminus of the coding region. This creates an in-frame insertion of 28 amino acids, as shown in “hMre11 (new).” These clones also contained a frameshift that generated a stop codon 28 amino acids before the anticipated stop. The new Mre11 amino acid sequence is compared to the previously published human sequence, “hMre11,” and the mouse Mre11 sequence, “mMre11” (GenBank).
(B) hMre11 and hRad50 were coexpressed using a baculovirus expression system. The proteins were purified on nickel-affinity and anion-exchange columns, and finally on a Superdex 200 HR 10/30 gel-filtration column (fractions shown here). A 1:1 complex of Mre11 and Rad50 elutes in the void volume (peak in lane 4), while the Mre11 alone is included in the column (peak in lanes 10 and 11). Comparison with sizing standards gives a size estimate of ∼350 kDa for Mre11. No protein was found in other parts of the eluted volume.
Molecular Cell 1998 1, DOI: ( /S (00) )

3. Figure 2 Characterization of Mre11 Exonuclease Activity
(A) 25 ng (0.3 pmol) of Mre11 was incubated with 0.05 pmol of double-stranded DNA labeled at the 5′ end, in the presence of various cations: 1 mM MnCl2, 5 mM MgCl2, or 5 mM CaCl2, for 30 min, and then run on a denaturing polyacrylamide gel.
(B) A time course of Mre11 activity was performed on the same substrate as in (A) in the presence of 1 MnCl2, with the time intervals as shown.
(C) 0.3 pmol of Mre11 or 0.3 pmol each of Mre11 and Rad50 (as indicated) was incubated with various substrates in the presence of 1 mM MnCl2 for 30 min as diagrammed in the figure. The single-stranded substrate and the top strand of the double-stranded substrates are shown with the 5′ end on the left, and the asterisk indicates the position of the [32P] label. DNA substrates in lanes 1–3: 5′-labeled single-stranded; lanes 4–6: 5′-labeled blunt, double-stranded; lanes 7–9: 3′-labeled blunt, double-stranded; lanes 10–12: 3′-labeled 5′ overhang, double-stranded; lanes 13–15: 5′-labeled 3′ recessed, double-stranded; lanes 16–18: 5′-labeled 3′ overhang, double-stranded. The [32P] cordycepin on the 3′-labeled substrates inhibited Mre11 and Mre11/Rad50 activity; thus, the extent of degradation on these substrates was decreased relative to the substrates labeled on the 5′ end.
(D) 0.3 pmol of Mre11 was incubated with either a 5′-labeled branched DNA substrate (lanes 1 and 2) or a 5′-labeled 3′ flap substrate (lanes 3 and 4), as shown in the diagram, with 1 mM MnCl2 for 30 min. The single-stranded portion of the labeled strand in lanes 1 and 2 is 16 nucleotides long; thus, the degradation by Mre11 is at the tip, not at the fork.
Molecular Cell 1998 1, DOI: ( /S (00) )

4. Figure 2 Characterization of Mre11 Exonuclease Activity
(A) 25 ng (0.3 pmol) of Mre11 was incubated with 0.05 pmol of double-stranded DNA labeled at the 5′ end, in the presence of various cations: 1 mM MnCl2, 5 mM MgCl2, or 5 mM CaCl2, for 30 min, and then run on a denaturing polyacrylamide gel.
(B) A time course of Mre11 activity was performed on the same substrate as in (A) in the presence of 1 MnCl2, with the time intervals as shown.
(C) 0.3 pmol of Mre11 or 0.3 pmol each of Mre11 and Rad50 (as indicated) was incubated with various substrates in the presence of 1 mM MnCl2 for 30 min as diagrammed in the figure. The single-stranded substrate and the top strand of the double-stranded substrates are shown with the 5′ end on the left, and the asterisk indicates the position of the [32P] label. DNA substrates in lanes 1–3: 5′-labeled single-stranded; lanes 4–6: 5′-labeled blunt, double-stranded; lanes 7–9: 3′-labeled blunt, double-stranded; lanes 10–12: 3′-labeled 5′ overhang, double-stranded; lanes 13–15: 5′-labeled 3′ recessed, double-stranded; lanes 16–18: 5′-labeled 3′ overhang, double-stranded. The [32P] cordycepin on the 3′-labeled substrates inhibited Mre11 and Mre11/Rad50 activity; thus, the extent of degradation on these substrates was decreased relative to the substrates labeled on the 5′ end.
(D) 0.3 pmol of Mre11 was incubated with either a 5′-labeled branched DNA substrate (lanes 1 and 2) or a 5′-labeled 3′ flap substrate (lanes 3 and 4), as shown in the diagram, with 1 mM MnCl2 for 30 min. The single-stranded portion of the labeled strand in lanes 1 and 2 is 16 nucleotides long; thus, the degradation by Mre11 is at the tip, not at the fork.
Molecular Cell 1998 1, DOI: ( /S (00) )

5. Figure 3 Mre11 Opens Hairpin Loops
(A) 3.1 pmol of Mre11 was incubated overnight at room temperature in the presence of 1 mM MnCl2 with a DNA dumbbell substrate containing two hairpins, one with a 14 nucleotide loop and the other fully paired, and then run on a denaturing polyacrylamide gel containing 20% formamide. The small amount of opened dumbbell in the control lane is due to spontaneous breakage during gel purification of the substrate. The asterisk indicates the location of the [32P] label.
(B) Mre11 was incubated as in (A) with substrates containing varying lengths of mismatched nucleotides at the hairpin tip. DNA substrates in lanes 1 and 2: 14 nucleotide loop; lanes 3 and 4: 4 nucleotide loop; lanes 5 and 6: no mismatches. All of the substrates contained a 5 bp 3′ overhang at the other end of the DNA to minimize exonuclease degradation. The cleavage sites are numbered such that a cut exactly at the tip is “0,” sites 3′ of this are positive, and sites 5′ of this are negative. Size markers are shown in lane M.
Molecular Cell 1998 1, DOI: ( /S (00) )

6. Figure 4
Mre11 Forms Stable Complexes with DNA Hairpins and 3′ Overhangs
0.3 pmol of Mre11 was incubated with 0.05 pmol of blunt-ended 5′-labeled DNA substrate in the presence of an equivalent amount of various competitor DNAs: hairpin dumbbell (lanes 3–5), linear DNA with 3′ overhangs (lanes 6–8), or linear blunt-ended DNA (lanes 9–11), and then run on a denaturing polyacrylamide gel. The competitor DNA was added either 5 min before, or at the same time, or 5 min after the addition of the labeled substrate, as indicated. All of the incubations were at 37°C, and the total incubation time of Mre11 with the labeled substrate was 45 min in each reaction.
Molecular Cell 1998 1, DOI: ( /S (00) )

7. Figure 5 Joining of Nonhomologous Ends Is Promoted by Mre11
(A) Diagram of the plasmid substrate with the positions of the PCR primers as shown and the positions of the restriction sites marked “X.”
(B) 4 ng of plasmid DNA cut with different combinations of restriction enzymes was incubated with 0.01 pmol of human ligase I and 0.06 pmol of Mre11 or 0.1 U of exonuclease III as indicated, and the products were then amplified by PCR and run on a native polyacrylamide gel. “Blunt” ends were made with HincII and ScaI, and “mismatched 5′ overhangs” were made with XhoI and BamHI.
Molecular Cell 1998 1, DOI: ( /S (00) )

8. Figure 6 Diagram of Junctions Formed by Mre11 and Ligase I
(A) Shown on the top line are both strands of the initial cut ends made by HincII (left) and ScaI (right). Only the relevant homologous sequence is shown, with the rest of the nucleotides symbolized by dashes and the sequence from the ScaI-cut end in bold. The two types of junction recovered are shown below with the number of nucleotides deleted, “Δ_,” and the number of sequenced junctions in parentheses.
(B) 5′ mismatched ends made by XhoI (left) and BamHI (right) are shown on the top line with the sequence from the BamHI-cut end in bold. Nucleotides and homologies are symbolized as in (A). The one type of junction recovered is shown below, in which only one copy of the GAGCT motif remains and the intervening sequence on the BamHI-cut end is deleted.
(C) Broken ends generated by HincII (left) and EcoRI (right) are shown on the top line, with only the relevant homologous sequences shown and the sequence from the EcoRI-cut end in bold. Below are the six types of junctions recovered, with the size of the deletion and the frequency of its occurrence indicated as in (A). The top five classes of junction utilized the AA dinucleotide in the EcoRI 5′ overhang, paired with TT dinucleotides in the HincII-cut end, with accompanying deletions of 21, 38, 39, 70, or 79 bp, respectively. The fifth class of junction shown in the very bottom line retains the HincII-cut end by joining at the CCAG homology, with a deletion of 13 bp in the EcoRI-cut end.
Molecular Cell 1998 1, DOI: ( /S (00) )