1. Volume 8, Issue 5, Pages 1117-1127 (November 2001)
Human Mus81-Associated Endonuclease Cleaves Holliday Junctions In Vitro 
Xiao-Bo Chen, Roberta Melchionna, Cecile-Marie Denis, Pierre-Henri L. Gaillard, Alessandra Blasina, Inez Van de Weyer, Michael N. Boddy, Paul Russell, Jorge Vialard, Clare H. McGowan 
Molecular Cell 
Volume 8, Issue 5, Pages (November 2001)
DOI: /S (01)

2. Figure 1 Identification of Human and Mouse Homologs of Mus81
Human and mouse cDNAs encoding open reading frames with similarity to the damage tolerance protein Mus81 from S. cerevisiae and S. pombe were identified. Alignment of Mus81 homologs was generated using the Clustal W program. Amino acid identities are highlighted in black, and conservative changes are shaded. Dotted lines indicate two putative helix-hairpin-helix motifs (Doherty et al., 1996). The motifs conserved in members of the XPF (Aravind et al., 1999) family are indicated by a solid line. Amino acid substitutions used to create inactive Mus81 are indicated above the human sequence. The GenBank accession number for human Mus81 is AF425646; the accession number for the mouse sequence is AF
Molecular Cell 2001 8, DOI: ( /S (01) )

3. Figure 2 Human Mus81 Interacts with Cds1
(A) HEK293 cells were transiently transfected with Flag.Cds1, HaMus81, or both. Forty-eight hours following transfection, lysates and Ha immune precipitates were probed for expression of Flag.Cds1. Flag.Cds1 was detected in Ha immune precipitates from cells that express HaMus81.
(B) GST-fusion proteins containing the indicated regions of Cds1 were expressed and purified from bacteria. The bead-bound proteins were incubated in extracts of HeLa cells expressing 3HaMus81. The beads were washed extensively, and retention of Mus81 was monitored by anti-Ha immune blotting.
Molecular Cell 2001 8, DOI: ( /S (01) )

4. Figure 3
Mus81 Is a Nuclear Protein Whose Abundance Increases after DNA Damage or Inhibition of Replication
(A) The abundance of p59Mus81 was monitored in extracts from asynchronous cells that were not treated (Asn), irradiated (γ), or incubated in the presence of agents that block DNA replication thymidine (Tdr), and hydroxyurea (HU) for 16 hr.
(B) Expression of 3HaMus81 was induced for 24 hr prior to the indicated treatment. Samples were harvested at the indicated time points, and the kinetics of 3HaMus81 abundance was monitored. Densiometric analysis of the immune blots showed that the 3HaMus81 signal is increased 4-fold following γ irradiation and 7- to 8-fold by UV or HU treatment. Similar results were obtained with two other cell lines.
(C) Indirect immune fluorescence using anti-Ha antibodies on cells expressing 3HaMus81 reveals that 3HaMus81 is nuclear.
Molecular Cell 2001 8, DOI: ( /S (01) )

5. Figure 4 Mus81 Has Associated Substrate-Specific Endonuclease Activity
(A) Schematic representations of the structure of the DNA substrate are shown above the gel. Each substrate contains a 5′ radiolabeled oligonucleotide (indicated by the dot). The indicated oligonucleotide substrates were incubated with preimmune sera (lane 1), anti-Mus81 immune precipitates (lane 2), anti-Mus81 immune precipitates in the presence of ATP (lane 3), Ha immune precipitates from cells that were transfected with 3HaMus81D/A plus ATP (lane 4), Ha immune precipitates of wild-type 3HaMus81 (lane 5), and Ha immune precipitates of wild-type 3HaMus81 plus ATP (lane 6). Substrate was incubated in buffer in lane 7. Cells were treated with 2 mM HU for 16 hr prior to harvesting. 3HaMus81D/A was created by substitution of aspartic residues at 338 and 339 with alanine. A + G and T + C sequencing ladders derived from oligonucleotide 1 were run in parallel. Reactions were analyzed on a 12% sequencing gel. Results are representative of five independent assays, using two preparations of substrate.
(B) The main sites of cleavage are indicated by arrows on relevant DNA structure. The shaded area represents the 12 nucleotide homology region that allows the junction to slide in structures X12 and PX12.
Molecular Cell 2001 8, DOI: ( /S (01) )

6. Figure 5
Resolution of the X12 Structure into Linear Duplex Products and Mapping of the Cleavage Sites on Each Arm of the X Structure
(A) The full X structure labeled on oligonucleotide 1 was incubated with buffer (lane 1), anti-Mus81 immune precipitate plus ATP (lane 2), anti-Mus81 immune precipitate (lane 3), preimmune precipitate plus ATP (lane 4), Ha immune precipitates from cells that were transfected with wild-type 3HaMus81 plus ATP (lane 5), Ha immune precipitates of wild-type 3HaMus81 (lane 6), and Ha immune precipitates from cells that were transfected with 3HaMus81D/A plus ATP (lane 7). Lane 8 was not loaded. Marker structures Y12 and Duplex X12 were run in lanes 9 and 10, respectively. The products of the reactions were analyzed by native PAGE gel.
(B) Four X12 structures, each labeled on a different oligonucleotide, were incubated with buffer (lane 1), preimmune precipitate (lane 2), anti-Mus81 immune precipitate (lane 3), Ha immune precipitates from cells that were transfected with wild-type 3HaMus81 (lane 4), and Ha immune precipitates from cells that were transfected with 3HaMus81D/A (lane 5) and analyzed on a denaturing 12% acrylamide gel as above. Results are representative of four independent assays, using two preparations of substrate.
(C) Marker structures X12 (lane 1), PX12 (lane 2), Y12 (lane 3), intact duplex (lane 4), nicked duplex (lane 5), duplex with a 2 nt flap (lane 6), and duplex with a 2 nt gap (lane 7) were analyzed by native PAGE.
Molecular Cell 2001 8, DOI: ( /S (01) )

7. Figure 6
Resolution of the X0 Structure into Linear Duplex Products and Mapping of the Cleavage Sites on the X0 Structure
(A) X0 structure labeled on oligonucleotide 1 was incubated with preimmune precipitate (lane1) and anti-Mus81 immune precipitate (lane 2). Lane 3 is blank. Marker structures PX0, Y0, and duplex DX0 were run in lanes 4, 5, and 6, respectively.
(B) Four X0 structures, each labeled on a different oligonucleotide, were incubated with preimmune precipitate (P) or with anti-Mus81 immune precipitate (I) and analyzed on a denaturing 12% acrylamide gel as above. A + G and T + C sequencing ladders derived from the labeled oligonucleotides were run in parallel. Results are representative of three independent assays, using two preparations of substrate.
Molecular Cell 2001 8, DOI: ( /S (01) )

8. Figure 7 Potential Holliday Junction Resolution Products
In (A), cutting on 2 and 4 occurs at the same position relative to the junction, and equivalent products that can be ligated are generated. In (B), cutting on 2 is closer to the junction than on 4, generating one product with a gap and a second product with an overhang. In (C), cutting on 4 is closer to the junction than on 2. The gapped products generated in (B) and (C) need to be filled in by a DNA polymerase prior to ligation. The products with overhangs need to be trimmed prior to ligation. All three patterns of cleavage allow resolution of the Holliday junction and precisely maintain the nucleotide sequence of the parental molecules.
Molecular Cell 2001 8, DOI: ( /S (01) )