Journal: Nucleic Acids Research

Article Title: Damage-dependent regulation of MUS81-EME1 by Fanconi anemia complementation group A protein

doi: 10.1093/nar/gkt975

Figure Lengend Snippet: Interaction of FANCA and MUS81 on ICL damage in living cells. ( A ) Western blot of FANCA knockdown. U2OS cells were transfected with an ON-TARGET Plus SMART Pool siFANCA, a control siRNA (siCtrl), and dH 2 O (Mock) through lipofectamine. Forty-eight hours later, 50 µg of whole cell protein extract was prepared for western blot analysis using a FANCA-specific antibody. Actin is the loading control. ( B ) U2OS cells were transfected with GFP-FANCA and treated with 8-MOP and/or a 405 nm laser beam as indicated. Laser passing path is indicated by yellow arrows. The panel is representative of 25 examined nuclei of each treatment. 25/25 showed the laser-induced FANCA stripes in the presence of 8-MOP. ( C ) Mock, control siRNA and siFANCA treated U2OS cells were transfected with GFP-MUS81 and treated with 8-MOP and/or a 405 nm laser beam as indicated. Laser passing path is indicated by yellow arrows. The panel is representative of 25 examined nuclei of each treatment. 25/25 showed the laser-induced MUS81 stripes in the presence of 8-MOP for the mock and control treatment. 25/25 of siFANCA nuclei did not show the laser-induced MUS81 stripes in the presence of 8-MOP.

Article Snippet: Complementary DNAs for human MUS81, EME1 and FANCA were obtained by polymerase chain reaction amplification from a universal complementary DNA pool (BioChain Institute, Inc.).

Techniques: Western Blot, Knockdown, Transfection, Control

Journal: Nucleic Acids Research

Article Title: Damage-dependent regulation of MUS81-EME1 by Fanconi anemia complementation group A protein

doi: 10.1093/nar/gkt975

Figure Lengend Snippet: Effect of FANCA on MUS81-EME1-mediated DNA incision. ( A ) Undamaged and psoralen ICL-damaged substrates were incubated with 1.5 nM of MUS81-EME1, respectively, and an increasing concentration of FANCA (0, 1.25, 2.5, 5, 10, 15 and 20 nM). DNA markers are shown in nucleotide on the left. Same marker as described in was used. ( B ) Quantitation of three independent experiments. MUS81-EME1 activity was calculated as percentage of incision products out of the input substrates. The experiment without FANCA was normalized to 0 and used to calibrate all other experiments with the indicated amount of FANCA. Error bars: standard deviation. Dashed line: Phase I and Phase II border. ( C ) Fold changes of the MUS81-EME1 regulation by FANCA. The experiment without FANCA was arbitrarily normalized to 0 for fold change and used to calibrate all other experiments with the indicated amount of FANCA. Error bars: standard deviation. Psoralen mono-adducted ( D ) and a benzo(α)pyrene diolepoxide deoxyguanosine adducted 3′ ssDNA branch structures ( E ) were incubated with 1.5 nM of MUS81-EME1 and an increasing concentration of FANCA (0, 1.25, 2.5, 5, 10, 15 and 20 nM). The schematic appearance of the products after incision are shown on the right. Letter P with a circle indicates 5′- 32 P labeling.

Article Snippet: Complementary DNAs for human MUS81, EME1 and FANCA were obtained by polymerase chain reaction amplification from a universal complementary DNA pool (BioChain Institute, Inc.).

Techniques: Incubation, Concentration Assay, Marker, Quantitation Assay, Activity Assay, Standard Deviation, Labeling

Journal: Nucleic Acids Research

Article Title: Damage-dependent regulation of MUS81-EME1 by Fanconi anemia complementation group A protein

doi: 10.1093/nar/gkt975

Figure Lengend Snippet: Effect of FANCA mutants on MUS81-EME1-mediated DNA incision. ( A ) Psoralen ICL-damaged (top) and ICL-undamaged DNA substrates (bottom panel) were incubated with 1.5 nM of MUS81-EME1 and 20 nM of purified WT and mutant FANCA proteins as indicated. DNA markers are shown on the left. The schematic appearance of the products after incision are shown on the right. Letter P with a circle indicates 5′- 32 P labeling. Asterisk indicates a decayed and uncross-linked species. ( B ) Quantitation of three independent experiments. Incision efficiency was normalized to WT-FANCA (arbitrarily assigned as 100%). Error bars: standard deviation. ( C ) Ability to discriminate ICL from undamaged DNA by FANCA mutants. The discrimination factor was calculated by dividing the relative incision rate of ICL of a protein in (B) by the relative incision rate of undamaged DNA of the same protein. The discrimination factor for FANCA was arbitrarily assigned as 100%.

Article Snippet: Complementary DNAs for human MUS81, EME1 and FANCA were obtained by polymerase chain reaction amplification from a universal complementary DNA pool (BioChain Institute, Inc.).

Techniques: Incubation, Purification, Mutagenesis, Labeling, Quantitation Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Damage-dependent regulation of MUS81-EME1 by Fanconi anemia complementation group A protein

doi: 10.1093/nar/gkt975

Figure Lengend Snippet: A working model for the regulation of MUS81-EME1 activity by FANCA. Blockage of DNA replication forks by an unknown damage (black box and question mark) initiates recruitment of FANCA (blue oval). FANCA recognizes ICL damage (red zigzag) and subsequently recruits MUS81-EME1 to incise the leading strand at the 5′ side of the ICL. If the damage only affects one DNA strand (green block) or there is no damage, FANCA will inactivate MUS81-EME1 to prevent unnecessary incisions. Red arrow: DNA incision; red cross: inactivation.

Article Snippet: Complementary DNAs for human MUS81, EME1 and FANCA were obtained by polymerase chain reaction amplification from a universal complementary DNA pool (BioChain Institute, Inc.).

Techniques: Activity Assay, Blocking Assay