Analyzing the molecular clamp mechanism of Mre11:Rad50 NBD 82

A major result of the structural analysis of T. maritima Mre11:Rad50NBD is that Rad50 blocks Mre11’s DNA binding sites in the ATP bound form (see section 4.3.2). However the possibility remains that, although the crystal structures of the open and closed complex explain the solution SAXS data (see sections 4.2.3 and 4.3.5), DNA induces an additional conformational change enabling it to bind to both Mre11 nuclease and capping domains and NBD-HLH module. For instance, a relatively moderate structural change could lead to double stranded DNA being sandwiched between Rad50 NBDs and the phosphodiesterase domains of Mre11.

To test this possibility, Mre11:Rad50NBD was disulfide bridged in the presence of single and double stranded DNA (ФX174 Virion and ФX174 RF II) under conditions where in EMSA most of the DNA is shifted by bound protein (Figure 28A-F). In both cases subsequent gel filtration failed to detect co-elution of DNA and protein. The fact that Mre11:Rad50NBD could not be crosslinked around internal DNA leads to the assumption that the complex does not bind the DNA by encircling it in a ring-like structure.

To confirm this statement DNA binding of T. maritima Mre11:Rad50NBD (S-S) was additionally investigated via EMSA and gel filtration with double stranded DNA oligos containing a 5′ fluorescein label (Möckel et al., 2011). A single-chain Fv fragment (scFv) of a fluorescein binding antibody can be used to block the DNA ends (Honegger et al., 2005) (Figure 29). To analyze if the disulfide bridged, trapped ATP bound form can still bind dsDNA with both ends blocked by the scFv fragment FITC-E2 (Figure 29A) DNA affinity of Mre11:Rad50NBD (S-S) was tested by adding the complex to the dsDNA with blocked ends and subsequent EMSA (Figure 29B). To exclude incomplete end blocking of the DNA, the interaction between antibody and fluorescein labeled DNA was validated by

4. Results 83 analytical gel filtration (Figure 29C). Nevertheless the disulfide bridged, ATP bound complex was still able to bind DNA with blocked ends. Thus, double stranded DNA is likely not to be encircled by Mre11:Rad50.

Figure 28: Biochemical analysis of the molecular clamp mechanism of T. maritima Mre11:Rad50NBD_.

(A) Gel filtration chromatogram of the disulfide bridged Mre11:Rad50NBD _{complex in the presence of ATP} and double stranded ФX174 RF II plasmid DNA was carried out onto a Superose 6 PC 3.2/30 gel filtration column. Disulfide bridging of the complex was carried out either in the presence (solid line) or absence (dashed line) of DNA, prior to gel filtration. The disulfide bridged Mre11:Rad50NBD _{did not co-elute with the} plasmid DNA fraction (peak 1), indicating that the complex cannot form a stable ring around internal DNA.

(B) Electrophoretic Mobility Shift Assay (EMSA) shows that the ФX174 RF II plasmid DNA was completely bound by the Mre11:Rad50NBD _{complex used in the above described gel filtration} chromatography experiment. (C) SDS PAGE presenting the disulfide bridged Mre11:Rad50NBD _complex from peak 2 of the gel filtration chromatogram in (A). (D) Gel filtration chromatogram of the disulfide bridged Mre11:Rad50NBD _{complex in the presence of ATP and single stranded ФX174 Virion plasmid DNA} was carried out onto a Superose 6 PC 3.2/30 gel filtration column. Disulfide bridging of the complex was carried out either in the presence (solid line) or absence (dashed line) of DNA, prior to gel filtration. The disulfide bridged Mre11:Rad50NBD _{did not co-elute with the plasmid DNA fraction (peak 1), indicating that} the complex cannot form a stable ring around internal DNA. (E) EMSA shows that the ФX174 Virion plasmid DNA was completely bound by the Mre11:Rad50NBD _{complex used in the above described gel} filtration chromatography experiment. (F) SDS PAGE presenting the disulfide bridged Mre11:Rad50NBD complex from peak 2 of the gel filtration chromatogram in (D).

A B C

4. Results 84

Figure 29: Details of the DNA binding mechanism of T. maritima Mre11:Rad50NBD (S-S) _{via the}

Antibody DNA Binding Assay (Möckel et al., 2011). (A) Illustration of the Antibody DNA Binding Assay described in Figure 29B–E. (B) Electrophoretic Mobility Shift Assay showing that the nucleotide bound, closed Mre11:Rad50NBD (S-S) _{complex can still bind to antibody blocked double stranded 40mer DNA.} Following protein concentrations were used: 0, 1.75, 3.50, 7.0 and 14.0 µM. (C) Gel filtration chromatogram of 5´fluorescein labeled ds40mer in the absence (solid line) and presence (dashed line) of the antibody fragment FITC-E2 verified complete blocking of the respective double stranded DNA. The presence of antibody scFV shifted the DNA to larger molecular weights (peak 1) compared to DNA alone (peak 2) and

A

B C

4. Results 85

free scFv (peak 3). (D) Gel filtration chromatogram of the 5´fluorescein labeled ds40mer in presence (solid line) and absence (dashed line) of Mre11:Rad50NBD (S-S)_{. Prior to gel filtration protein and DNA were} incubated under conditions where in EMSA most of the DNA is shifted by bound protein. The gel filtration retention volumes were subsequently analyzed by agarose gel electrophoresis. The agarose gel lanes are aligned with the respective fractions of the gel filtration elution. (E) Gel filtration chromatogram of the DNA-antibody mixture (dashed line) and the ternary DNA-(Mre11:Rad50NBD (S-S)_{)-antibody complex (solid} line). Analysis of the respective elution fractions by agarose gel electrophoresis indicates that the complex could not be trapped on double stranded DNA. All gel filtration experiments were carried out using a S200 5/150 GL column.

To further validate this model, fluorescein labeled dsDNA was first incubated with Mre11:Rad50NBD (S-S), followed by blocking of the 5´ ends by scFv. Subsequently the DNA:protein complex was analyzed by analytical gel filtration and agarose gel electrophoresis (Figure 29D-E). Since the gel filtration retention volume of DNA bound to scFv and/or Mre11:Rad50NBD (S-S) would be shifted in comparison to that of free DNA, it should be possible to detect encircling of MRNBD (S-S) _{around DNA. However, we did not}

see any change in retention volume or evidence for a ternary DNA:(Mre11:Rad50NBD (S-S)) :antibody complex. Therefore it is unlikely that Mre11:Rad50NBD forms a ring around dsDNA (Figure 29B-E).