Mst77F tightly compacts long DNA in vitro

Within the nucleus of spermatids the carrier of the genetic information is several orders of magnitude longer than the very short DNAs that have been used for the previous experiments. During the postmeiotic maturation process of Drosophila spermatids the DNA adapts a 200-fold higher compaction status compared to somatic cells. This state requires extraordinary tight arrangement of the DNA.

According to the data I generated in the course of this study which clearly show Mst77Fs potential to aggregate and interconnect even short DNAs, I proposed a massive structural impact of the Mst77F wild-type protein on longer DNA. This effect

should be discriminative from the 100N multimerization mutant that displays less potential to link up individual DNA molecules, the Mst77F 60C binding mutant, hH1.4 and xPR-Set7.

To investigate the structural effects that Mst77F and the other proteins exert on longer, linear DNA I conducted again AFM with DNA that is 2434 bp in lenght. The structural properties of Mst77F wild-type DNA complexes were compared to DNA complexes that are shaped by the Mst77F 100N multimerization mutant and the Mst77F 60C DNA binding mutant. Histone hH1.4 as a member of the H1 family proteins that have been reported to structurally impact DNA/chromatin, was used to discriminate the Mst77F effect on DNA from the effects of well-studied structural proteins (Dootz et al., 2011; Shen et al., 1995). Previous experiments within this study showed clear functional differences between Mst77F and hH1.4 and on this basis I expected also structurally different complexes. Last but not least, xPR-Set7 that was not implicated in direct DNA binding, though displayed certain DNA binding properties in EMSA assays, was used as a further control. The structural properties of the DNA were imaged in the presence of 4-fold, 20-fold and 100-fold molar excess of the indicated proteins over DNA (Fig. 3.9).

At little molar excess of protein over DNA (4:1) the observed effects were limited to single molecules. Compared to free DNA imaging that showed DNA molecules in their extended but flexible conformation, a 4-fold molar excess of Mst77F wild-type structurally induced bending and looping of a fraction of molecules. Mst77F formed hubs that interconnected parts within one molecule (top row 1:4). Importantly this effect was intra-molecular and limited to individual molecules. A similar effect was observed for the 100N mutant that also exerted intra-molecular connections between parts of the same DNA molecule. DNA complexed with Mst77F 60C was indistinguishable from DNA “only” structures reflecting its mutant DNA association.

Histone H1 has previously been shown to form fibrillar, branched and network-like structures (“tramtracks”) with DNA mediated through sandwiching of DNA molecules by the protein (Lu et al., 2009a; Lucius et al., 2001). At a molar ration 1 : 4 the fibrillar and branched species were seen with low frequency. The predominant form was indistinguishable from DNA “only” images. As expected for a non DNA binding protein xPR-Set7 DNA complexes showed similar results to DNA only at the lowest molar DNA protein ratio.

Increased molar DNA-protein rations of 1:20 uncovered the specific Mst77F effect on DNA that can be attributed to the N-terminal domain of the protein (Fig. 3.9 ; 1:20 column). Individual DNA molecules clustered and formed a “nucleus” with protruding DNA “tentacles” that seemed to be free of protein. Within this “nucleus”

substructures were not recognizable suggesting a tightly packed, dense DNA protein entity. With lower frequency the second observable species was individual DNA molecules that displayed, by the majority high intra-molecular associations.

Interestingly, the DNA structures built up by the Mst77F 100N mutant were very much alike the structures formed by hH1.4 suggesting a similar interaction mode with the proteins packaging DNA molecules in a parallel aligned manner. Both proteins converted individual DNA molecules into fibrillar, branched complexes consisting of several DNAs. The recorded differences between Mst77F wild-type, Mst77F 100N and hH1.4 nicely reflected the specific function that extend the simple charge mediated binding to DNA. Again, as predicted no significant differences between DNA incubated with Mst77F 60C, xPR-Set7 and DNA “only” species became obvious.

Further increase of the molar DNA protein ratios to 1:100 led to an amplification of the formed structures (Fig. 3.9 ; 1:100 column). The “nucleus” structures elicited by the Mst77F wild-type protein expanded their dimensions in the Z plane. Individual DNA molecules could not be detected anymore. A major change from scattered fibrillar, branched towards tightly arranged, network like structures was seen for the

100N mutant possibly reflecting the impact of an excess of flexible, highly charged peptide moieties on their counter-ion polymer. Even though hH1.4 has a similar charge density to the 100N mutant and showed previously similar results, the formed complexes with long DNA were distinct. At the highest monitored molar ratio fibrillar, branched conformations were still evident but the major species constituted spacious, parallel (and maybe lateral) interconnected DNA molecules with a small, central high-density entity. Fundamentally, this structure was very different from the structures built up by Mst77F wild-type. As previously shown for the lower molar ratios there was also no effect of the Mst77F 60C protein on DNA structure at the 1:100 molar ratio. However, a 100-fold molar excess of xPR-Set7 led to branched interconnection of DNA molecules distinct from structures seen for lower molar ratios

of Mst77F 100N or hH1.4. I reasoned this is attributed to the attractive charges of the protein towards DNA.

Fig. 3.9 AFM uncovers structural differences caused by Mst77F from effects triggered by other charged proteins

Mst77F wild-type, Mst77F 100N, Mst77F 60C, hH1.4 and xPR-Set7 were complexed with 12 x 200 x 601 DNA at 4-fold, 20-fold and 100-fold molar excess of the protein over DNA. Images were recorded in

tapping mode. The scale bar represents 500 nm. The height scale of all images is 3 nm but for Mst77F wild-type 1:100: due to illustration the height scale was increased to 20 nm.

Taken together, Mst77F tightly compacted DNA in vitro. In very good agreement with the centrifugation fractionation and DNA crosslinking assays this effect can be attributed to the N-terminal region of the protein that constitutes a multimerization module. Consequently, mutants that were impaired in DNA binding show no structural effect on DNA. The Mst77F mutant that lacked the N-terminus induced network like complexes with long DNAs. Importantly, histone H1 proteins that have been reported to elicit structural effects on DNA showed different structures.