The ParABS system

The Type I partition system ParABS was discovered and originally defined as the DNA segregation apparatus for low copy number plasmids [Gerdes et al., 2010]. To-day, Type I is known as the most widely present system for bacterial chromsome

72 4.1. Introduction

Figure 4.2:ParB spreading model from parS. ParB dimerizes at the C-terminal domain and binds DNA as dimer recognizing the parS sequence by the helix-turn-helix motif located at the N-terminal domain. DNA binding promotes dimerization trough the N-terminal domain and the recruitment of additional ParB units by dimerization with the C-terminal domain.

Adapted from Leonard et al. [2004].

segregation [Wang et al., 2013]. The ParABS system comprises ParB, a DNA binding protein, its binding sequence termed parS, and ParA protein. ParA is a DNA binding protein with ATPase activity. The ParB-parS complex forms a centromere-like struc-ture which is then recognized and segregated by ParA. A homologous apparatus is important for chromosome segregation during cell division or sporulation in many bacteria including Bacillus subtilis [Mierzejewska and Jagura-Burdzy, 2012; Yamaichi and Niki, 2000]. In B. subtilis there are eight functional parS sequences (consensus sequence TGTTTCACGTGAAACA) located near the ori, resulting in an acumulation of ParB –also called Spo0J in B.subtilis– at these regions [Lewis and Errington, 1997;

Lin and Grossman, 1998]. ParB binds non-specifically to DNA sequences in the vicin-ity of parS sites for several kilobases [Lin and Grossman, 1998; Breier and Grossman, 2007]. Based on structural data, Leonard et al. [2004] proposed a model where af-ter dimerization through the C-af-terminal domain ParB binds specifically to the parS sequence (Figure 4.2). The specific binding is dependent on the helix-turn-helix motif located at the N-terminal domain. The N-terminal domain forms a secondary dimerization site necessary for DNA binding. After binding the parS sequence, the dimerization domain at the C-terminal interacts with the C-terminal domain of a different ParB protomer, promoting the binding to adjacent non-specific DNA sites.

Therefore, the parS sequence acts as a nucleation site for the formation of a ParB fil-ament that spreads to non-specific DNA regions. This phenomenon has been shown to be important for the biological function in several cases [Breier and Grossman, 2007; Rodionov and Yarmolinsky, 2004; Murray et al., 2006]. It is known that null mutants in ParB gene are lethal or produce anucleate cells depending on the species

[Mohl et al., 2001; Bartosik et al., 2009]. In B. subtilis the spreading mutation R80A results in an abnormal nucleoid and are defective in sporulation [Autret et al., 2001].

In fact, non-specific binding from parS sequences is a general feature of ParB pro-teins [Schumacher, 2008]. However, the mechanism of ParB spreading from parS sequences and the implications of spreading to hallmark chromosome segregation are not understood.

Remarkably, the interaction between B. subtilis ParB and parS is implicated in the recruitment of the SMC complex to the chromosome, providing an intriguing struc-tural link between chromosome partitioning and condensation [Gruber and Erring-ton, 2009; Sullivan et al., 2009]. The details of how ParB proteins assemble to form higher order segrosome complexes with their DNA targets remain to be determined and may differ, even between ParB proteins that are related at the level of primary structure [Schumacher, 2008]. In addition, how ParB triggers the loading of SMC proteins and how the activity of the condensin is enhanced by ParB proteins around the ori remains unknown.

Electrophoretic mobility shift assays (EMSAs) on short oligonucleotide substrates, as well as footprinting assays, have demonstrated specific binding of parS by B. sub-tilisParB in vitro but do not provide evidence for the lateral spreading that had been expected based on the in vivo behaviour [Breier and Grossman, 2007; Murray et al., 2006]. Moreover, several previous studies using EMSAs with longer DNA substrates showed an apparent laddering effect that was consistent with non-specific coating of DNA by ParB, but did not directly address the role of parS sequences in (potentially) nucleating these structures [Murray et al., 2006; Sullivan et al., 2009]. Recently, sin-gle molecule imaging studies have revealed that several different ParB proteins are able to condense flow-stretched DNA, and it has been suggested that this is the result of protein-mediated bridges between distant non-specific DNA segments [Graham et al., 2014]. The possible role of parS in this activity was unclear because the study focussed on substrates lacking specific sites, and ParB localization at parS was not observed. Based in part upon this work, modelling of ParB binding to DNA has suggested a two-state nucleation model for ParB binding and condensation of DNA around parS [Broedersz et al., 2014]. In this model, a combination of 3D bridging and lateral spreading protein-protein interactions form a network of ParB protomers that together condense DNA, and it is proposed that such networks are most efficiently nucleated by parS-bound ParB molecules.

In this chapter, we show using EMSA assays that Bacillus subtilis ParB binds to DNA both specifically and non-specifically. Magnetic tweezers (MT) show that ParB is capable of packaging non-specific DNA with a low condensing force of ≈2 pN. This condensation activity is reversible and due to the formation of loops be-tween non-specific DNA segments that are bridged in cis by multiple ParB proteins.

Bridging in trans was also observed in experiments involving multiple-tethered DNA molecules. The large nucleoprotein complexes are presumably formed by a combi-nation of ParB-ParB interactions together with non-specific ParB-DNA interactions.

We speculate that non-specific binding may occur at an unidentified site on the pro-tein, such that the nucleoprotein condensates could be anchored around parS by an

74 4.2. Materials and Methods