In response to DNA damage, cells activate checkpoint signaling cascades to control cell-cycle progression and elicit DNA repair in order to maintain genomic integrity. The sensing and repair of lesions is critical for Bacillus subtilis cells entering the developmental process of sporulation as damaged DNA may prevent the cells from completing spore morphogenesis. We report the identification of the protein DisA (DNA integrity scanning protein, annotated YacK), which is required to delay the initiation of sporulation in response to chromosomal damage. DisA is a nonspecific DNA binding protein that forms a single focus, which moves rapidly within the bacterial cell, pausing at sites of DNA damage. We propose that the DisA focus scans along the chromosomes searching for lesions. Upon encountering a lesion, DisA delays entry into sporulation until the damage is repaired.

Emerging evidence indicates that the global organization of the bacterial chromosome is defined by its physical map. This architectural understanding has been gained mainly by observing the localization and dynamics of specific chromosomal loci. However, the spatial and temporal organization of the entire mass of newly synthesized DNA remains elusive. To visualize replicated DNA within living cells, we developed an experimental system in the bacterium Bacillus subtilis whereby fluorescently labeled nucleotides are incorporated into the chromosome as it is being replicated. Here, we present the first visualization of replication morphologies exhibited by the bacterial chromosome. At the start of replication, newly synthesized DNA is translocated via a helical structure from midcell toward the poles, where it accumulates. Next, additionally synthesized DNA forms a second, visually distinct helix that interweaves with the original one. In the final stage of replication, the space between the two helices is filled up with the very last synthesized DNA. This striking geometry provides insight into the three-dimensional conformation of the replicating chromosome.Bacillus subtilis ͉ bacterial cell biology ͉ DNA replication ͉ nucleoid ͉ replisome F aithful DNA replication and segregation are essential for all prokaryotic and eukaryotic cells to maintain their genomic integrity. The topological features of DNA, combined with its enormous length, raise the fundamental questions of how replication is organized spatially and what is the geometry of the replicating chromosome. These questions are even more crucial for bacteria, because they are capable of initiating a new round of DNA replication before the previous round has been completed, resulting in multifork replication (1). Moreover, bacteria lack a nucleus compartmentalizing their DNA; instead the bacterial DNA mass (nucleoid) occupies the majority of the cytoplasmic space. Nevertheless, the nucleoid appears as a well organized structure in which chromosome replication and partitioning are carried out with remarkable spatial and temporal regulation (2-9).Like many bacteria, Bacillus subtilis has a single circular chromosome (Ϸ4,200 kilobase pairs), where DNA replication is initiated from a single origin (oriC) and proceeds bidirectionally. Visualization of specific B. subtilis chromosomal loci has revealed that, in general, the oriC regions are duplicated at the cell center and then move toward opposite poles, whereas the unduplicated terminus region remains centrally located throughout the replication process (10-14). The replication machinery (replisome) is composed of two complexes, one for each replication fork, that tend to remain at midcell during chromosome duplication (15,16). It has been proposed that unduplicated DNA is pulled into the replisome and newly synthesized DNA is extruded bidirectionally from the complex (4, 16).The spatial and temporal organization of the replicating bacterial chromosome has been elucidated mainly by observing the localization a...