Control of the replication initiator DnaA by an anti-cooperativity factor

Abstract

Summary Proper coordination of DNA replication with cell growth and division is critical for production of viable progeny. In bacteria, coordination of DNA replication with cell growth is generally achieved by controlling activity of the replication initiator DnaA and its access to the chromosomal origin of replication, oriC. Here we describe a previously unknown mechanism for regulation of DnaA. YabA, a negative regulator of replication initiation in Bacillus subtilis, interacts with DnaA and DnaN, the sliding (processivity) clamp of DNA polymerase. We found that in vivo, YabA associated with the oriC region in a DnaA-dependent manner and limited the amount of DnaA at oriC. In vitro, purified YabA altered binding of DnaA to DNA by inhibiting cooperativity. Though previously undescribed, proteins that directly inhibit cooperativity may be a common mechanism for regulating replication initiation. Conditions that cause release of DnaN from the replisome, or overproduction of DnaN, caused decreased association of YabA and increased association of DnaA with oriC. This effect of DnaN, either directly or indirectly, is likely responsible, in part, for enabling initiation of a new round of replication following completion of a previous round.

The Primosomal Protein DnaD Inhibits Cooperative DNA Binding by the Replication Initiator DnaA in Bacillus subtilis

“…Binding was highly cooperative and had a Hill coefficient of 8. These results are consistent with previous findings (40).…”

“…We found that the ATPase activity of DnaA was not needed for these effects by DnaD, indicating that DnaD is not regulating the ATPase activity of DnaA. These effects of DnaD on the ability of DnaA to bind DNA are similar to the effects of YabA (40) and Soj (55), two other regulators of B. subtilis DnaA and replication initiation, and further substantiate the notion that modulation of cooperative binding and oligomerization of DnaA to DNA might be a common mechanism of regulation (40,55).…”

“…In contrast, DnaD had a marked effect on the ability of DnaA to bind DNA. Binding of DnaA-ATP to DNA fragments that contain multiple binding sites is normally highly cooperative (40,41). We found that in the presence of DnaD, binding of DnaA to DNA was no longer cooperative and the apparent dissociation constant (K d ) for DnaA and DNA was reduced.…”

“…Some factors that affect the activity of DnaA, predominantly in E. coli, are known to alter its nucleotide-bound state (16,26,64). In contrast, regulators of B. subtilis DnaA, (e.g., YabA, Soj, and SirA) are known to alter its DNA binding properties (40,53,55,66). Using purified proteins, we tested for effects of DnaD both on the ability of DnaA to bind DNA and on nucleotide exchange.…”

The Blueprint of a Minimal Cell: MiniBacillus

“…Rtp is required for the correct termination of DNA replication and the subsequent segregation of daughter chromosomes (47). YabA, on the other hand, controls replication initiation by inhibiting the oligomerization of the initiator protein DnaA (48). Finally, DNA polymerase I fulfills an essential function, the removal of RNA primers that initiate the synthesis of Okazaki fragments.…”