oriD structure controls RepD initiation during rolling-circle replication

Abstract

Bacterial antibiotic resistance is often carried by circular DNA plasmids that are copied separately from the genomic DNA and can be passed to other bacteria, spreading the resistance. The chloramphenicol-resistance plasmid pC221 from Staphylococcus aureus is duplicated by a process called asymmetric rolling circle replication. It is not fully understood how the replication process is regulated but its initiation requires a plasmid-encoded protein called RepD that nicks one strand of the parent plasmid at the double-stranded origin of replication (oriD). Using magnetic tweezers to control the DNA linking number we found RepD nicking occurred only when DNA was negatively supercoiled and that binding of a non-nicking mutant (RepDY188F) stabilized secondary structure formation at oriD. Quenched-flow experiments showed the inverted complementary repeat sequence, ICRII, within oriD was most important for rapid nicking of intact plasmids. Our results show that cruciform formation at oriD is an important control for initiation of plasmid replication.

Single-molecule measurements reveal that PARP1 condenses DNA by loop stabilization

“…A lookup table for calculating z position was created by stepping the objective using a piezo actuator (P721 PiFoc, Physik Instrumente). Bead positions were tracked, in real time, using a previously described algorithm ( 71 ). The force, F , was calibrated according to the formula F = k b Tl /< x 2 >, where k b is the Boltzmann constant, T is the absolute temperature, l is the DNA extension, and < x 2 > is a variance in bead x position (parallel to the magnetic field lines).…”

Interaction of Proteins with Inverted Repeats and Cruciform Structures in Nucleic Acids

“…Cruciforms have also been demonstrated to influence various aspects of DNA replication. A range of studies confirmed cruciform formation in the origins of replication in bacteria, yeast, and mammalian cells [ 15 , 103 ]. Furthermore, several proteins involved in replication bind to cruciform structures, such as S16, MLL, WRN, and 14-3-3 ( Table 1 ).…”

Microfluidic flow-cell with passive flow control for microscopy applications

“…The bead position was tracked by video microscopy using a Nikon TE-2000 microscope, high powered LED illuminator and 60x Nikon air objective. The z-height of the bead was computed by using a look-up table for the diffraction ring pattern [21]. The microscope drift was corrected by subtracting the position of a fiducial silica bead which was bound to the surface.…”