Interaction with single-stranded DNA-binding protein localizes ribonuclease HI to DNA replication forks and facilitates R-loop removal

Abstract

DNA replication complexes (replisomes) routinely encounter proteins and unusual nucleic acid structures that can impede their progress. Barriers can include transcription complexes and R-loops that form when RNA hybridizes with complementary DNA templates behind RNA polymerases. Cells encode several RNA polymerase and R-loop clearance mechanisms to limit replisome exposure to these potential obstructions. One such mechanism is hydrolysis of R-loops by ribonuclease HI (RNase HI). Here, we examine the cellular role of the interaction between Escherichia coli RNase HI and the single-stranded DNA-binding protein (SSB) in this process. Interaction with SSB localizes RNase HI foci to DNA replication sites. Mutation of rnhA to encode an RNase HI variant that cannot interact with SSB but that maintains enzymatic activity (rnhAK60E) eliminates RNase HI foci. The mutation also produces a media-dependent slow-growth phenotype and an activated DNA damage response in cells lacking Rep helicase, which is an enzyme that disrupts stalled transcription complexes. RNA polymerase variants that are thought to increase or decrease R-loop accumulation enhance or suppress, respectively, the growth phenotype of rnhAK60E rep::kan strains. These results identify a cellular role for the RNase HI/SSB interaction in helping to clear R-loops that block DNA replication.

Keywords: DNA replication; genome maintenance; transcription.

Figure 1.. SSB-mediated localization of RNase HI to the replication fork.

Fluorescence microscopy studies of E. coli strains expressing the fluorescent fusion proteins RNase HI-YPet and β-clamp-mCherry. (A) Representative images showing RNase HI-YPet (left), β-clamp-mCherry (middle) and a merged image showing the overlap of RNase HI-YPet and β-clamp-mCherry (right) in strain VV11. Digital contrast enhancement was used for presentation purposes. Scale bar in the right image is 2 μm. (B) Detectable RNase HI-YPet and β-clamp-mCherry foci per cell are plotted as the frequency for the cell population. (C) Radial distribution function for RNase HI-YPet and β-clamp-mCherry representing colocalization (blue line). As comparison, g(r) is plotted for a set of randomly distributed spots in cells (grey line). Inset shows the distribution of nearest-neighbor distances between spots of RNase HI-YPet and β-clamp-mCherry. (D) Representative fluorescent image of showing RNase HI K60E-YPet fusion protein distributed throughout strain VV08. Imaging conditions and digital contrast enhancement used were as in 1B. Scale bar is 2 μm.

Figure 2.. The rnhAK60E rep::kan strain is sensitive to growth on rich medium plates.

(A) Dilutions of overnight cultures grown in minimal medium (56/2) and plated on minimal (left) or LB media (middle and right). Plates were incubated at 37°C for 24 or 48 hours. The images are representative of plating experiments performed in triplicate. Strains are CP65, CP58, CP84, and CP86 from top to bottom. (B & C) The CFU/mL of each strain (normalized to OD₆₀₀) is plotted from overnight cultures diluted and plated on minimal (squares) or LB (circles) media. Colonies were quantitated after growth at 37°C for 24 or 48 hours. Each symbol is a single culture and the mean CFU/mL for each strain is represented by a black line. Strains are CP65, CP58, CP84, and CP86 from left to right. Error bars indicate the standard deviation. ** = p-value < 0.005 and n.s = p-value > 0.05 using two-tailed t-test.

Figure 3.. The rnhAK60E rep::kan strain has elevated SOS levels in rich medium.

(A) The relative fluorescent units (RFU) from plasmid-borne GFP driven by a recN promoter is plotted for each strain at mid-log phase and normalized to CFU/mL at the time of data collection. Bars represent the mean RFU/CFU/mL from biological and technical replicates of strains were grown in minimal medium (56/2) and LB. Error bars display the standard deviation of the mean. Mean values are written above each bar. Strains in this figure are CP127, CP128, CP129, and CP126 (from left to right). (B) Representative phase contrast images from strains grown in minimal medium (left) or LB (right). The strains were grown to early log phase and subsequently spotted on 2% agarose pads for imaging using a Nikon Eclipse Ti microscope equipped with a Photometrics CoolSNAP HQ2 charge-coupled-device (CCD) camera. (C) The cell lengths captured by MATLAB of microscope images. Cells were grown in minimal medium or LB and treated as in (B).

Figure 4.. The plating efficiency of rnhAK60E rep::kan rpoB mutants on minimal and rich media.

The CFU/mL of each strain (normalized to OD₆₀₀) is plotted from overnight cultures diluted and plated on minimal (squares) or LB (circles) media. Colonies were quantitated after growth at 37°C for 24 or 48 hours. Each symbol is a single culture and the mean CFU/mL for each strain is represented by a black line. The strains are CP65, CP86, CP119, and CP124 from left to right. The error bars indicate the standard deviation. Significant difference levels (p-values) were determined between rnhAK60E rep::kan strains +/− rpoB mutants grown in the same medium using two-tailed t-test. * = p-value < 0.05; ** = p-value < 0.005; *** = p-value < 0.0005; n.s = p-value > 0.05

Figure 5.. Schematic model for RNase HI and Rep helicase localization and action at sites of replication/transcription collision.

RNase HI (purple) is localized to the DNA replication fork by interaction with SSB (yellow). Rep helicase (grey) is localized by interaction with DnaB (orange). SSB-Ct tails are shown explicitly for only one SSB tetramer for clarity. DNA strands are shown in blue, RNA strands are shown in red, and DNA polymerases are shown in green. Several replisome components have been omitted or separated for clarity.